Contents

Plenary Lectures: ...................................................................................................................... 1

White Biotechnology

Session 1: Nanotechnologies in industrial processes ............................................................................. 3 Chair: Zygmunt Kowalski

Session 2: Development of renewable energy in biotechnology .......................................................... 13 Chair: Henryk Kołoczek

Session 3: Bioeconomy ............................................................................................................................... 22 Chair: Stanisław Bielecki

Session 4: Bioplastics and biobased polymers ........................................................................................ 26 Chair: Andrzej Kononowicz, Krzysztof Pielichowski

Session 5: Pharmaceutical biotechnology ............................................................................................... 33 Chair: Alicja Józkowicz, Katarzyna Kieć-Kononowicz

Green Biotechnology

Session 6: Plant molecular breeding......................................................................................................... 60 Chair: Dariusz Grzebelus

Session 7: Environmental biotechnology ................................................................................................ 75 Chair: Katarzyna Turnau

Session 8: Plant genetic engineering ........................................................................................................ 125 Chair: Rafał Barański

Session 9: Animal biotechnology in biomedicine .................................................................................. 134 Chair: Zdzisław Smorąg, Ryszard Słomski

Session 10: Animal biotechnology in agriculture ..................................................................................... 148 Chair: Krystyna Koziec, Arieh Gertler

Session 11: Legislation for biotechnologists .............................................................................................. 165 Chair: Tomasz Twardowski

Satellite Panels

Session 1: Induced pluripotent stem cells: a future of biomedicine ..................................................... 168 Chair: Alicja Józkowicz

Session 2 : Business Session – Life Science Open Space Chair : Kazimierz Murzyn

Preface by the Organizing Committee

Dear Madame/Sir,

It gives me great pleasure to welcome you, on behalf of the Organizing Committee, to the V Central European Congress of Life Sciences – 2013 Eurobiotech in the Royal City of Crakow. The Congress is held under the honorary patronage of the Voivode of the Malopolska Voivodeship, the Marshal of the Malopolska Voivodeship and the Mayor of the Royal Capital City of Crakow.The Congress has been organized by Crakow University of Technology (member of the Organizing Committee for the first time this year), the University of Agriculture, the Jagiellonian University, Biotechnology Committee of Polish Academy of Sciences, Polish Federation of Biotechnology, Malopolska Center of Biotechnology, Life Science Center in Crakow, Jagiellonian Center of Innovation Ltd. and Targi w Krakowie Ltd. Satellite sessions devoted to stem cells and bio-business discussions will precede the opening ceremony of the Congress. The scientific programme of the Eurobiotech 2013 will combine white and green biotechnology topics as well as legislation problems in the biotechnology sector.Outstanding scientists from Europe, USA and other countries have been invited to deliver lectures on such important issues as nanotechnologies, renewable energy, environmental biotechnology or animal biotechnology in biomedicine and agriculture and many others.We hope that the V Eurobiotech Congress and the accompanying exhibitions will enable you to exchange your scientific opinions and to present the latest biotechnological achievements and future trends in the area of white and green biotechnologies. We are convinced that the Congress being of great value to the Polish science will encourage the invited scientists and businessmen to start long-term scientific and business cooperation.We warmly welcome you to the Congress hoping that its main goal, providing you with a scientific programme of the highest quality, will be achieved and that you will spend wonderful time in our vibrant city with great cultural and historical dignity.

On behalf of the Congress Organizing Committee

Henryk KoloczekCongress ChairCrakow University of TechnologyCrakow, October 2013

Plenary lectures

PL1

Synthetic Plant Biotechnology Tools for Precise Transgene Expression C Neal Stewart, Jr.

Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA

Synthetic biology tools have the ability to greatly increase the precision and amount of gene expression in transgen-ic plants. The presentation will discuss synthetic promot-ers for inducible gene expression and how they are being utilized in our phytosensors research program. Here, we are producing phytosensor tobacco plants with an orange fluorescent protein reporter gene that is induced upon pathogen infection. This research is designed to fit into precision agriculture systems to allow farmers to treat for plant pathogens where needed and prior to when disease symptoms are observed. We are also studying how syn-thetic transcription activator-like effectors (TALEs) can be used as transcription factors to increase gene expres-sion. We are using designer TALE-TFs that bind to the flanking regions of the TATA-box motif on the CaMV 35S promoter for the purpose understanding the engineera-ble “hot-spots” for increasing transgene expression. We demonstrated that the de novo-engineered TALEs could increase reporter gene expression by up to 3 fold in stable transgenic tobacco harboring an orange fluorescent pro-tein reporter gene under the control of synthetic induci-ble, minimal, or full-length 35S promoters. These results provide novel insights into the potential applications of synthetic promoters and TALE-TFs for targeted gene ac-tivation of transgenes in plants.

PL2

Oxidative Stress Signal Transduction in plants Frank Van Breusegem

VIB Department of Plant Systems Biology, UGent Technologie Park 927 B-9052 Gent, Belgium

Different biotic and abiotic stresses adversely affect plant growth and development. A common theme within these environmental factors is the perturbation of reactive ox-ygen species (ROS) homeostasis. The signal transduction mechanisms of the oxidative stress response in plants are poorly understood. We conducted genetic and chemical screens, which combined with proteomic approaches will lead to a more comprehensive overview on the com-ponents of the network that govern the oxidative stress response. These efforts resulted in the identification of a  number of genes as new members of the oxidative stress gene network in plants. In our recent studies, we have identified a novel transcriptional regulator of the mitochondrial retrograde regulation and are assessing the modulation of cellular NAD(P) homeostasis as a new strategy to improve plant performance in stress condi-tions.

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PL3

Teaching from the Plant Kingdome; Energy, Economics and the EnvironmentStanisław Karpiński

Warsaw University of Life Sciences,Department of Genetics, Breeding and Plant Biotechnology,Nowoursynowska 159, 02-776 Warsaw, Poland

Almost all of the modern infrastructure of our civilization relies on fossil fuels for energy. Despite clear evidence that the combustion of fossil fuels over the 20th and the beginning of 21st century has led to significant increase in atmospheric carbon dioxide that is altering the global environment, there has been little collective political will to alter the energy sources of the largest industrial societies. This economic positive feedback is further exacerbated by the abundance of large quantities of fossil fuels, especially coal and natural gas, remaining in known reservoirs. To alter this trajectory in the 21st century, concerted investment in alternative, clean energy technologies must be made both by governments and private industries. In this lecture, I will discuss potential of plant and photosynthesis biotechnologies, and I will present lesson of economy from the Plant Kingdome for encouraging investment by government and private industries.

PL4

Luuk A.M. van der Wielen

Delft University of Technology, The Netherlands, Industrial Biotechnology (tentative title)

Nanotechnologies in industrial processes

LecturesL1.1

The use of biofiltration in the rendering plants L. Tymczyna1, A. Chmielowiec-Korzeniowska1, Z. Paluszak2, M. Banach3, J. Pulit3

1Uniwersytet Przyrodniczy w Lublinie; 2Uniwersytet Technologiczno-Przyrodniczy w Bydgoszczy; 3Politechnika Krakowska

The study evaluated the effectiveness of air biofiltration of rendering plants. The biofilter material comprised com-post soil (40%) and peat (40%) mixed up with coconut fiber (medium A) and oak bark (medium B). During bio-filtration average reduction of VOCs was 88.4% for me-dium A and 89.7% for medium B. There was a positive relationship of the reduction of aldehydes from material humidity (r = 0.502, α < 0.05). Other parameters of the biomaterial does not have a significant impact on the effi-ciency of the treatment.

L1.2

Use of titanium dioxide for purification, self-cleaning and disinfection in urban infrastructuresAgata Markowska-Szczupak1, Beata Tryba1, Antoni W. Morawski1

1Institute of Chemical and Environment Engineering, West Pomeranian University of Technology, Szczecin

Titanium dioxide is commonly used as a white pigment for paints and lacquers (about 70% of its total produc-tion). Recently it has emerged as an excellent photocata-lytic material for environment purification, self-cleaning materials and disinfection. In this review, current prog-ress in this area of TiO2 applications is presented.Titanium dioxide, particularly in the anatase form, can be use as a photocatalyst when it is excitated with the UV light. The overall photocatalytic activity of titanium diox-ide particles depends on number of parameters such as: method of preparation, phase composition, ratio between the anatase and rutile crystal phases, anatase crystallite size, excitation light wavelength and many others. The strong oxidative potential of hydroxyl radicals which are generated on the surface of TiO2 particles after its exci-tation is responsible for oxidation a huge number of or-ganic and inorganic compounds such as: nitrogen oxides, aromatic hydrocarbons, aldehydes, ketones, VOCs, drugs, pesticides etc. [1].The development of new photocatalysts for the optimiza-tion of photocatalytic performance is promoted in various directions. To achieve improved photocatalytic activity of TiO2, particularly under visible light excitation, different modifications are performed such as: doping of both, metal or non-metal ions, creating of nonstructured ma-terials (e.g. nanotubes, nanowires, nanoshells etc.), and formation of co-catalysts.New generation photocatalysts based on titanium diox-ide are added to some building materials (e.g. paints, ce-ments, gypsum, glasses, tiles etc.) in order to form their self-cleaning properties. The most effective use of photo-catalytic materials can be seen on large-scale construction projects, in which the large surfaces are exposed to the light promoting self-cleaning and anti-pollutant perfor-mance. For this reason facades, roadways and pavements in the traffic urban areas became the target for the first ex-amples of utilisation these photocatalytic materials. Such applications are already in progress in Japan and Europe and are recently initiated in the United States.Under solar or UV light irradiation TiO2 exhibits the su-perhydrophilic properties by reducing the water contact

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angle, thus gives increased self-cleaning capacity and cooling effect. It was found the application in production of anti-fogging windows, car mirrors, glasses. The cooling properties let to reduce the amount of energy consump-tion used for operation of air-conditioners and also artifi-cial heat emission in glass buildings [2].Titanium dioxide is internationally recognized as one of the new sterilization materials, which can kill wild spec-trum of organisms including viruses, bacteria, fungi and algae. It has been widely used in places with high steril-ization demanding such as hospitals, institutions, schools etc. Self-cleaning fabrics could revolutionize the sport and army apparel industry [3].The titania nano-biotechnology is considered as alterna-tive method of cancer therapy, which targets only cancer cells and does not affect normal living tissue [4].Titanium dioxide is used in numerous applications be-cause of its compatibility with modern technology. New applications involving TiO2 can improve our lives in areas such as water treatment, indoor and outdoors purifica-tion and energy saving.AcknowledgementsThis work was supported by National Centre for Science and Ministry of Science and Higher Education of Poland under project No. MNiSW/DPN/4878/TD/2010. References[1] Carp O., Huisman C.L., Reller A. (2004) Photoinduced reactivity of titanium dioxide, Prog. Solid State Chem. 32: 33–177.[2] Nakata K., Fujishima A., (2012) TiO2 photocatalysis: design and applications. J. Photochem. Photobiology C: Photochem. Rev. 13: 169–189.[3] Markowska-Szczupak A., Ulfig K., Morawski A.W. (2011) The application of titanium dioxide for deactivation of bioparticulates: an overview, Catal. Today, 169: 249–257.[4] Rozhkova E., Ulasov I., Lai B., Dimitrijevic N.M., Lesniak M.S., Rajh T. (2009) Nanobio photocatalyst for targeted brain cancer therapy, Nano Lett. 9: 3337–3342.

L1.3

Nanosilver – a new product for disinfecting treatmentMarcin Banach1, Jolanta Pulit1, Leszek Tymczyna2, Anna Chmielowiec-Korzeniowska2

1Institute of Inorganic Chemistry and Technology, Crakow University of Technology; 2University of Life Sciences in Lublin

The human body is exposed to microorganisms such as bacteria, viruses or fungi. Micro-organisms and their remnants, may cause imbalance of enzymatic homeosta-sis in one’s organism and may exert immunotoxic effects on the immune system [1].The amount of chemicals used for disinfecting is limit-ed due to their negative impact on the body and because of the difficulties with their solubility and with the pos-sibility of direct application. In addition, the ecological consumer lifestyle forces to use only those chemicals that occur naturally in nature, or whose use does not pose a threat to the environment. Organic acids and their salts, 1-Hexadecylpyridinium chloride, sodium orthophos-phate, hydrogen peroxide and sodium bicarbonate are a large group of compounds among preferred additives destroying bacterial flora. However, not all of these com-pounds appear to be fully effective, so new methods of microorganisms disposing still have been looked for. Sil-ver nanoparticles, which, according to the literature ex-hibit good biocide effect, may be an alternative for those disinfectants [2–4].Nanostructured silver is an effective destructing agent against a broad spectrum of Gram-negative and Gram-positive bacteria, not excluding antibiotic-resis-tant strains [5]. It is characterized by a specific surface and a high content of surface atoms which induces a rare physicochemical properties. The results suggest that met-al which reacts with a thiol group (–SH) binds with a pro-tein of the microorganism, which leads to its inactivation [4, 6].Therefore, nanotechnology takes a considerable chal-lenge, which aims to reduce pathogenic bacterial activity.However, nanometals toxicity against eukaryotic cells, in-cluding the cells of vertebrates is an important issue. Re-sults of in vitro studies presented in the literature confirm nanometals cytotoxicity, particularly nanostructured silver which is destroying to the rat liver cells, neurons, human lung epithelial cells and murine stem cells. Silver nanopar-ticles may also act genotoxic. Some in vivo studies have also shown nanometals effect on the cardiovascular sys-tem, respiratory system, central nervous system and liver. However, these data are incomplete and their confirmation requires further research in this direction [6].The work presents results of research related to obtaining nanostructured silver, its characteristics, biocidal properties and uses for disinfection purposes.AcknowledgementsThe scientific work is financed for the years 2010–2013 as research project 4111/B/H03/2010/39.

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References[1] Burrell R. (1995) Ann. Agric. Environ. Med. 2, 11.[2] Kowalski Z., Banach M., Powałka E. (2009) Przem. Chem. 88, 478.[3] Konopka M., Kowalski Z., Wzorek Z. (2009) Arch. Environ. Prot. 35, 107.[4] Banach M., Kowalski Z., Wzorek Z. (2007) Chemik 9, 435.[5] Wright J.B., Lam K., Hansen D., Burrell R.E. (1999) Am. J. Infect. Control 27, 344.[6] Banach M., Pulit J. (2013) Przem. Chem. 92, 6, 1056.

L1.4

Pig manure treatment by filtrationZygmunt Kowalski1, Agnieszka Makara1, Dalibor Matýsek2, Józef Hoffmann3, Krystyna Hoffmann3

1Institute of Chemistry and Inorganic Technology, Crakow University of Technology, Warszawska 24, 31-155 Crakow, Poland; 2Institute of Geological Engineering, VŠB-Technical University of Ostrava, 17. listopadu 15, 708 33 Ostrava-Poruba, Czech Republic; 3Institute of Inorganic Technology and Mineral Fertilizers, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland

Numerous livestock farms in Europe import fodders in-cluding the nitrogen (N) and phosphorus (P)-bearing ingredients that these fodders contain. This results in local nutrient surpluses because home-grown crops take up less N and P than available in the manure. We have developed in our study new pig manure treatment and filtration process. The advantage of worked out technolo-gy is the method of incorporation in organic phase of the manure of 40–59% of crystalline phase. This resulted in achievement of high filtration rate over 1000 L/m2/h with the used pressure filters and good quality of filtrate. The method is able to maintain an overall average pollutant removal performance ~90% for the chemical oxide de-mand COD, > 99% for the suspended solids SS, to 47% for the total nitrogen content determined with Kjeldahl’s method TKN. Preliminary investigation indicated that the mineralization process eliminates over 75% of the odor intensity coming from the filtrate obtained in com-parison to odor intensity coming from the pig manure. AcknowledgementsThis study has been carried out in the framework of the Development Project No. 14-0003-10/2010 granted by the National Centre for Research and Development.

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L1.5

Phosphorus cycle –possibilies of its rebulidngKatarzyna Gorazda, Zbigniew Wzorek, Anna K. Nowak, Barbara Tarko

Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, PolandInstitute of Inorganic Chemistry and Technology

The recovery of phosphorus from waste can take place in many ways: from the waste water streams, sewage sludge, animal waste, industrial waste and other indus-trial emissions. It is proposed, that thermal processing of sewage sludge, where phosphorus is removed by chem-ical precipitation, will be a preferable method for their utilisation. Obtain product in the form of ash could be potentially used as a substitute of natural ore. Developed by Cracow University of Technology and patented meth-od of phosphorus recovery by acid extraction methods allows for the phosphorus recovery efficiency between 80–96%. The average phosphorus content in Polish ash-es is around 9.7%. 3000 to 4000 tons of phosphorus per year can be recycled and introduced to the environment. That amount is equivalent to 30 770 t of phosphorus ore. The proposed technology allowed finding out alternative sources of phosphorus for individual industries.

L1.6

Nanoemulsion as a base to controlled release of forskolinMałgorzata Jaworska1, Elżbieta Sikora1, Maria Jose Garcia-Celma3, Elvira Escribano3, Conxita Solans4, Jan Ogonowski1

1Faculty of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Crakow University of Technology; 2Dept. of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Barcelona, Spain; 3Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain

Forskolin is one of labdane diterpenoids. It is isolated from the roots of Coleus Forskohlii, an aromatic herb belongs to the family of mints and lavenders originated from India. It is characterized by many biological effect e.g. stimulates adenylate cyclase, resulting in increase of cAMP levels which is related to biochemical mechanism of maintaining or increasing lean body mass [1]. More-over, its applied in the treatment of atopic dermatisis and used as an antiallergic agent [2]. Nanoemulsions are a new form of cosmetic and pharmaceutic products. They show some advantages comparing to classic emulsions: relatively easily penetrate into the skin, exhibit a high degree of hydration and soften the skin and have a very good user properties. They are one of the most promising formulations used to enhance percutaneous penetration of active substances as well as their bioavailability [3, 4]. The aim of this work was to determine the release of for-skolin from nanoemulsion. The nano-emulsion based on caprylic/capric triglycerides and stabilized by Polisorbate 80 was obtained. The formulation was prepared by step-wise addition of water to a mixture of polisorbate/oil/forskolin, at room temperature. Drug release study of for-skolin was carried out using the Spectra/Por Standard Regenerated Cellulose (RC). The concentration of the active in the receptor solution (i.e. Ethanol/PBS mixture) was analyzed by means spectrophotometer UV-VIS and HPLC. The results have shown that nanoemulsions can be used as carriers for controlled release of forskolinAcknowledgementsThe research (work) was supported by the European Union through the research scholarship “Doctus – Małopolski fundusz stypendialny dla doktorantów”.References[1] Bagchi D., Preuss H.G. (2012) Obesity: Epidemiology, Pathophysiol-ogy and Prevention, Taylor and Francis Group.[2] Ciotonea C., Cernatesul C. (2001) Biological active effects of Forskolin extract, Buletinul InstitutulviPolitehnic DIN IASI, 4, 95–106.[3] Gugliemini G. (2006) Evaluating droplet size in nanoemulsions from a novel emulsifier system, Cosmetics and Toiletries. 121 (12), 67–72.[4] Tharwat Tadros, Izquierdo P., Esquena J., Solans C. (2004) Formation and stability of nano-emulsions, Advances in Colloid and Interface Science.108-109, 303–318.

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Posters

P1.1

Preparation of Ag nanoparticles using plant extractsZygmunt Sadowski, Hanna Hyż, Szcepan Gorzynik

Wroclaw University of Technology, Chemical Engineering Department

Present study reports a green chemistry approach for the synthesis of silver nanoparticles. A simple proce-dure using Aloe vera and Pelargonium graveolens leaf extracts was used to produce of silver nanoparticles. The extract from dried powder of Punica granatum was also used as reducining agent. The silver nanoparticles syn-thesized using the plant extracts were characterized by UV-vis spectroscopy. The results from UV-vis show that the reduction mechanism of silver ions was affected by the extract concentration and precursor solution (silver nitrate). The temperature of extract preparation was re-sponsible for the synthesis of silver nanoparticles. The bioreduction of silver ions occurred at room temperature in a few hours. The kinetics of reduction reaction was investigated. Biosynthesis of silver nanoparticles using plant extract is considered to be a novel, effective and eco-friendly method. AcknowledgementsZlecenie Nr S20072/Z0307.

P1.2

Studies on the formation of O/W nano-emulsions, by low-energy emulsification method, suitable for cosmeceutical applicationsMałgorzata Jaworska1, Elżbieta Sikora1, Michał Zielina2, Jan Ogonowski1

1Faculty of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Crakow University of Technology; 2Faculty of Environmental Engineering, Institute of Water Supply and Environmental Protection, Crakow University of Technology

Nanoemulsions offer several advantages for cosmeceu-tical use. They are easy to prepared, they characterized by long term stability, high solubilization capacity for hydrophilic and lipophilic actives and additionally im-proved transdermal active delivery [1, 2]. The formation of O/W nano-emulsions suitable for cosmeceutical ap-plication was studied. Nano-emulsions were prepared by using phase inversion composition (PIC) method, one of low-energy emulsification methods. The process consist of stepwise water addition to oil/surfactant mixture, at 25°C. Caprylic/capric triglycerides, propylene glycol di-caprylate/dicaprate and oleic acid were applied as an oil phase. Polisorbate 80 was used as the surfactant. Kinetic stability of the nano-emulsions was analyzed by measur-ing droplet diameter as a function of time for different oil/surfactant ratio. The particles size distribution was analyzed by means DLS measurement technique (Dy-namic Light Scattering), using Zetasizer Nano ZS (Mal-vern Instruments). One of triterpenoic acid, practically non-water soluble substance was selected as an active and incorporated into the stable formulation. The obtained results proved that the nanoemulsion based on caprylic/capric triglycerides was the most stable one and addition-ally showed the highest solubilisation for the triterpene. The maximum concentration of the active achieved in oil/surfactant mixture (20/80) was 2,5 mg/ml.AcknowledgementsThe research (work) was supported by the European Union through the research scholarship “Doctus – Małopolski fundusz stypendialny dla doktorantów”.References[1] Maghraby G.M., Transdermal delivery of hydrocortisone from eucalyptus oil microemulsion: effects of cosurfactants. International Journal of Pharmaceutics, 2008, 355(1–2), 285–292.[2] Peltola S., Saarinen-Savolainen P., Kiesvaara J., Suhonen T.M., Urtii A., Microemulsions for topical delivery of estradiol, International Journal of Pharmaceutics, 2003, 254, 99.

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P1.3

Influence of process parameters on properties of Nanostructured Lipid Carriers (NLC) formulationElwira Lasoń, Elżbieta Sikora, Jan Ogonowski

Institute of Organic Chemistry and Technology, Crakow University of Technology, Poland

Nanostructured lipid carriers (NLC) are stabile colloidal formulations with notable advantages for drug delivery systems, i.e. physicochemical stability, biocompatibility, biodegradability and controlled drug release. NLC are de-rived from o/w emulsions by simply replacing the liquid lipids (oils) by a solid lipids. Due to controlled nanostruc-ture, the particles offers enough space to accommodate the active ingredient [1, 2]. The particles are produced through a mixing of solid and liquid lipid with water phase in the presence of surfactants. The most preferred NLC production methods are high pressure homogeniza-tion and ultrasound technology.The aim of the study was preparation and characteriza-tion of nanostructured lipid carriers (NLC). Both, effect of process parameters and effect of preemulsion compo-sition on the NLC properties were investigated. In the work, different type of surfactants (i.e. decyl glucoside, Poloxamer188, Tween 80, sodium cholate) and their com-binations were used to stabilize NLC dispersions. More-over, several kinds of solid lipids (modified beeswax, glic-eryl behenate, cetyl palmitate and berry wax) and liquid lipids (caprilic/capric triglyceride and decyl oleate) were applied. An ultrasonication method using a probe type sonicator (Sonics Vibra-Cell, Sonics & Materials, INC.), were used to obtained NLC, the time and energy of the process were modifying. The physicochemical properties of the formulations, such as particle size, size distribu-tion, polidispersity index and zeta potential were studied using dynamic light scattering (DLS) method (Malvern Zetasizer instrument).The obtained results showed that the process parameters and the composition of preemulsion significant influ-ence on the nanoparticles structure. Stable formulation, of particles size approximately 80 nm, were prepared by ultrasonified the mixture for 10 min at 35W, maintaining the temperature at least 5°C above the lipid melting point. As a stabilizing agent alkylpolyglucoside and its combina-tion with some other surfactants give the best effect.AcknowledgementsThe research (work) was supported by the European Union through the European Social Fund within “Cracow University of Technology development program – top quality teaching for the prospective Polish engineers; University of the 21st century” project (contract No. UDA-POKL.04.01.01-00-029/10-00). References[1] Müller R.H., Petersen R.D., Hommoss A., Pardeike J. (2007) Nanostructured lipid carriers (NLC) in cosmetic dermal products, Adv. Drug Deliv. Rev. 59: 522–530.[2] Radtke M., Souto E., Müller R.H. (2005) Nanostructured lipid carriers: a novel generation of solid lipid drug carriers, Pharm. Technol. Eur. 17: 45–50.

P1.4

Nanometals as tool in fight against plant pathogensAgnieszka Sobczak-Kupiec1, Dagmara Malina1, Bożena Tyliszczak2, Zbigniew J. Burgieł3

1Institute of Inorganic Chemistry and Technology, Crakow University of Technology, 24 Warszawska Str., 31-155 Crakow, Poland; 2Department of Chemistry and Technology of Polymers, Crakow University of Technology, 24 Warszawska St., 31-155 Crakow, Poland; 3Department of Plant Protection, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

In recent years, a great interest in nanotechnology has been observed. Nanostructured metallic particles be-cause of their unusual properties, have been the subject of numerous intensive research due to their unusual properties, which are dependent on the nanometric siz-es. Products based on nanoparticles are applied in the electronic, optic, chemical, textile industries, as well as in pharmacy, cosmetology, and medicine. Moreover, certain types of nanometals (e.g. silver and gold nanoparticles) also demonstrate strong antimicrobial activity against bacteria, fungi and viruses.The main aim of this study was to investigate antifun-gal activity of selected metallic nanoparticels towards common pathogenic fungi of crop species. For this pur-pose suspensions of nanosized Ag and Au, proceeded by chemical reduction using a suitable stabilizer, were added to the medium for fungi growth. In the next step, media with appropriate amount of nanoparticles were inoculat-ed with mycelial fragments of selected fungi strains.Nanometals applied in research exhibit high fungicidal activity against all fungi tested independently of con-centration. Based on the obtained results it is assumed that in the future, pesticides harmful for plants may be replaced by nanoparticles free from negative effects on plant growth.AcknowledgementsThe research was funded from the budget for science for years 2013–2014, No. IP2012 062872.

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P1.5

Carbon aerogels based on natural starches – synthesis and characterizationMonika Bakierska, Marcin Molenda, Małgorzata Maria Zaitz, Roman Dziembaj

Faculty of Chemistry, Jagiellonian University

Introduction. Carbon aerogels are a novel class of nano-structured and porous carbon materials which exhibit unusual properties and can be used in a wide variety of applications (eg. energy storage, catalysis, biomedicine etc.) [1–3]. These materials can be obtained by the py-rolysis of organic aerogels at elevated temperatures under inert atmosphere. The most widely used organics for the fabrications of the aerogels via the sol-gel polycondensa-tion are resorcinol and formaldehyde [4]. However, the use of natural polysaccharides and their derivatives is considered to be more attractive because of their stability, availability, renewability, low cost and non toxicity [5–7].The purpose of this study was to prepare and character-ize carbon aerogels based on natural starches of different botanical origin (potato, maize and rice). Moreover, to decrease the process cost and shorten processing time, an ambient pressure drying instead of supercritical drying was applied.Experimental. Carbon aerogels were prepared by the carbonization of organic aerogels. Four main steps can be distinguished in the preparation of these materials, and the first was the gelation process in which different types of starches (potato, maize and rice) were dissolved in water and stirred while maintaining the appropriate gelatinization temperature for each type of starch. The second step was the solvent exchanging carried out ei-ther by soaking the gel directly in the new solvent – eth-anol. The third step was the wet gel drying performed at 50°C for 1 day under ambient pressure and the fourth was the carbonization of dried gel under constant flow of argon at 600°C and after that carbon aerogels were obtained.Optimal conditions for carbonization process of organic aerogels were determined by thermal analysis methods (TGA/DTG/SDTA/EGA-QMS). The structure and the morphology of the prepared carbon aerogels were inves-tigated using X-ray powder diffraction (XRD) and low temperature N2 adsorption measurements (N2-BET). Electrical properties of the obtained aerogels were ex-amined by electrical conductivity studies (EC) using 4-probe method within temperature range of –20°C to +40°C.Conclusions. To conclude, starch-based carbon aero-gels were synthesized by the pyrolysis of organic aero-gels, which were prepared in gelatinization process of natural starches (potato, maize and rice). This method made it possible to obtain nanostructured materials with well-developed porosity and high surface area. The electrical conductivity measurements proved that pre-

pared materials provide good electrical properties. The obtained carbon aerogels seem to be perspective mate-rials which can be widely used in many fields of science and industry.Keywords: carbon aerogels, starches, gelatinization process, textural properties, conductivityAcknowledgementsThis work is supported by the European Institute of Innovation and Technology under the KIC InnoEnergy NewMat project. References[1] Mirzaeian M., Hall P.J. (2009) Electrochimica Acta 54: 7444.[2] Moreno-Castilla C., Maldonado-Hódar F.J. (2005) Carbon 43: 455.[3] García-González C.A., Alnaief M., Smirnova I. (2011) Carbohydrate Polymers 86: 1425.[4] Pekala R.W. (1989) Journal of Materials Science 24: 3221.[5] Aaltonen O., Jauhiainen O. (2009) Carbohydrate Polymers 75: 125.[6] Tsioptsias C., Michailof C., Stauropoulos G., Panayiotou C. (2009) Carbohydrate Polymers 76: 535.[7] Chang X., Chen D., Jiao X. (2010) Polymer 51: 3801.

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P1.6

Characterization and properties of silver nanoparticles for agro-engineering applicationsAgnieszka Sobczak-Kupiec1, Katarzyna Bialik-Wąs2, Bożena Tyliszczak2, Dagmara Malina1, Zbigniew Burgieł3

1Institute of Inorganic Chemistry and Technology, Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, Poland; 2Department of Chemistry and Technology of Polymers, Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, Poland; 3Department of Plant Protection, Agricultural University

Nowadays nanomaterials and nanotechnology are very important in medicine, pharmacy, tissue engineering as well as in the world of agriculture. Today protect plants against pests is carried out mainly by chemical methods that use synthetic pesticides. Its advantage is the effective action and thus possibility of preventing the occurrence epidemic of plant diseases. However this method is very detrimental to the human health and environment be-cause of several active substances with high toxicity [1].It turns out that formulations containing nanometals par-ticles can be particular useful, because they characterize higher biochemical activity than the macro-structures and more effective influence on the bacteria or fungi. According to the literature, silver and gold nanoparticles have high fungistatic activity and biocidal properties. The biocidal activity of the silver nanoparticles is dependent on the size and shape of the particles [2–4].In carried out investigations were developed stable sus-pensions of silver nanoparticles containing degradable polymer dispersant. The use of polymers for the prepara-tion of dispersions improve the homogeneity, rheological properties and stability of nanoparticles. The agrochem-ical formulations containing metallic nanoparticles of silver, were developed. The physicochemical characteri-zation of the obtained materials was carried out: particle size distribution, stability and sedimentation, analysis of the morphology of the particles.AcknowledgementsThe research was funded from the budget for science for years 2013–2014 – No. IP2012 062872.References[1] Weber Z., Kryczyński S. (red.) (2010) Fitopatologia, PWRiL, Poznań.[2] Nair R., Varghese S.H., Nair B.G., Maekawa T., Yoshida Y., Kumar D.S. (2010) Plant Science, 179: 154–163. [3] Rai M., Yadav A., Gade A. (2009) Biotechnology Advances, 27: 76–83.[4] Zielinska A., Skwarek E., Zaleska A., Gazda M., Hupka J. (2009) Procedia Chemistry 1: 1560–1566.

P1.7

Silver nanoparticles as an effective biocidal factorJolanta Pulit1, Marcin Banach1, Renata Szczygłowska2, Mirosław Bryk3

1Crakow University of Technology, Institute of Inorganic Chemistry and Technology, Warszawska 24 St., 31-155 Crakow, Poland; 2Silesian Environmental Ph.D. Studies Centre, Central Mining Institute, Gwarkow Sq., 1, 40-166 Katowice, Poland; 3Implementing Company

Nanosilver is one of the most thoroughly investigated nanomaterials, and owes its popularity to its biocid-al properties [1]. Its antimicrobial activity is associated with the characteristic structure of nanoparticles. They are characterized by a high fraction of surface atoms so that nanoparticles have a greater affinity for interactions with thiol groups, after which the destruction of the bac-teria follows [2]. Biological activity is therefore a very im-portant factor, so that nanosilver is used where asepsis is highly desirable. Mostly, this applies in biomedical en-gineering, cosmetology, food packaging industry, textile industry, crops, animal husbandry etc. [3].Recently, using nanosilver as biological agent has become increasingly common. Such an idea aims at the synthesis of nanometric silver in accordance with the principles of “green chemistry”. The development of nanosilver prepa-ration processes while respecting “green chemistry” prin-ciples was the intention of the authors. It was decided to find a solution that would allow the use of one substance acting as both a stabilizing and a reducing agent. This assumption was carried out using a raspberry extract, which largely reflects the ideas of environmental technol-ogies. The use of raspberry extract as a source of ellagic acid offers the possibility of direct silver ion reduction, as it has strong antioxidant effects, similar to many other polyphenols. Furthermore, minor amounts of other com-pounds with antioxidant properties, such as gallic acid, ascorbic acid, and quercetin that also contribute to effi-ciently carrying out the chemical reduction are also pres-ent in raspberry extract. The presence of other organic compounds from the group of polyphenols, tannins and flavonols allows to stabilize emerging suspensions with no need to add other factors that inhibit the growth of metallic silver agglomerates [4].Authors also raise the problem of asepsis. Microbial colonization, for instance, in painted buildings causes aesthetic problems and may lead to degradation of the coating and splintering [5]. Much more dangerous ef-fects may be caused by the influence of mould on human health. Spores and mycelial fragments escaping from the ground, together with bacteria and dust, form bioaerosols that are a source of air pollution. They may contain tox-ic compounds called mycotoxins, which cause allergies. Penicillium spp., Aspergillus spp. and Alternaria spp. are significantly responsible for the formation of many of the allergic symptoms and respiratory diseases in people ex-posed to high levels of mould spores and allergens.

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Thus, according to the varying degrees of effectiveness of the antimicrobial properties of nanosilver obtained via green technology and the need to recognize the ap-plication in the coating field, researches were undertaken whose aim was to evaluate the antifungal activity of sil-icon paint, plaster and ground plaster containing silver nanoparticles as well as perform a mycological efficacy resistance analysis of tested preparations in relation to different concentrations of nanostructured silver. The use of nanoparticles with antimicrobial properties in construction materials can bring numerous potential ad-vantages, such as reinforcing their hygienic properties, preventing microbial growth and maintaining their me-chanical properties.The work presents the way of obtaining an aqueous rasp-berry extract, its physicochemical and analytical charac-teristic as well as the method of preparation of nanosilver suspensions based on raspberry extract. Moreover, re-sults of microbiological studies on biocidal effect of ob-tained nanosilver against Cladosporium cladosporoides, Aspergillus Niger, Rhodotorula mucilaginosa, Aspergillus niger and Geotrichium candidum are described.AcknowledgementsThe work is part of a project “Synthesis and application of innovative nanomaterials with antimicrobial properties” supported by National Centre for Research and Development under the contract No. LIDER/03/146/L-3/11/NCBR/2012 for the period of 2012–2015.References[1] Vaidyanathan R., Kalishwaralal K., Gopalram S., Gurunathan S., Nanosilver – the burgeoning therapeutic molecule and its green synthesis. Biotechnol. Adv. 2009, 27, 924.[2] Sadeghi B., Jamali M., Kia S., Amininia A., Ghafari S., Synthesis and characterization of silver nanoparticles for antibacterial activity. Int. J. Nano. Dim. 2010, 1, 119.[3] Pulit J., Banach M., Kowalski Z., Nanosilver – making difficult decisions. Ecol. Chem. Eng. S. 2011, 18, 185.[4] Hakkinen S. H., Karenlampi S.O., Mykkanen H.M., Heinonen I.M., Torronen A.R., Ellagic acid content in berries: Influence of domestic processing and storage. Eur. Food Res. Technol. 2000, 212, 75.[5] Bellotti N., Salvatore L., Deyá C., Del Panno M.T., del Amo B., Romagnoli R., The application of bioactive compounds from the food industry to control mold growth in indoor waterborne coatings. Colloids Surf. B, 2013, 104, 140.

P1.8

The impact of industrial nanoaerosols on the pulmonary surfactantDorota Kondej1, Tomasz R. Sosnowski2

1Central Institute for Labour Protection – National Research Institute/ Department of Chemical, Aerosol and Biological Hazards/ Czerniakowska 16/ 00-701 Warsaw/ Poland; 2Warsaw University of Technology/ Faculty of Chemical and Process Engineering/ Warynskiego 1/ 00-645 Warsaw/ Poland

The development of nanotechnologies and the related increasing use of nanomaterials create a potential risk of workers’ exposure to inhalation of nanoparticles realised during industrial processes. The aim of this study was to evaluate the impact of selected mineral nanoparticles (bentonite PGV, halloysite HN, surface-modified mont-morillonite I.28E) used as nanofillers in the production of polymer nanocomposites on the surface activity of the pulmonary surfactant (PS). PS being the first barrier sep-arating the inhaled air present in pulmonary alveoli from the lung tissue plays an important role in the physiology of the respiratory system. PS is responsible for a signifi-cant reduction of the surface tension of the liquid cover-ing the lung tissue which has important implications for the mechanics of the respiratory cycle and mass transfer processes in the lung. The research was done using two complimentary techniques (the Langmuir-Wilhelmy film balance and the pulsating bubble method) allow-ing to study different aspects of the influence of mineral nanoparticles on the interfacial activity of model pulmo-nary surfactants (DPPC and Survanta). It was found the mineral nanoparticles alter the surface activity of model pulmonary surfactants under dynamic conditions relat-ed to lung behaviour during breathing. The presence of PGV and HN nanoparticles causes a reduction in the activity of model pulmonary surfactants which may be attributed to the adsorption of PS molecules on the sur-face of the nanoparticles investigated. Surface-modified montmorillonite I.28E nanoparticles induce increase of the surface pressure in the system which can be explained by a release of surface-active compounds from I.28E par-ticles. Inhalable nanoparticles used in the production of polymer nanocomposites may cause the disturbance of surface activity of PS. By this mechanism industrial na-noaerosols may contribute to adverse health effects in the respiratory system of workers involved in the production process.AcknowledgementsThis paper has been prepared on the basis of the results of research project No. I.B.10 carried out within the National Programme “Improvement of safety and working conditions” partly supported in 2011–2013 within the scope of research and development by the Ministry of Science and Higher Education. CIOP-PIB has been the Programme main coordinator.

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P1.9

The impact of nanosilver addition on element ions release form light-cured dental composite and compomer into 0,9% NaClKrzysztof Sokołowski1, Małgorzata Iwona Szynkowska2, Aleksandra Pawlaczyk2, Jerzy Sokołowski3

1Department of Conservative Dentistry, Medical University of Lodz, Pomorska 251, 92-213 Lodz, Poland; 2Institute of General and Ecological Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland; 3Department of General Dentistry, Medical University of Lodz, Pomorska 251, 92-213 Lodz, Poland

Nowadays, a wide spectrum of dental materials is avail-able, material properties depend on the basic material type e.g. metals, ceramics or polymers. In fact, a detailed understanding of the key concept for each of them gives an insight into how each class of material can behave as a restorative dental material, as well as an idea of the potential of these materials if some of their limitations can be overcome. There is no surprise that only certain specific material properties determine their selection for application in restorative dentistry. Basing on that knowledge, it is possible to make a proper selection of the material being the best compromise of desired prop-erties versus inherent limitations. Due to the fact that any class of basic type of presented materials possesses all the desired properties, in many cases they are used in combination limiting their usefulness [1]. The main ob-jective of this study was to assess the release of metal ions from dental fillings into 0.9% NaCl solution in different time intervals. The investigated dental materials were samples of flowable composite material with nanosilver addition and flowable compomer material with nanosil-ver addition, both being a light-cured dental restorative material with flow characteristics, which makes it ideal material for filling small cavities in anterior and poste-rior teeth. Composite restorations seem to represent ex-cellent aesthetics properties, but due to the fact that they undergo polymerization shrinkage on setting, they are associated with marginal leakage, which causes bacterial penetration and, in consequence, the potential damage to the tooth. Notwithstanding, fluoride releasing mate-rials in combination with silver ions can influence the surrounding micro environment, involving bacteria. Each of the samples was first washed with ethanol and then placed in a test-tube in 10 mL 0.9% NaCl solution. A number of counts of metal ions released into NaCl solution from the dental materials were determined us-ing Optimass 8000 ICP-TOF MS spectrometer (GBC Scientific Equipment, Australia) after a week, a month and three months time of storage. The results confirmed the significant increase in the number of counts of metal ions originating form the main components of studied dental material with the time of storage. The gathered spectra were also compared with some previous out-

comes obtained for other restorative materials not con-taining silver in their composition.AcknowledgementsThe financial support of this work by the Polish Scientific Research Council (grant N N209 343237 ) is gratefully acknowledged.

Development of renewable energy in biotechnology

Lectures

L2.1

Metabolic Engineering of Non-Conventional Yeasts for Construction of the Advanced Producers of Lignocellulosic EthanolAndriy A. Sibirny1, 2

1Department of Molecular Genetics and Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine; 2Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow 35-601, Poland

Non-conventional yeasts are attractive ethanol pro-ducers from lignocellulose. One of the most promising ethanol producer from lignocellulose is Hansenula pol-ymorpha which belongs to the best studied non-conven-tional yeasts. Its genome has been sequenced and pub-licly available (http://genomeportal.jgipsf.org/Hanpo2/Hanpo2.info.html) and methods of molecular genetics and cell biology are well developed. It is a popular host system for expression of heterologous proteins and some metabolites as well as favorite model organism for stud-ying peroxisome biogenesis and autophagic degrada-tion, methanol catabolism and stress response. Besides, H. polymorpha is apparently the most thermotolerant yeast known with maximal growth temperature at 50°C. Several years ago, we have found that H. polymorpha is capable of high-temperature xylose alcoholic fermen-tation though ethanol production is quite low. At the same time is effectively ferments glucose and cellobiose (Ryabova et al., 2003, Ishchuk et al., 2009). More recent-ly, it was shown that H. polymorpha can effectively pro-duce ethanol from glycerol, the byproduct of biodiesel production (Suvannarangsee et al., 2010). To construct effective ethanol producers from xylose, overexpression of the modified gene XYL1m (encoding xylose reductase) and the native genes XYL2 (xylitol dehydrogenase), XYL3 (xylulokinase) and PDC1 (pyruvate decarboxylase) and additionally, the isolation of 3-bromopyruvate-resistant mutants from metabolically engineered strains have been conducted (Dmytruk et al., 2008; Ishchuk et al., 2008). Using mentioned approaches, the strains of H. polymor-pha have been constructed which accumulate elevated amounts of ethanol from xylose, up to 10–12 g/L or near 22 g/L after correction for ethanol evaporation. However, to be economically feasible, available level of ethanol syn-thesis from xylose has to be increased for several times, up to 30–35 g/L. We have found that increase in ethanol production from xylose can be achieved by either disrup-

tion or knock out of ATG13 gene involved in autophagy and pexophagy. Increase in xylose conversion to ethanol could be also achieved by overexpression of genes coding peroxisomal enzymes dihydroxyacetone synthase (DAS1) and transaldolase (TAL2). DAS1 overexpression resulted in increase in ethanol production from xylose both on the backgrounds of the wild-type strain and previously isolated metabolically engineered strain with enhanced ethanol synthesis. Other promising organism for xylose alcoholic fermentation is Pichia stipitis. Ethanol accumu-lation in the last organism could be also increased using methods of classic selection and metabolic engineering. References[1] Dmytruk O.V. et al. (2008) Microb Cell Fact. 7, 21.[2] Ishchuk O.P. et al. (2008) FEMS Yeast Res. 8, 1164–1174.[3] Ishchuk O.P. et al. (2009) Biotechnol. Bioeng. 104, 911–919.[4] Ryabova O.B. et al. (2003) FEMS Yeast Res. 4, 157–164.[5] Suvannarangasse S. (2010) Appl. Microbiol. Biotechnol. 88, 497–507.

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Posters

P2.1

The influence of phytase addition during the enzymatic starch hydrolysis process with the use of basic amylolytic enzymes on yield and the course of alcoholic fermentation processDawid Mikulski, Grzegorz Kłosowski

Department of Biotechnology, Institute of Experimental Biology, Kazimierz Wielki University, 85-667 Bydgoszcz, Chodkiewicza 51 St., Poland

Phytic acid (myo-inositol hexa-phosphoric acid, IP6) is the major phosphorus storage compound of most cere-al grains. Phosphorylated form of inositol due to ionized phosphates groups has strong chelating properties. Phyt-ic acid creates complexes, the so-called phytin complexes with divalent cations (Mg2+, Ca2+, Fe2+, Zn2+), with proteins and polysaccharides, i.e. starch [1]. These complexes limit the availability of the divalent cations, proteins and starch for organisms which do not produce the phosphohydro-lases enzymes. The applications of phytase, the enzyme hydrolysing phytin complexes during the preparation of the fermentation medium, should result in the increase of availability of biogenic compounds [2]. The aim of the study was to determine the influence of phytin hydroly-sis before and after starch hydrolysis process present in high gravity maize mashes (HG mashes) on yield and the course of alcoholic fermentation process. The raw mate-rial used in preparation of HG mashes according to pres-sureless liberation of starch technology (PLS technology) was milled maize grain (starch concentration – 53.34%, phytic acid concentration – 2.76 mg/g, total phosphorus concentration – 2.93 mg/g). The process of enzymatic starch hydrolysis was conducted with heat-stable α-am-ylase (Termamyl SC DS preparation) and glucoamylase (Saczyme preparation) produced by Novozymes® com-pany. Phytin complexes hydrolysis was conducted with the use of Phytase 10000L preparation produced by Erb-sloh® company. During the alcoholic fermentation the basic technological indicators (alcohol concentration, productivity, yield and fermentation energy) and indi-cators of the physiological condition of yeast (number, viability, phosphorus concentration in yeast cells) were determined. Alcoholic fermentation volatile by-products were determined with capillary gas chromatography (GC method). The analysis of variance (significant level α3/100 kg of starch was observed in the variant with phytin hy-drolysis before starch hydrolysis process. In the initial fermentation phase irrespectively of the research variants the viability of yeast was ca. 98%. In subsequent phases the increase of ethanol concentration caused the decrease of viability and number of yeast cells in variants with ad-dition of phytase in relations to the control variant. The highest concentration of acetaldehyde (524.1±25.6 mg/L)

and higher alcohols (4144.2±154.0 mg/L) was observed in the distillate obtained in the fermentation medium with phytin hydrolysis before hydrolysis of starch. AcknowledgementsThe study financed from the science resources in the years 2013–2014, as an experimental study of the National Science Centre (No. 2012/05/N/NZ9/02436). References[1] Garcia-Estepa R.M., Guerra-Hernández E., Garcia-Villanova B. (1999) Phytic acid content in milled cereal products and breads. Food Research International 32, 217–221.[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and biotechnology. Biotechnology Letters, 25, 1787–1794.

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P2.2

Determination of the impact of lactose suplementation of synthetic cellulolytic media on the activity of cellulase complex Dorota Macko, Beata Miklaszewska, Dawid Mikulski, Grzegorz Kłosowski

Department of Biotechnology, Institute of Experimental Biology, Kazimierz Wielki University, 85-667 Bydgoszcz, Chodkiewicza 51 St., Poland

Cellulose is linear β-D-glucopyranose polymer, in which individual units (8-12 thousand) are connected with β-1-4-glycosidic bonds, no other chemical compound of the polysaccharides group is not produced in the nat-ural environment, in such large amounts. Cellulose is a renewable, and potential source of glucose for the pro-duction of ethanol. Effective enzymatic hydrolysis of this polymer depends on proper activation of lignocellulotic biomass (pre – treatment method) [Mosier et al. 2005], and also on the composition, concentration, and activity of enzymes comprising the cellulase complex: endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase and 1,4-ß-D-glu-cosidase. Great importance on the polymer degradation has a process of induction of cellulase secretion in fungal hyphae, it is followed by natural activators such as oligo-saccharides: cellobiose, sophorose, gentobiose [Suto and Tamita, 2001]. The aim of the study was to evaluate the influence of lactose supplementation of culture medium on enzymatic activity of endoglucanase (CMC-ases), ce-lobiase, and total celulase activity (FPaza) produced by isolate of Penicillium sp. selected from cellulolytic in-dustry materials. The strain was cultivated by liquid fer-mentation on medium contained different substrates of cellulose: microcrystalline (AVICEL 2%), carboxymethyl cellulose high viscosity (CMChv 1%), and carboxymeth-yl cellulose low viscosity (CMClv 2%) in Vogels medium (according to Ahmed et. al 2009). The experiment was carried out in two different variants: on pure Vogels me-dium with and without addition of 10 g lactose per one liter of medium. All analyses of the enzymatic activity and concentrations of reducing sugars were conduct-ed according to the IUPAC procedure (International Union of Pure and Applyed Chemistry). The analysis of variance (significant level α deviation was carried out by using STATISTICA software, ver. 10). Statistical signifi-cant influence on FPase and CMCase activity by supple-mentation medium with lactose was detected. During the fermentation reaction in medium contained with AVI-CEL, total FPase activity grew from 0,690 (without lac-tose addition) to 4,893 mg of glucose/ min/ mg of protein (with lactose addition). Similar the activity of CMCase increased from 11,910 to 43,796 mg of glucose/ min/ mg of protein, reaching a maximum value.References[1] Suto M, Tomita F. (2001) Induction and catabolite repression mechanisms of cellulase in fungi, Journal of Bioscience and Bioengineering, Vol. 92, No. 4, 305–311.

[2] Mosier N., Wyman Ch., Dale B., Elander R., Lee Y.Y., Holtzapple M., Ladisch M. (2005) Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technology 96: 673–686.[3] Ahmed S., Bashir A., Saleem H., Saadia M., Jamil A. (2009) Production and purification of cellulose-degrading enzymes from a  filamentous fungus, Trichoderma harzianum Pakistan Journal of Botany 41(3): 1411–1419.

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P2.3

Perspectives of biofuel production from cellulosic crop wasteDariusz Dziga1, Dominika Jagiełło-Flasińska2

1Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Crakow, Poland; 2Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Crakow, Poland

Cellulose is a major component of plant biomass and could be applied in the production of biofuels, especially bioethanol. An alternative approach is to produce hydro-gen from cellulosic biomass. In this paper an innovatory model of cellulosic waste utilization has been proposed to verify the possibility of cellulose degradation product utilization by purple non-sulfur bacteria. The concept is based on a two-step process of microbial cellulose con-version in order to obtain an organic acid mixture. In the next stage such products are consumed by Rhodobacter sphaeroides which is capable of hydrogen production in a photofermentation process using different organic compounds. It has been documented that R. sphaeroi-des employed in this process can effectively consume organic acids released from straw by Cellulomonas uda and Lactobacillus rhamnosus and that it is able to grow rapidly in the presence of these substrates. Additionally, an enhanced nitrogenase activity of R. sphaeroides has been shown when bacteria were cultivated in the pres-ence of cellulose derivatives which suggest that hydrogen production occurs. These promising results indicate the necessity to carry out further research using the proposed model of cellulose transformation into biohydrogen. The key aim of further experiments is to confirm whether R. sphaeroides is able to produce hydrogen effectively us-ing a mixture of organic acids originating from cellulose.

P2.4

Biogas generation from sewage sludge in the anaerobic digestion process Mariusz Małkowski, Paweł Wolski, Lidia Wolny

Institute of Environmental Engineering, Czestochowa University of Technology

Anaerobic digestion is a process used in the most of sew-age treatment plants. Volume reduction in relation to cost is a big opportunity in the sludge management, both in relation to less volume of digesters and less final product. Volume reduction can be achieved by converting more of the sludge to biogas and also by better mechanical dewa-tering of sludge.Those aims are achieved with advanced digestion. Ultra-sonic field as physical agent is very effective pretreatment method to enhance the biodegradability of sludge in the anaerobic digestion. Decomposition of organic (nutrient) substances is limited mainly by the velocity of the first step of methane fermentation process named as hydro-lysis. During this hydrolysis the organic compounds can be decomposed into liquid and finally becomes methane and carbon dioxide. Intensification of the methane fer-mentation hydrolytic phase and the increase of biogas generation depends on dispersion of sewage sludge solid phase.The aim of investigations was the preliminary treatment process evaluation before the anaerobic digestion of ex-cess sludge influencing on the amount of biogas genera-tion and the energetic balance. The ratio of energy costs during the sludge ultrasonication process to the amount of biogas was evaluated. Excess sludge from the wastewa-ter treatment plant of paper industry was the substrate of investigations. For sewage sludge ultrasonication the ultrasonic disintegrator VCX-1500 (Sonics Company) of frequency 20 kHz was used. Ultrasonication of tested sludge was made at four vibration amplitudes 16, 24, 32 and 39 µm in the time of 300 seconds. The anaerobic di-gestion of preliminary modified sewage sludge with ul-trasonic field was conducted in the digestion chamber at temperature 37°C during 25 days.In the paper the effective preliminary method of sludge pretreatment was presented, which improve degree of sludge disintegration and biogas output. In the anaero-bic digestion of tested excess sludge after this preliminary treatment two – time increase of biogas generation was observed.Keywords: anaerobic digestion, sewage sludge, ultrasonic field, biogas

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P2.5

Zeolite nano-adsorbents for fuel ethanol dehydratioTomasz Tokarz

Wydział Inżynierii I Technologii Chemicznej Politechniki Krakowskiej, Instytut Chemii i Technologii Organicznej, Katedra Technologii Organicznej i Procesów Rafineryjnych

Anhydrous ethanol from fermentation process of bioma-terials is widely used in chemical, pharmaceutical and cosmetic industries. This is also used as a fuel additive in the internal combustion engines. Technologically eth-anol production of a high concentration is a major prob-lem, due to the presence in the system of ethanol/water azeotropic point (p = 1,013 bar, T = 78,15°C, 4,43% m/m H2O). High energy consumption in the traditional azeo-tropic distillation is a main disadvantage of this process. One of the most widely used alternative method is Pres-sure Swing Adsorption (PSA) in which zeolite nano-ad-sorbent is applied. The aim of this study is to find rela-tions between surface area and pore structure for mass production zeolite nano-adsorbents with their efficiency in dehydration of raw ethanol. Keywords: PSA, adsorption, separation, zeolite, ethanol

P2.6

Production of butane-2,3-diol by Bacillus licheniformis NCIMB 8059 in batch and fed-batch cultures and its up-scalingJoanna Krysiak1, Ewa Gromek1, Halina Kalinowska1, Marianna Turkiewicz1, Aneta Białkowska1, Siegmund Lang2

1Institute of Technical Biochemistry Lodz University of Technology;2Institute for Biochemistry, Biotechnology and Bioinformatics Braunschweig University of Technology

2,3-butanediol (2,3-BD) is a colorless and odorless poly-hydroxyalcohol with a chemical formula C4H10O2. This liquid is characterized by a high boiling temperature and a low melting temperature. Because of attractive physical properties and feasibility of diverse chemical conversions this alcohol has found multiple applications in cosmetic, pharmaceutical and fuel industries etc. 2,3-BD has been hitherto produced from crude oil by chemical technologies, but due to the depletion of fossil fuels and their growing prices its biosynthesis by micro-organisms has recently become an interesting alterna-tive. It is to note that the majority of microorganisms synthesizing appreciable amounts of this compound are pathogens, which cannot be used in industry. Therefore, investigations on improvement of 2,3-BD biosynthesis by non-pathogenic bacteria like Bacillus strains are so important.This study aimed at maximizing 2,3-BD biosynthesis by a  non-pathogenic Bacillus licheniformis strain through fed-batch cultures and process up-scaling using bioreac-tors with volumes of 0.75 and 30 L. Tested carbon sources used in 2,3-BD production by this strain were by-products from food process-ing such as molasses and an enzymatic hydrolysate of apple pomace. Fermentations conducted in 0.75 L bioreactors enabled selection of the most appropriate carbon source and optimum agitation intensity (test-ed rotation rates of a stirrer: 150, 250 or 450 rpm). These results enabled efficient 2,3-BD production in fed-batch cultures conducted in 30 L bioreactors. The most suitable carbon source both in batch and fed-batch cultures (irrespective of the scale) was found to be molasses. Concentration of 2,3-BD synthesized by the B. licheniformis strain in 30 L bioreactor  (fed-batch cultures, fed 4-times with 50% glucose solution) reached 80 g/L after 72 h. When carbon sources were mixtures of the apple pomace hydrolysate and pure glucose, the maximum 2,3-BD concentration was only slightly lower (73 g/L) but it was achieved after 96 h of fermentation. These results are comparable to 2,3-BD yields presented in literature for its most efficient pathogenic producer – Klebsiella pneumoniae strains. We found that among tested by-products from food processing the most suit-able for 2,3-BD production in industrial scale is molasses from sugar beets. Advantages of this raw material are its low price and good availability as well as, in contrast to

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the apple pomace, the lack of necessity of its earlier hy-drolysis before fermentation.AcknowledgementsPresented study was accomplished with the scope of the international project “Production and upgrading of 2,3-butanediol from biomass”.

P2.7

Biogas from physical modified excess sludge of pulp industryMariusz Barański, Lidia Wolny, Iwona Zawieja

Institute of Environmental Engineering, Czestochowa University of Technology

Physical conditioning methods of sludge as for example ultrasonic field operate mainly to the disintegration of sludge, which can intensify the hydrolysis process as the first stage of anaerobic digestion. Ultrasonication before anaerobic digestion causes that hydrolytic phase is short-er as well as the degree of organic compounds decompo-sition is better in comparison to not conditioned sludge. The aim of investigations was the evaluation of excess sludge disintegration and biogas generation in the an-aerobic stabilization after ultrasonic generator VCX-750 (Sonics Company) application. Tested substrate was excess sludge from paper industry. Results obtained af-ter anaerobic stabilization of ultrasonically conditioned sludge were compared with the results of unconditioned sludge.The feed/inoculum ratio in the anerobic digestion was 9/1. Two series of investigations were conducted.: for unconditioned excess sludge (mixture A) and for condi-tioned excess sludge (mixture B) at vibration amplitude of ultrasonic field 35% and 60% (mixture C). In both cas-es the sonication time was 360 seconds. The most advan-tageous results of ultrasonic field action were observed at amplitude of 60% and sonication time 360 seconds. For this parameters the 70% degree of fermentation was obtained and 50% increase of unitary biogas production (3.54dm³/g s.m.org.) in comparison to unconditioned sludge.

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P2.8

Polymer-enzyme catalyst systems for the hydrolysis of cellulose Joanna Świder1, Katarzyna Gawełek1, Ewa Witek1, Edyta Podstawka- Proniewicz1, Anna Konieczna-Molenda2

1Faculty of Chemistry, Jagiellonian University, Ingardena 3 St., 30-060 Crakow, Poland; 2Department of Chemistry and Physics, University of Agriculture, Balicka 122 St., 30-149 Crakow, Poland

Immobilization of enzymes used in the food, pharmaceu-tical and biofuels synthesis on polymeric supports sig-nificantly reduces the cost and improves the hydrolysis of polysaccharides [1, 2].The study involved polymer carriers based on crosslinked N-vinyl formamide (NVF) synthesized by the reaction of free radical suspension polymerization of N,N’-meth-ylenebisacrylamide (NMBA), optionally with ethylene glycol dimethacrylate (EGDMA) [3].Developed optimum parameters for copolymerization NVF/NMBA and NVF/EGDMA in inverse suspension giving spherical polymer grains having a low coefficient of swelling in water. It has been shown that the most ef-fective medium in this process is a silicone oil.Selected six most preferred polymeric carriers char-acterized by physicochemical parameters and used it to immobilize the enzyme mixture from the group of carbohydrases under the trade name Viskozyme L® (Sigma-Aldrich). Received systems used in the test reac-tions of hydrolysis microcrystalline cellulose and hydro-lysis of wood pulp as application process. Immobilization of enzyme was carried out by means of glutaraldehyde, as well as without its participation. The morphology of carriers determined based on microscopic images. Physi-co-chemical methods for the characterization of obtained catalytic polymer-carrier systems included CHN analysis, FTIR spectroscopy and Raman spectroscopy.It has been shown that reactive formamide groups do not provide a permanent binding medium glutaralde-hyde-enzyme, hence the catalytic tests showed less than the immobilized enzyme activity of native enzyme.AcknowledgementsThe authors wish to thank the Ministry of Science and Higher Education for the funding of the research. The grant N N204 354840.References[1] Konieczna-Molenda A., Kochanowski A., Walaszek A., Bortel E., Tomasik P. (2009) Chemical Engineering Journal, 146: 515–519 [2] Farrell A.E., Plevin R.J., Turner B.T., Jones A.D., O’Hare M., Kammen D.M. (2006) Science, 311 (5760), 506–508[3] Witek E. (2008) Polimery, 53: 477–480.

P2.9

Effect of cellulase immobilization upon digestion of celluloseKatarzyna Gawełek1, Agnieszka Tąta1, Ewa Witek1, Edyta Podstawka-Proniewicz1, Anna Konieczna-Molenda2

1Faculty of Chemistry, Jagiellonian University, Ingardena 3 St., 30-060 Crakow, Poland; 2Department of Chemistry and Physics, University of Agriculture, Balicka 122 St., 30-149 Crakow, Poland

Cellulases play an important role in specialized com-mercial applications such as fabric modifications, paper and pulp industry, food industry [1] etc. Enzymatic hy-drolysis of cellulose has been extensively studied in the past decades since the utilization of cellulosic biomass as a renewable resource has great potential for reducing emissions of carbon dioxide and thereby prevents global warming [2]. Cellulase can be used as the biocatalyst for cellulose hydrolysis, but its activity is rather lower than that of other hydrolases.Cellulase was immobilized on N-vinylformamidecross-linked with divinylbenzene as a result of copolymeriza-tion in reverse suspension [3]. The morphology of car-riers determined based on microscopic images. Physi-co-chemical methods for the characterization of obtained catalytic polymer-carrier systems included CHN analysis, FTIR spectroscopy and Raman spectroscopy.The enzyme Novozym 476 (Novozym®) was covalently bound directly to the supports or using glutaraldehyde as a spacer [4]. The immobilization procedure was opti-mized involving such factors as temperature, pH, time, sequence of reactions, and kind of carrier employed [5].Received systems used in the test reactions of hydrolysis microcrystalline cellulose and hydrolysis of paper as ap-plication process. The results of the immobilization were evaluated in base on analyses of the enzyme activity and stability prior and after immobilization, as well as on the immobilization yield and stability.Highly active biocomposites were designed that provided their multiple application for cellulose hydrolysis.AcknowledgementsThe authors wish to thank the Ministry of Science and Higher Education for the funding of the research. The grant N N204 354840.References[1] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2007) Bioresearch Technology, 98: 1366–1372.[2] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2004) J. Chem. Eng. Jpn., 37: 680–684.[3] Witek E. (2008) Polimery, 53: 477–480.[4] Bryjak J., Bachmann K., Pawłów B., Maliszewska I., Trochimczuk A., Kolarz B.N. (1997) Chem. Eng. J.,65: 249.[5] Konieczna-Molenda A., Kochanowski A., Grabiec, A., Bortel E., Tomasik P. (2008) PTTZ-Malopolska Branch, Krakow, Ch. 8: 103–120.

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P2.10

Application of wastes from alcohol distillation in two-step process of biohydrogen productionRoman Zagrodnik

Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland

Recent years showed that the cost of obtaining energy from traditional sources is increasing in a manner dif-ficult to predict. The solution to these problems is utili-zation of renewable energy sources such as biomass. The necessity of recycling and use of organic compounds contained in waste materials and wastewater is inevitable. However, regardless of the source, the energy has to reach the user through an efficient carrier, that can be used in a wide range of applications. Hydrogen is considered to be one of the most promising fuels of the future, and is widely recognized as a potential substitute for fossil fuels.Biological processes of hydrogen production are found to be the most environmentally friendly processes of hy-drogen production, and dark and photo fermentations of organic wastes are considered as the most promising pro-cesses for biological hydrogen production. These process-es are operated at ambient temperature and atmospheric pressures, thus are less energy intensive and more envi-ronmentally friendly as compared to thermochemical and electrochemical processes. Moreover, the biological hydrogen generation can be carried out as a continuous process, which is appealing to large scale systems.In this work the hybrid system applying both dark fermen-tation and photofermentation was tested. At first, the fer-mentation of wheat distillation effluent containing certain amounts of simple sugars was conducted in bioreactor. Active sludge from municipal wastes was a source of mi-croorganisms in dark fermentation. Here, starch and simple sugars were transformed into hydrogen and liquid metab-olites (lactic acid, acetic acid, simple alcohols – effective substrates for photo fermentative bacteria). This step was followed by photofermentation process with Rhodobacter sphaeroides bacteria. Here, flat plate photobioreactor of our own construction, working under continuous conditions was applied. Continuous condition were provided by two peristaltic pumps. The COD of waste water from wheat dis-tillation was 45.7 g O2/l and after dark fermentation 27.8 g O2/l. The amount of the lactic and acetic acid during photo-fermentation process was reduced by 98 and 88%, respec-tively. Average hydrogen production rate in the case of pho-tofermentation was 10,8 ml H2/l medium/h, and was slight-ly lower than in the case of dark fermentation – 97,2 ml H2/l medium/h. Experiments with effluent from dark fermen-tation as the source of organic carbon proved that wastes originating from food industry can be efficiently applied for hydrogen production in photofermentation process.AcknowledgementsThe project was financed by the National Science Centre granted on the basis of decision DEC-2012/05/N/NZ9/01577.

P2.11

Electrochemical studies of chemically modified and native Sinorhizobium meliloti laccase Anna Pawlik, Grzegorz Janusz, Jerzy Rogalski

Maria Curie-Skłodowska University, Faculty of Biology and Biotechnology, Department of Biochemistry

Laccases (EC 1.10.3.2) are propably the most numerous members of the multicopper protein family, being dis-tributed in all domains of life. Because of their broad sub-strate specifity and interesting chemical properties (e.g.: high thermostability, alkaline pH-stable activity), laccas-es harbor great potential to biotechnological processes. A significant number of reports have been published in the past decade that have focused on the biochemical properties of these proteins and/or on their applications in the paper and pulp, food, cosmetic, textile and petro-chemical industry, nanobiotechnology, medical diagnos-tics and for bioremediation and clinical purposes. The use of laccase in electrochemical biosensors and biofuel cells has been the subject of basic as well as applied research [1]. Wild laccases are not always suitable for industrial purposes. For example, they suffer from several limits: in some cases, their activity needs the presence of a redox mediator, their pH activity is acidic and does not corre-spond to environmental conditions. Chemical modifica-tion may provide recombinant enzymes with wider sub-strate specificity, higher oxidation potential and neutral pH of activity.The present work mainly focused on electrochemical measurements of chemically modified and native bacte-rial laccases.The crude enzyme extract of Sinorhiozbium meliloti lac-case was purified by means of chromatographic tech-niques. Laccase modification was performed as described elsewhere [2]. µAUTOLAB instrument and GPES soft-ware was used for electrochemical measurements and analysis of modified and native enzymes.The use of modified enzyme allowed to determine the effect of chemical modifications on laccase activity and stability. The basic bioelectrochemical studies of modified and native S. meliloti proteins enable further implementa-tion research and application of laccases in bioenergetics.AcknowledgementsThe research was supported by the the Polish Scientific Project BS/ZBioch/Maria Curie-Skłodowska University.References[1] Kunamneni A., Plou F.J., Ballesteros A., Alcalde M. (2008) Laccases: a patent review. Recent Pat Biotechnol. 2: 10–24.[2] Kucharzyk K.H., Janusz G., Karczmarczyk I., Rogalski J. (2012) Chemical modifications of laccase from white-rot basidiomycete Cerrena unicolor. Appl Biochem Biotechnol. 168: 1989–2003.

21Eurobiotech 2013

P2.12

Micelle-mediated extraction of elderberry blossom by whey protein and naturally-derived surfactantsKarolina Śliwa, Anna Tomaszkiewicz-Potępa, Elżbieta Sikora, Jan Ogonowski

Faculty of Chemical Engineering and Technology, Crakow University of Technology

Classical methods for the extraction of active ingredi-ents from the plant material are expensive, complicated and often environmentally unfriendly. Popular methods of extraction require the use of an expensive equipment, a large quantity of electricity, heat, and large volumes of flammable and toxic organic solvents such as methanol, ethyl acetate or n-propanol [1, 2].The micelle-mediated extraction method (MME) seems to be a good alternative. The extraction temperatures are relatively low and there is no need to apply organic sol-vents. In addition, this method enables to receive a higher yield of polyphenols than the standard methods. The use of surfactants that are suitable for human consumption could also reduce costs of the process. The additional ad-vantages of micelle-mediated extraction are no need for separation of used surfactants from the polyphenols con-taining extract and protection of the polyphenols against oxidation [2].The aim of this work was the study of the possibility of whey protein application as extraction agent. In this work, extractions of elderberry blossoms (Flos Sambuci) were performed. The micelle-mediated method using several popular surfactants and whey protein was applied. The results were compared with those obtained for extraction with methanol. Antioxidative properties of the extract and collected, after separation on the chromatographic column, fractions were analyzed. Two different methods were applied: reaction with DPPH reagent, Follin’s meth-od. Futhermore, the flavonoid content was determined.The results confirmed that the MME method with using whey protein might be an alternative and a convenient method for the preparation of rich in natural antioxidants plant extracts.Reference[1] Katsoyannos E., Chatzilazarou A. (2006) Fresenius Environmental Bulletin, 15: 1122.[2] Chatzilazarou A., Katsoyannos E. (2010) Journal of The Air & Waste Management Association, 60: 454.

P2.13

Biosynthesis of 2,3-butanediol from waste biomass by Raoultella Planticola CECT 843Mariusz Lesiecki, Adrian Czerniak

Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poland

Introduction. 2,3-Butanediol (2,3-BD) is a chiral biva-lent alcohol which exhibits a wide range of potential utili-zations as a solvent, liquid fuel or precursor for 1,3-buta-diene – an intermediate for synthetic rubber production. Several microorganisms are able to synthesis 2,3-BD. So far the highest yields were reached using risk class 2 strains e.g. Klebsiella species, however an industrial pro-cess with risk class 1 microorganisms are cheaper and less recalcitrant. Based on this fact, the present studies deal with the production of 2,3-BD from sugar beet pulp hy-drolysates by risk class 1 bacteria, namely Raoultella plan-ticola CECT 843 strain.AIM. The main study objective was to statistically opti-mise the biosynthesis process of 2,3-BD from sugar beet pulp hydrolysates by Raoultella planticola CECT 843 strain.Materials and methods. Response surface methodology (RSM) was used in this work to optimise the biosynthe-sis of 2,3-Butanediol by Raoultella planticola CECT 843 strain. Sugar beet pulp hydrolysates were used as a me-dium culture. The optimisation experiment was planned according to D-optimal design and consisted of 25 runs. The effect of yeast extract (YE) and concentration of CH3COO–, Fe2+

and Mg2+ ions was evaluated on the final lev-el of 2,3-butanodiol. Sugars and other carbon compounds concentration were determined preferably by HPLC or GC. Results. Obtained results showed statistically significant influence (p < 0,05) of the yeast extract, CH3COO– and Mg2+ ions concentration on the studied process. On the basis of statistic analysis, optimal values of analysed vari-ables were determined as follows (g·dm–3): yeast extract – 4; CH3COONH4 – 4; FeSO4·7H2O – 0,1; MgSO4·H2O – 0,3; that corresponded to 19,7 g·dm–3 final 2,3-Butanediol concentration.Conclusions. Under optimised conditions Raoultella planticola CECT 843 can effectively convert fermentable sugars from sugar beet pulp hydrolysates to 2,3-BD. Pro-duction of diol achieved nearly 20 g·dm–3 and efficiency of production reached 0,42 g·g–1

glucose in 24h of cultivation. It shows that risk class 1 bacteria strains are promising direction in research of microbial production of 2,3-Bu-tanediol.AcknowledgementsThis work was supported by ERA-IB EU-project called “Production and Upgrading of 2,3-Butanediol from Biomass” (ERA-NET-IB/03/2009).

Bioeconomy

Lectures

L3.1

Agri-food industry – a key element of Polish bioeconomyLech Michalczuk

Research Institute of Horticulture, Skierniewice, Poland

Agri-food industry is one of the most important sectors of Polish economy. In 2011 total value of its production sold exceeded 37 bln € (app. 22% of total industrial production sold) and gross value added (GVA) amounted to 10 bln €, which accounted for 12,7% of total industrial gross value added and was 1.2 times higher than GVA in mining in-dustry, 2.2 times higher than GVA in car manufacturing, 4.1 times higher than GVA in fuel and petrochemical in-dustry and 6.5 times higher than GVA in electronics and computer manufacturing. Polish agri-food industry has also stronger position in the European Union than the whole Polish economy; its GVA accounts for 6.5% of total industrial gross value in EU, compared with 4.1% share of Polish GDP in Gross European Product. The main challenges facing Polish agri-food industry are low labour productivity (83% of mean labour productiv-ity in Polish economy and 50% of mean labour produc-tivity in European Union) and relatively low innovative-ness. Serious problems are also with soil degradation and shrinking water resources for irrigation, which are affect-ing yield and crop quality in primary agricultural produc-tion. Results of foresight project “Food and nutrition in 21 century”, completed during 2009–2011 show that the best strategy for further development of Polish agri-food in-dustry is investment in innovative technologies increasing food quality and safety, especially those based on biotech-nology, nanotechnology, nutigenomics and nutrigenetics. Much efforts shall be also done to ameliorate prognosed effects of global climate changes affecting agricultural production. Especially important is prevention of further soil degradation, which shall involve both basic research on soil biology and developing technologies aimed at in-creasing abundance and diversity of soil microflora and organic matter content.

L3.2

Biomass as a source of raw materials for the preparation of environmentally friendly polymer materialsAndrzej Okruszek

Lodz University of Technology, Institute of Technical Biochemistry

The aim of the project (acronymBIOMASA) is utiliza-tion of various kinds of plant biomass and textile waste materials by their transformation with biotechnological methods, involving either enzymatic or microbial pro-cesses, into fibrous polymer materials. The intermediate products in those transformations are: cellulose nanofi-bres, tactic polylactide and aliphatic-aromatic co-poly-esters, which all are known to be important raw-materi-als for the production of biodegradable fibrous materials as well as other kinds of biodegradable polymer com-posites.For the preparation of cellulose nanofibres, a cellu-lose-rich plant biomass is being utilized, including grass and straw of various cereals as well as waste fibres from textile industry (cotton, linen). The biomass is first pre-treated with physical and/or chemical methods including boiling, steam-explosion or treatment with certain chem-icals. Multienzyme complex obtained from Aspergillus niger mould is utilized as the main enzymatic tool. The fibrous materials and composites prepared within this project on the basis of abovementioned intermediates will be further utilized for obtaining new functional textiles and nonwovens with potential sanitary or technical appli-cations, such as sweat-absorbing textile inserts, sanitary textiles, filtration materials, geotextiles and agrotextiles. Within this project, the processes of ageing and controlled biodegradation of prepared materials will be studied, as well as the conditions of their recycling and possible use of degradation products in agriculture.The synthesis of tactic polylactide is being performed by chemical polymerization of L,L-lactide, prepared from L-lactic acid. The latter is obtained by stereoselective fer-mentation of plant biomass, after its saccharization by ppropriate enzymes (Aspergillus niger preparations). The microorganisms (bacteria), used for the fermentation, were selected by classical microbiology methods from the environment. In this case patatoes, cereal grains or beet pulp are employed as starting biomass. The tactic polylac-tide will be further utilized for fiber formation and  her-moforming.The third path involves utilization of various oil-plant bio-mass, which on sequential treatment with lipase prepara-

23Eurobiotech 2013

tions obtained from Mucor circinelloides and Mucor race-mosus moulds (transesterification with 2-methylbutanol) and dimerization of obtained esters (cycloaddition) are transformed into dimeric esters containing fatty acid residues. These will be co-polymerized with appropriate reagents in order to produce new biodegradable aliphat-ic-aromatic co-polyesters. The polyesters will be utilized as components in the preparation of various fibrous poly-mers and composites.The project is being realized by nine research groups from Poland belonging to four different research estab-lishments, with the Lodz University of Technology be-ing the leader. The elaboration within this project new methods of preparation of polymer fibrous materials and composites will positively influence development of sci-ence-based economy and will increase the innovativeness of connected areas of research and production. The main recipients of elaborated methods will be producers of fi-bers and nonwovens from thermoplastic materials, sani-tary textiles, filtration materials, geotextiles, agrotextiles and packing materials.AcknowledgementsThis research is realized within the project BIOMASA (POIG 01.01.02-10-123/09) and partially financed by the European Union from the European Regional Development.

L3.3

Biotransfromations as a tool for BiorafineriesPawel Kafarski

Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland

The biorefinery concept is analogous to petrolium refin-ery, which produce multiple fuels and products from pe-troleum. At the beginning this term was used to describe a facility that integrates biomass conversion processes and equipment to produce fuels, power, heat, and value-add-ed chemicals. Athough still being related to biofuel pro-duction in a broader sense biorefining is the sustainable processing of biomass into a spectrum of marketable products, especially high-value fine chemicals. Ideally it is focused on integrated production for food, feed and non-food applications, while aiming for zero waste and getting maximum added value from co-products. It is be-lieved that in 2030 many key chemicals using biological and/or chemical processes.Biotransformations are chemical reactions that are cata-lyzed by microorganisms in terms of growing or resting cells or that are catalyzed by isolated enzymes. Because of the high stereo- or regioselectivity combined with high product purity and high enantiomeric excesses, bio-transformations are considered as technically superior to traditional chemical synthesis. They appeare to be a su-periour tool in production of high-value chemicals upon biorefining processes. For example, the Department of Energy, USA has identified 30 platform chemicals com-posed of one to six carbon atoms as potential candidates for biobased production with biotransformations being leading processes in this respect.AcknowledgementsThis work was supportedby the project “Biotransformations for pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-158/09, which is partly-financed by the European Union within the European Regional Development Fund for the Innovative Economy.

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L3.4

Bionidustry in Poland – current status, major obstacles, driving force and open opportunitiesTomasz Kapela

Biotechnika

The main goal of the presentation is to outline in gen-eral the current shape of bioindustries in Poland, brief-ly define what are the foundations of this new industri-al branch and try to outline what pushes it forward and what holds it back. An author will try, basing on his own experience, to draw a picture of current biobusinesses in Poland and characterise major fields of activities. On one hand Poland is a country of great opportunities and ex-pectations in terms of its bioresources. But on the other it is still struggling numerous problems of various nature (legal, financial, public awareness – related, etc.). Hope-fully the presentation will flash some light on positives as well as those issues that still need to be tackled in order to ensure, that Poland takes its chance to built strong, bio-based economy. Several case studies will help the guests to have a complete picture of discussed ideas

L3.5

Biobased production an answer to societal challenges? The importance of sustainability and communicationPatricia Osseweijer

Delft University of Technology, Department of Biotechnology

There are many reasons why we should replace our pres-ent fossil-based products such as plastics, chemicals and carpets to products made from biorenewable sources. One of them is that we have to be much more sustainable if we wish to mitigate climate change and feed a grow-ing world population. In that sense biobased production does contribute to societal challenges. However, the way we do this will matter, so far integrated approaches for production of materials, chemicals and energy do seem to provide the best economical, environmentally sustain-able and social benefits. However to introduce such practices successfully, we not only require innovation in industrial biotechnology to deliver the best routes to develop new materials from waste products but also a better management of our ag-ricultural production and novel collaboration between agricultural, chemical and energy sectors. In order to achieve this and the adequate policy measures to support this we need to have the support of citizens and consumers as well. That is not straightforward as for example the debate on food versus (bio)fuel shows. The presentation will address the different views on sustain-ability and the importance of (scientists to be involved in) communication to achieve a supported and well gov-erned transition to a biobased society.

25Eurobiotech 2013

Posters

P3.1

Isolation of bacterial strains capable of converting glycerol into succinic acidMarcin Podleśny, Piotr Jarocki, Jakub Wyrostek, Tomasz Czernecki, Zdzisław Targoński

Department of Biotechnology, Human Nutrition and Food Commodities, University of Life Sciences in Lublin

Succinic acid is currently used mainly in industries pro-ducing food, pharmaceutical products, detergents, sur-factants and ingredients stimulating plant and animal growth [1]. Enormous versatility of succinic acid and its derivatives applications leads to treatment of this di-carboxylic acid as one of the top twelve building block chemicals of great interest according to the United States Department of Energy [2]. Development of econom-ically efficient biotechnology process for succinic acid production could extend application market for this chemical to biodegradable plastics and green solvents. Present production scheme of succinic acid is based on petroleum as the starting source. Attempts for petro-leum substitution by renewable low-cost raw material are intensively made [3, 4]. These promising efforts are inseparable connected with the usage of high-yield pro-ducing microorganisms. Therefore searching for micro-organisms capable of efficient utilization of renewable low-cost substrates during succinic acid production is of the great interest [5]. Among considered inexpensive compounds for succinic acid production glycerol gained significant attention. This byproduct generated during biodiesel production process is desirable substrate be-cause having the same level of reduction as succinic acid. Bioconversion of glycerol to succinic acid seems therefore a redox balanced pathway [6].Present study describes isolation and screening of mi-croorganisms for succinic acid production on glycer-ol. Isolation of bacteria from different eight eco-niches was carried out. A total number of 187 bacterial isolates were obtained using microbiological techniques. After 96 hours of anaerobic cultivation  on glycerol containing medium isolates were screened for succinic acid produc-tion by HPLC. Succinic acid was not detected in nearly half of the examined bacterial strains. About eight per-cent of bacterial isolates screened showed succinic acid production rates exceeding 2 g/l. Succinic acid was not the only product of glycerol utilization in anaerobic con-dition. Ethanol and acetic acid were the most common byproducts followed by formic acid. Isolate KRBGAL, a facultative anaerobe isolated from Holstein Freisian cow rumen fluid, showed maximum yield of 4,6 g/l of succinic acid from 20 g/l of glycerol. The glycerol utilization rate during growth of KRBGAL isolate was nearly 68%. This isolate was identified as Escherichia coli by 16S rDNA se-quence analysis followed by biochemical identification using API 20E. This strain may become a candidate for

further genetic improvement of succinic acid production because of relatively high succinic acid yields and glycerol utilization rate.AcknowledgementsThis study was co-funded by The European Union project No. PO IG 01.01.02-00-074/09 from The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007–2013.References[1] McKinlay J.B., Vieille C., Zeikus J.G. (2007) Prospects for a bio-based succinate industry. Appl Microbiol Biotechnol 76: 727–40.[2] Werpy T., Petersen G. Eds. (2004) Top Value Added Chemicals from Biomass. US DoE.[3] Du C., Lin S.K.C., Koutinas A., Wang R., Webb C. (2007) Succinic acid production from wheat using biorefining strategy. Appl Microbiol Biotechnol 76: 1263–70.[4] Liu Y.P., Zheng P., Sun Z.H., Ni Y., Dong J.J., Zhu L.L. (2008) Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Biores Technol 99: 1736–42.[5] Scholten D., Daegele D. (2008) Succinic acid production by a newly isolated bacterium. Biotechnol Lett 30: 2143–46.[6] Yazdani S.S., Gonzalez R. (2007) Anaerobic fermentation of glicerol: a path to economic viability for the biofuels industry. Current Opinion in Biotechnology 18: 213-19.

Bioplastics and biobased polymers

Lectures

L4.1

Novel bioplastic fibres for tissue engineering and biodegradable packaging materialsMagdalena Wróbel-Kwiatkowska1, Jan Szopa1, 2, 3

1Linum Foundation, Wroclaw, Poland; 2Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland; 3WCB EIT+, Wroclaw, Poland

Flax (Linum usitatissimum L.) is best known as a plant cul-tivated for industrial purposes. Flax seeds are the source of oil used as the basal component or an additive for vari-ous paints or polymers, flax stems are the source of fibres that can be used in the textile and paper industries. A new generation of novel bioplastic fibres contain polyhydroxy-butyrate (PHB) were obtained from transgenic flax plants. Transgenic plants were generated by introduction of three bacterial genes necessary for PHB synthesis [1]. Biochem-ical analysis of bioplastic fibres revealed presence of sev-eral antioxidative compounds of hydrophilic (phenolics) and hydrophobic (cannabidiol CBD, lutein) nature, indi-cating their high antioxidant potential.IR spectra of transgenic flax fibres revealed structural changes in comparison to unmodified fibres, i.e. observed higher ordering of cellulose in transgenic fibres resulted from the intra- and/or intermolecular hydrogen bonds of glucopyranosyl residues and from the formation of the PHB-cellulose composite [2]. The presence of PHB nat-urally incorporated into the fibres increases their com-patibility with other polymers of biodegradable (polyhy-droxyalconate) and non-biodegradable (polypropylene, polystyrene) nature.Natural bioplastic flax fibres with stronger physical and higher bioactive properties may serve as the source of material for industry (automotive industry, packaging materials) or for biomedical application (tissues scaffold, implant).Thus biodegradable and bioactive composites with poly-lactic acid (PLA) or poly-e-caprolactone (PCL) as the matrix and bioplastic flax fibres as reinforcement were prepared and analyzed [3]. FTIR analysis of composites showed intermolecular hydrogen bonds between the constituents in composite PLA-flax fibers which were not detected in PCL-based composite. Mechanical anal-ysis of prepared composites revealed improved stiffness and a decrease in tensile strength. The viability of human dermal fibroblasts on the surface of composites made of PLA and transgenic flax fibres was the same as for cells

cultured without composites and only slightly lower (up to 9%) for PCL-based composites. The amount of plate-lets and Escherichia coli cells aggregated on the surface of the PLA based composites was significantly lower than for pure polymer. Thus, composites made of PLA and trans-genic flax fibres seemed to have bacteriostatic, platelet an-ti-aggregated, and non-cytotoxic effect.The composites made of PLA and transgenic bioplastic flax fibres implanted into rat musculus latissimus dorsi [4] did not have any negative effect on muscle function and genes expression (i.e. vascular endothelial growth factor, insulin like growth factor 1, insulin like growth factor 2, collagen-1, collagen-2 and myostatin). The composites also did not cause any inflammation response after inser-tion to the muscles and exhibited good biocompatibility to the muscle.The promising effect of bioplastic fibres which enhanced polypropylene resin was also detected. The preliminary data showed that composite of polypropylene and bio-plastic fibres may serve as novel packaging material with increased rate of degradation in soil.References[1] Wróbel M., Żebrowski J., Szopa J. (2004) Polyhydroxybutyrate synthesis in transgenic flax. J. Biotech. 107: 41–54.[2] Wróbel-Kwiatkowska M., Zuk M., Szopa J. et al. (2009) Poly-3-hydroxy butyric acid interaction with the transgenic flax fibers: FT-IR and Raman spectra of the composite extracted from a GM flax. Spectrochim Acta A Mol Biomol Spectrosc 73: 286–294.[3] Wróbel-Kwiatkowska M., Czemplik M., Kulma A., Żuk M., Kaczmar J., Dymińska L., Hanuza J., Ptak M., Szopa J. (2012) New biocomposites based on bioplastic flax fibres and biodegradable polymers. Biotech. Prog. 1336–1316.[4] Gredes T., Kunert-Keil C., Dominiak M., Gedrange T., Wróbel--Kwiatkowska M., Szopa J. (2010) The influence of biocomposites containing genetically modified flax fibers on gene expression in rat skeletal muscle. Biomedizinische Technik. Biomedical engineering 55(6): 323–9.

27Eurobiotech 2013

L4.2

Bacterial nanocellulose scaffolds for regenerative medicineKarolina Ludwicka, Marzena Jedrzejczak-Krzepkowska, Katarzyna Kubiak, Marek Kolodziejczyk, Stanislaw Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, Poland

Bacterial nanocellulose (BNC), synthesized by Gluconac-etobacter strain, is the natural biopolymer of exceptional purity and outstanding biocompatibility. Bionanocellu-lose is the material of known properties being already in-vestigated in a wide range of industrial and medical appli-cations, including clinical trials for wet wound dressing and as a potential implant e.g. in vascular surgery (small vascular grafts), bone, dental fillings, peripheral nerves regeneration and many others. Many publications con-firm that as a nondegradable biomaterial BNC implants fulfil their function by appropriate high hydrophilicity, inertness, lack of cytotoxicity, outstanding biocompati-bility and stability in a wide range of temperatures and pH. It was recently investigated that the neurotubes made of BNC are of very good biocompatibility and allow the accumulation of neurotrophic factors inside, thus, facili-tating the process of peripheral nerves regeneration. The successful application of BNC fragments in trachea re-construction was also described. Following the obtained results the direction of the current research is set to the improvement of BNC scaffolds in vivo functionality. A new method of production of novel composites made of BNC and polymeric or titanium meshes  has been de-veloped and investigated. The potential application of such constructs include cranioplasty and herniorraphy. Also a cartilage-like BNC scaffolds mechanical properties were evaluated together with in vitro biocompatibility confirmation. The results suggest the possible application of such a material in cartilage reconstruction. Although still, the stable connection with the growing cells is cru-cial for the effective tissue regeneration. Since the prop-erly adjusted structure of BNC membrane determines the success of such constructs adaptation, it is necessary to improve the manufacturing process to produce a number of both suitably dense and porous BNC scaffolds, in order to create the efficient drug reservoir system, as well as the proper conditions for the proliferation of host and in vitro introduced tissue-engineered cells.ReferencesKowalska-Ludwicka K. et al. (2013) Arch Med Sci 9(3), 527–534.Bielecki S. et al. (2012) In Bacterial Nanocellulose: A Sophisticated Multifunctional Material; Eds. Boca Raton, Florida: CRC Press, 157–174.Kolodziejczyk M. et al. (2010) Military Pharmacy and Medicine 3(1), 62–65.

L4.3

Biomedical applications of dendrimersBarbara Klajnert-Maculewicz

University of Lodz, Department of General Biophysics

Dendrimers are a family of synthetic macromolecules of considerable interest as a platform for biomedical re-search because they can be designed and synthesized hav-ing specific desirable properties. Dendrimers are charac-terized by a globular shape which is a result of their in-ternal structure. All bonds in dendrimers emerge radially from a central core to which monomers are attached. This unique molecular architecture means that dendrimers have a number of distinctive properties that distinguish them from other polymers. Dendrimers are monodisperse macromolecules with plenty of surface groups and internal cavities. The specific structure makes dendrimers suitable for many biomedi-cal applications.Due to a large number of terminal groups to which drug molecules can be conjugated, one molecule of dendrimer is capable of carrying drugs at a high density. Encapsulat-ing drugs inside dendrimers offers potential benefits such as prolonging the circulation time in the blood, protect-ing unstable drugs from the environment, enhancing sol-ubility of poorly soluble drugs, and achieving controlled release.Dendrimers, in addition to being excellent carriers for active compounds, have biological activity in their own right. They can be therapeutic agents in the prevention of neurodegenerative disorders and viral or bacterial in-fections.However, there is still a substantial gap of knowledge in understanding how dendrimers interact with biological structures, which has considerable consequences for the translation of research from bench to bedside.

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L4.4

Bionanocellulose production control from molecular point of viewKatarzyna Kubiak, Marzena Jędrzejczak-Krzepkowska, Marta Kurzawa, Karolina Ludwicka, Marek Kołodziejczyk, Stanisław Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, Stefanowskiego 4/10 St., 90-924 Lodz, Poland

Bionanocellulose (BNC) is an innovative bio-material tested in numerous applications, among which medical ones are on main focus of research conducted at IBT TUL. Development of new products is limited by diver-sity in Ga. xylinus strains productivity and time needed for whole process. Most challenging aspect of BNC pro-duction is planning the structural composition of fibrils building up the BNC membrane. The product parameters may be modulated by technological modifications done during or after bacteria culture only to some extent. The mentioned above aspects limiting technology develop-ment (productivity of a strain, time needed for biosyn-thesis and BNC structure control) are goals which may be reached after understanding of molecular processes responsible for control over polysaccharides synthesis and secretion, cell motility and rate of bacteria growth. In order to find molecular targets for productive strain modifications we performed genome sequencing and comparative transcriptomic analysis. Conclusions driven from results of both projects have opened a number of promising lines of molecular research. At the moment we are investigating the most intensively one of them, name-ly cellular stress response mechanisms. Initial results of BNC production with genetically engineered strains evi-dence that molecular control over production of BNC is realistic in a near future.

L4.5

Functionalized microfibrillar cellulose for bio-based polyamide compositesKrzysztof Pielichowski, Agnieszka Leszczyńska

Department of Chemistry and Technology of Polymers, Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, Poland

Crystalline cellulose nanoparticles, such as microfibril-lated cellulose (MFC), have been recently considered for manufacturing of short fiber reinforced polymeric com-posite materials as an alternative for artificial fibers, es-pecially glass fibers. Due to submicronic dimensions and crystalline structure cellulose nanofibers present higher mechanical strength and better thermal stability as com-pared to natural fibers. However, initial degradation tem-perature of MFC is still lower than processing tempera-tures of numerous engineering polymers and restrain wide implementation of green composites in structural applications. With respect to that problem a  heteroge-neous surface modification of never dried MFC was carried out by acetylation with acetic acid as well as es-terification with hexanoyl and decanoyl chlorides. The effect of molar ratio of total cellulose hydroxyl groups to modifying agent (OH:MA) and time of reaction on the thermal stability, structure, morphology and surface properties of cellulose fibrils were discussed. As a result of modification an increase in thermal stability of MFC was achieved while crystalline structure and fibrous morphol-ogy of the cellulose filler was preserved. Moreover, mod-ified MFC displayed significantly improved compatibility with a new bio-based engineering polymer polyamide 4,10. Thus blending of modified MFC with polymer melt was possible at temperatures as high as 285ºC. PA 4,10/acetylated MFC composites showed significantly better structural and morphological properties than polyamide 4,10/neat MFC system. The composite structure clearly depended on the processing conditions.AcknowledgementsAuthors are grateful to the Polish National Science Center for financial support under contract No. DEC-2011/01/M/ST8/06834. J. Rettenmeier & Shöne GmbH +CO KG is kindly acknowledged for sending free samples of microfibrillated cellulose.

29Eurobiotech 2013

Posters

P4.1

Oxidative polymerization of lignins by laccase in water-organic solvent systemIonita Firuta Fitigau1, 4, Francisc Peter1, Carmen Gabriela Boeriu2, 3

1University “Politehnica” of Timişoara, Faculty of Industrial Chemistry and Environmental Engineering, C. Telbisz 6, 300001 Timişoara, Romania; 2Wageningen UR Food & Biobased Research, Bornse Weilanden 9, 6708 WG Wageningen, Netherlands; 3University “Aurel Vlaicu” of Arad, Department of Life Sciences, str. E. Dragoi nr 2, Arad, Romania; 4National Institute of Research-Development for Electrochemistry and Condensed Matter, A. P. Podeanu 144, 300569 Timişoara, Romania

The enzymatic oxidative polymerization of five techni-cal lignins with different molecular properties, i.e. Soda Grass/Wheat straw Lignin, Organosolv Hardwood Lig-nin, Soda Wheat straw Lignin, Alkali pretreated Wheat straw Lignin, and Kraft Softwood was studied. All lig-nins were previously fractionated by acetone/water 50:50 (v/v) and the polymerisation of the low molecular weight fractions (Mw < 4000 g/mol) was carried out in the same solvent system. Reactivity of lignin substrates in lac-case-catalysed reactions was determined by monitoring the oxygen consumption. The oxidation reactions in 50% acetone in water mixture proceed with high rate for all tested lignins but the enzyme lost its catalytic activity at acetone concentration above 55%. The syringyl type Or-ganosolv Hardwood Lignin had the highest reactivity and the reaction proceeded with 19,66 (μM/min) initial rate. Polymerisation products were analysed by Size Exclusion Chromatography, Fourier Transform Infrared Spectros-copy, 31P-NMR. The results provide evidence of important lignin modifications after incubation with laccase. Struc-tural oxidation and a notably molecular weight increase (Mw up to 17500 g/mol) were attained. 31P-NMR spectral analysis revealed a decrease of the total phenolic hydroxyl groups and a substantial increase of the condensed phe-nolic OH content for all the polymerisation products. The obtained polymers have potential for applications in bio-plastics, adhesives and polymeric dispersants production.AcknowledgementsThe work of I.F. Fiţigău was partially supported by the strategic grant POSDRU 107/1.5/S/77265, inside POSDRU Romania 2007–2013 co-financed by the European Social Fund – Investing in People. The work at WUR FBR was carried out in the frame of the ERA-IB project “Products from Lignocellulose” (EIB 10.013).

P4.2

Biocatalytic synthesis of gluconolactone and ε-caprolactone copolymersAnamaria Todea1, Lajos Nagy2, Valentin Badea1, Sandor Keki2, Francisc Peter1

1University “Politehnica” of Timişoara, Faculty of Industrial Chemistry and Environmental Engineering, C. Telbisz 6, 300001 Timisoara, Romania; 2Department of Applied Chemistry, University of Debrecen, H-4010 Debrecen, Hungary

Synthesis of copolymers based on bio-derived monomers by in vitro enzymatic catalysis has attracted many research interests in the past years. The biodegradability and bio-compatibility properties of ε-caprolactone homopoly-mers place it as a valuable raw material, particularly for controlled drug delivery and tissue engineering applica-tions. However, the usefulness of such materials is limit-ed by their low hydrophylicity and slow biodegradation rate. In order to improve polycaprolactone properties and functionalities, copolymerization of ε-caprolactone with gluconolactone was investigated. Since enzymatic reac-tions involving sugars are usually hindered by the low sol-ubility of these compounds in common organic solvents, finding the best reaction medium was a major objective of this research.The optimal copolymerization conditions of the reactions were carried out by using different organic solvents (solvents mixtures), ionic liquids or solvent free systems that are able to dissolve (completely or partial-ly) sugars and are nontoxic for enzymes. Native and im-mobilized lipases by different immobilization techniques from Candida antarctica B and Mucor miehei have been used as biocatalyst and the reaction temperatures were set up to 80°C. Although the main copolymer amount was synthesized in DMSO:t-BuOH=20:80 medium, the high-est polymerization degrees, up to 16 for the copolymer product, were achieved in solventless conditions. The reaction product analysis revealed the formation of the cyclic products that could be probably the major imped-iment of further increase of the chain length. The prod-ucts, cyclic and linear polyesters, have been characterized by FT-IR, MALDI-TOF-MS and NMR analysis. Carbo-hydrate derivatives represent an important monomer source and insertion into non-polysaccharide backbones can lead to functional polymers with specific properties and application.

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P4.3

New methods of poly(itaconic acid) synthesisSzczepan Bednarz, Diana Błażejewska, Alicja Błaszczyk, Aleksandra Nowak, Piotr Krzywda

Crakow University of Technology, Faculty of Chemical Engineering and Technology

Itaconic acid (IA) is a one of twelve most important plat-from chemicals that can be produced from sugars via biological conversions. Large scale fermentation pro-cesses using molasses as carbohydrate source and fun-gus Aspergillus Terreus have been developed. The acid and its derivatives are frequently used in production of various polymers, which are viable alternatives to acryl-ic and methacrylic acid based polymers. Recently, due to non-toxic nature of the acid and biocompatibility, IA-based copolymers have been synthesized in form of hydrogels for biomedical applications. Itaconic acid is a  well-know, unsaturated monomer which polymerizes via radical mechanism with difficulties. Persulfates and water are used as a standard initiators and the most com-mon solvent, respectively. However, the process takes quiet a long time and for that reason, persulfate activators such as amino alcohols (N,N-dimethylenethanoloam-ine) or sodium hypophosphite have been developed to increase the polymerization rate or decrease the process temperature. As stated above, polymerization IA is usu-ally carried out in aqueous phase and to the best of our knowledge application of ionic liquids as the polymer-ization medium or the polymerization catalysts has not been previously reported. In this study, a preliminary re-sults of application choline salts or choline-based ionic liquids as polymerization catalyst or solvents, respective-ly, were shown. The catalytic effect of choline cation on decomposition rate of the polymerization initiator was demonstrated. Additionally, possibility of polymerization of ionic liquid – choline hydrogen-itaconate to obtain poly(itaconic acid) was presented. AcknowledgementsThis work was supported by the European Union Funds under Human Capital Programme within “Bioinżynier chemiczny (BINC)” project (contract number POKL.04.01.02-00-217/11-00).

P4.4

Medium engineering for 2-methylbutyl-1ol esters synthesisMirosława Szczęsna-Antczak, Emilia Grabarczyk, Agnieszka Borowska, Jakub Szeląg, Katarzyna Chudzik, Magdalena Żakowicz-Siuda, Tadeusz Antczak

Institute of Technical Biochemistry, Lodz University of Technology, Stefanowskiego 4/10 St., 90-924 Lodz, Poland

This study aims at the development of chemo-enzymatic methods of conversion of oleaginous biomass into diols – components of alkyl-aryl co-polyesters. It is one of objec-tives of the Biomass project realized by Ludz University of Technology. The main concept of the proposed (within the scope of task 3.2) procedure is based on chemo-enzy-matic conversions of triacylglycerols (TAGs), contained in various oils (mainly rapeseed, sunflower and waste oils), into dimers of fatty acid esters, followed by their conversion into diols and polymerization of the latter.In the first step plant oils are subjected to enzymatic alco-holysis by a branched alcohol (e.g. 2-methylbutan-1-ol) and resulting esters are physico-chemically converted into dimers (cycloaddition reaction), which are then converted into diols. Enzymatic step of this procedure is mainly based on application of Mucor circinelloides mem-brane-bound lipase, as whole-cell preparations, which are predestined to catalysis in non-aqueous media [1–4].Presented study focused on medium engineering (mo-lar ratio of substrates, type of organic medium and water content in reaction medium, as well as addition of some polar substances modifying the lipase micro-environ-ment such as diethylamine, triethylamine and ectoine [4] in concentration of 0.5–60 mM) of enzymatic alcoholysis of rape and sunflower oils in batch and semi-continuous processes.Water content was controlled by water quantification by Karl Fischer method and water activity measurement (Rotronik probe). Reaction progress was assayed by GC method (yield of esters) as well as by TLC plates anal-ysis using JustTLC program (Sweday) that enables esti-mation of percentage content of esters, mono-, di- and triacylglycerols and free fatty acids in reaction mixtures. Free fatty acids were additionally assayed by copper soaps method [5]. The reaction mixtures were also analyzed by HPLC (RP-C18 column, NQAD detector) method. Also the stability of lipases (by residual lipolytic activity mea-surement) was estimated.AcknowledgementsThe study was realized within the scope of the project POIG 01.01.02-10-123/09 “Application of biomass in production of environmentally friendly polymer materials” – tasks 3.2., co-financed from the funds of European Fund of Regional Development within the frames of Operation Program Innovative Economy 2007–2013.References[1] Szczesna-Antczak M. et al. (2009) Renew. Energy 34, 1185–1194.[2] Szczesna-Antczak M. et al. (2006) Enzyme Microb. Technol. 39, 1214–1222.[3] Szczesna-Antczak M. et al. (2004) J. Mol. Cat. B, Enzymatic, 29, 163–171.[4] Antczak T. et al. (2002) J. Mol. Cat. B Enzymatic, 19-20, 287–294.[5] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470–472.

31Eurobiotech 2013

P4.5

An influence of water content on enzymatic transesterificationJakub Szeląg, Mirosława Szczęsna-Antczak, Łukasz Stańczyk, Małgorzata Piotrowicz-Wasiak, Tadeusz Antczak

Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10 St., 90-924 Lodz, Poland

Water strongly affects reaction efficiency in non-aqueous systems. An impact of water content was studied for sun-flower oil and 2-methylbutan-1-ol transesterification re-action catalyzed by immobilized lipases: Mucor circinel-loides (mycelial preparation from ITB) and Thermomyces lanuginosa (from Novozymes).The influence of water content in reaction mixture on esters yield and stability of lipases preparations (residu-al lipolytic activity measurement) was determined. The transesterification processes were carried out using either only a mixture of substrates (without any additional sol-vent) or with the addition of petroleum ether. Water con-tent in reactions mixtures, which was controlled by Karl Fischer method, varied from 0 to 3% in the first variant and from 0 to 5% in the variant with petroleum ether. Products of transesterification reaction (esters, mono-, di- and triacylglycerols and free fatty acids) were quan-tified using JustTLC analysis program (Swedey) and the yield of esters was determined also by GC method. Free fatty acids were additionally assayed by a colorimetric method [1]. Water content was found to be a key fac-tor deciding of transesterification efficiency. The highest yield of 2-methylbutan-1-ol esters was achieved at ~0.5% water concentration for both M. circinelloides (67.5%, without petroleum ether) and T. lanuginosa (80%, also in the absence of petroleum ether) lipases. The decrease in lipolytic activity of both immobilized lipase preparations was the lowest at 1.5% water concentration.AcknowledgementsThe study was realized within the scope of the project POIG 01.01.02-10-123/09 “Application of biomass in production of environmentally friendly polymer materials” – task PZ 3.2., co-financed from the funds of European Fund of Regional Development within the frames of Operation Program Innovative Economy 2007–2013.References[1] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470–472.

P4.6

Synthesis of fluorescent polyesters from citric acidWiktor Kasprzyk, Szczepan Bednarz, Dariusz Bogdał

Chair of Biotechnology and Physical Chemistry, Faculty of Chemical Engineering and Technology, Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, Poland

Novel and carefully prepared biomaterials can facilitate growing demands of medicine. Excellent examples of these include fluorescent proteins, fluorescent organic dyes, lanthanide chelates, and quantum dots as well as recently developed carbon nanodots (CD). Carbon nan-odots can be easily synthesized from low-temperature py-rolysis of mixtures of citric acid with amines or diamines. Furthermore, relatively high quantum yields (up to 62%) have also been achieved for biodegradable photolumi-nescent polyesters (cys-BPLP). This was accomplished through introduction of different L-amino acids into the polyester structure made of biocompatible monomers of citric acid and aliphatic diols. Thus produced polyester solutions, films, scaffolds and nanoparticles emit strong and tunable fluorescence (up to 725 nm). Moreover, BPLPs demonstrate in vitro cytocompatibility, minimal chronic inflammatory responses in vivo, controlled deg-radability, good photostability and desired mechanical properties. Initial studies on the fluorescence mechanism of BPLPs have indicated morpholine-2,5-dione as the structure responsible for the fluorescent properties. The fluorescence mechanism of this ring was explained based on its planarity and hyperconjugation theory, though it is not fully understood yet.Here we present results of our investigations of chemical structure of the moiety responsible for cys-BPLP lumi-nescence. We found that as a result of polycondensation reaction of citric acid and amino acids, among many oth-ers, luminescent derivatives of 2-piridone are formed. In turn, the luminescent compound (5-oxo-2,3-dihy-dro-5H-[1,3]thiazolo[3,2-a]pyridine-3,7-dicarboxylic acid – TPA) was synthesized directly from citric acid and L-cysteine as well as being separated from cys-BPLP hy-drolyzate. Furthermore, attempts of introducing TPA to poly(diol citrate) films revealed that it can be incorpo-rated into the polyester chain through formation of es-ter bonds and even minor quantities led to luminescent materials.AcknowledgementsThe authors gratefully acknowledge the support for this work from the National Centre of Science of Poland (grant number: 2011/01/N/ST5/05591). We are also grateful to Marek Żylewski and Paweł Żmudzki for performing NMR and ESI-MS analyses, respectively.

32 Eurobiotech 2013

P4.7

Application of selectively-hydrogenated rapeseed oil for modification of flexible foamsSylwia Dworakowska1, Dariusz Bogdal1, Federica Zaccheria2, Nicoletta Ravasio2

1Department of Biotechnology and Physical Chemistry, Crakow University of Technology, Warszawska 24, Crakow, Poland; 2CNR-Institute of Molecular Science and Technologies, Via Golgi 19, Milan, Italy

Recently, renewable resources are getting more and more interesting because of decreasing fossil feedstocks and their higher prices. Natural oils have been shown to be a  potential bio-renewable feedstock since they can be derivatized towards different monomers for polymer chemistry e.g. for various polyurethane materials.Selective hydrogenation represents a valuable tool for standardization of vegetable oils’ composition. The use of selectively hydrogenated oil (with increased monoenic chains in the triglycerides) constitutes promising way of normalisation of vegetable oils [1–3].This work demonstrates synthesis of flexible polyurethane foams modified with rapeseed oil-based polyols being 30 wt.% replacement of petroleum polyols [4]. The aim of this study was to apply rapeseed oil stabilized through a selective hydrogenation process for synthesis of polyols and afterwards obtained flexible foams. Two-stage meth-od for preparation of selectively hydrogenated oil-derived polyols was presented. The bio-based polyols with differ-ent hydroxyl values were used for modifications of flexi-ble foams which allowed obtaining tailored properties of the final products for bedding and furniture applications i.e. increased compressive strength as well as elongation at break and tensile strength in comparison to reference ones made from 100% petrochemical polyol [5].AcknowledgementsThe authors thank the UBIOCHEM COST Action CM0903 for Short Term Scientific Missions of S. Dworakowska and F. Zaccheria. Authors also gratefully acknowledge Regione Lombardia for financial support through the VeLiCA Project and the European Union through the European Social Found within „Cracow University of Technology development program – top quality teaching for the prospective Polish engineers; University of the 21st century” project (contract No. UDA-POKL.04.01.01-00-029/10-00).References[1] Zaccheria F., Psaro R., Ravasio N., Bondioli P. (2012) Eur. J. Lipid Sci. Technol. 114: 24–30.[2] Zaccheria F., Psaro R., Ravasio N. (2009) Green Chem. 11: 462–465.[3] Trasarti A.F., Segobia D.J., Apesteguia C.R., Santoro F., Zaccheria F., Ravasio N. (2012) J. Am. Oil Chem. Soc. 89: 2245–2252.[4] Dworakowska S., Bogdal D., Prociak A. (2012) Polymers 4: 1462–1477.[5] Dworakowska S., Bogdal D., Zaccheria F., Ravasio N., Catal. Today (in press).

P4.8

Microwave-enhanced chemical modification of chitosanMarek Sławiński, Marek Piątkowski

Chair of Biotechnology and Physical Chemistry, Crakow University of Technology, Crakow, Poland

Recently in many research centres intense efforts towards obtaining new biobased polymers of high purity applica-ble in biomaterial engineering, are made. Such macromo-lecular compounds have to fulfil specific requirements, such as non-toxicity, biocompatibility and biodegrad-ability. Both polymers and products of their decompo-sition inside organism must be non-toxic, cause no in-flammation states or other immune responses, as well as any changes in body fluids. Currently, derivatives of lactic acid and poly(ethylene glycol)s are used for this purpose. In addition, due to specific properties, various kinds of chitosan derivatives are more often applied in biomedical engineering [1–3].An innovative and original method of the synthesis of chitosan derivative in form of copolymer with aspartic acid and various glycols under microwave radiation, was developed. The use of microwave technology allows the complete elimination of toxic catalyst, significantly re-duces reaction time and improves the yield. Moreover, in aqueous solution chitosan-based copolymer forms hydrogel selectively sorbing heavy metal ions, especially from very diluted solutions. The swelling properties are dependent on synthesis parameters as well as type of diol used in reaction.Presented research comprises synthesis route and also determination of properties of novel biobased materi-als with use of Fourier Transform Infrared Spectrosco-py (FT-IR), gel permeation chromatography (GPC) and UV-Vis Spectroscopy.References[1] Dee K.C., Puleo D.A., Bizios R. (2002) An Introduction To Tissue-Biomaterial Interactions, Wiley-Liss Inc.[2] Ratner B.D., Hoffman A.S., Schoen F.J., Lemons J.E. (2004) Biomaterials science, Elsevier Academic Press.[3] Bronzino J.D. (2000) The Biomedical Engineering Handbook: Second Edition, Boca Raton: CRC Press LLC.

Pharmaceutical biotechnology

Lectures

L5.1

Biosynthesis of delta-9-tetrahydrocannabinolic acid – from biotransformation studies in Cannabis sativa L. to biotechnological prodution in S. cerevisiaeOliver Kayser

Technical University Dortmund

delta-9-Tetrahydrocannabinolic acid (THCA) is an im-portant drug from Cannabis sativa L. with psychomi-metic activities. THCA and related herbal products are mostly misued and illegal use is dominating despite the fact that THCA has a significant medicinal value (e.g. che-motherapy, multiple sclerosis, anorexia). Today, breeding and cultivation of high content plants is not legal for ex-traction, but also organic synthesis is too expensive and not efficient to obtain high amount of THCA and it cor-responding decarboxylated THC derivative for industrial production. Biotechnology may provide new avenues for biosynthesis and production strategies, but the biosynthe-sis in planta is not fully eleucidated, why heterologous ex-pression of biosynthetic genes is complicated. Herein we provide an overview of recent developments on the eluci-dation of the pathway towards cannabinoids and updated attempts for construction of a recombinant host for the production of THCA and its main precursors CBGA and GPP. In short plants genetics, transciptomics and metabo-limcs of trichomes where biosynthesis is localised will be discussed [1], principles of gene expression of the main important genes (CBGA-Synthase, THCA-Synthase) will be explained, and into basic concepts of bioengineering of yeasts as potential hosts will be introduced [2].AcknowledgementsThis research was funded by Deutsche Bundesstiftung Umwelt (DBU, Förderkennzeichen 13252) and Arbeitsgemeinschaft Industrielle For-schung (AIF). Further information on heterologous THC production on www.pharma-biotechnologie.de.References[1] Happyana N. et al. (2013) Analysis of cannabinoids in laser microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR. Phytochermistry 87: 51–59.[2] Muntendam R et al. (2009) Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts Appl. Microbiol Biotechnol. 84: 1003–1019.

L5.2

How to pick the right pocket - the where and how of ligand binding Anke C. Schiedel, Dominik Thimm, Sonja Hinz, Benjamin Seibt, Farag Sherbini, Christa E. Müller

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der Immenburg 4, 53121 Bonn, Germany

G protein-coupled receptors (GPCRs) are among the most prominent drug targets and more than 30% of the drugs on the market are targeting GPCRs. For the development of new, highly potent, and subtype-selective drugs it is of considerable interest to know the receptor’s binding pocket and the exact binding modes of different ligands as well as amino acids involved in subtype selectivity and receptor activation. Although X-ray structures have re-cently been published for several GPCRs in complex with agonists or antagonists a combination of homology mod-eling and mutagenesis studies is still a powerful approach to facilitate rational drug design, especially since for the majority of receptors and ligands structural data are not available and will not be accessible in the near future.In the present study we investigated the A2B adenosine receptor (A2BAR), which plays a role in cancer, inflam-mation, and pain, and has been proposed as a new drug target. We analyzed the A2BAR’s binding pocket, and the subtype selectivity of several ligands versus its closest ho-molog, the A2AAR [1]. Using 3D homology models, based on crystal structures of the A2AAR, we predicted sever-al amino acids to be involved in ligand binding and/or receptor activation [2]. In addition we analyzed the role of the extracellular loop (ECL) 2, which contains four cysteine residues [3, 4] The predicted amino acid resi-dues were exchanged for alanine, or in case of cysteine, for serine. The mutants were stably expressed in Chinese hamster ovary (CHO) cells and characterized by radi-oligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine and aminopyridine derivatives (agonists). Our studies re-vealed that diverse ligands have distinct, yet overlapping binding pockets. Trp2476.48, Val2506.51, and Ser2797.42 were shown to be important for binding of nucleosidic ago-nists, while non-nucleosidic agonists did not interact with Ser2797.42 and showed weaker interactions with Trp2476.48

and Val2506.51. Leu813.28, Asn1865.42, and Val2506.51 were found to be crucial for binding of the xanthine-derived antagonist PSB-603.1,5 In addition, Leu813.28, which is not conserved among the adenosine receptors, was found to be important for subtype-selectivit [1]. We also investi-gated all four cysteine residues located in the ECL2 and

34 Eurobiotech 2013

were able to show that in the A2BAR only the highly con-served C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation [4]. We additionally ex-changed the whole ECL2 of the A2BAR for the ECL2 of the A2AAR and demonstrated that the ECL2 is involved in ligand binding and subtype selectivity and modulates ag-onist-bound receptor conformations, thereby controlling signaling efficacy [3]. The interdisciplinary approach pre-sented in this study using both, experimental data and computational predictions, provides valuable informa-tion for the rational design of desired highly potent and selective ligands, which are required to validate and ex-ploit their therapeutic potential, and to further elucidate the (patho)physiological roles of GPCRs.References[1] Thimm et al., Biochemistry, 2013, 52, 726–740.[2] Sherbiny et al., J. Comput. Aided Mol. Des., 2009, 23, 807–28.[3] Seibt et al., Biochem. Pharmacol., 2013, 85, 1317–1329.[4] Schiedel et al., Biochem. Pharmacol., 2011, 82, 389–399.[5] Borrmann et al., J Med Chem, 2009, 52, 3994–4006.

L5.3

Monoclonal antibodies targeting angiogenesis and lymphangiogenesis in cancer therapiesMonika Bzowska, Małgorzata Kulesza, Tomasz Klaus, Karolina Ossysek, Renata Mężyk-Kopeć, Joanna Bereta

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University

Monoclonal antibodies (mAbs) are one of the fastest de-veloping branches of the pharmaceutical industry. Due to a very high affinity and selectivity of binding to antigens, mAbs are used for specific targeting of certain molecules, e.g. soluble cytokines or tumor-associated antigens ex-pressed by cancer cells. Presently, therapeutic mAbs are applied: (i) to prevent transplant rejection, (ii) to inhib-it the progression of chronic inflammatory diseases and (iii) to fight cancer (usually in combination with other therapies). Apart from mAbs that directly target cancer cells, another approach which aims at angiogenesis has also been introduced to the clinic to inhibit tumor growth and spreading (metastasis). However, long-term studies indicate limited beneficial effects of this anti-angiogenic approach. Since tumor cells can escape from the primary site by entering not only blood- but also lymphatic ves-sels, the attempts to inhibit lymphangiogenesis in order to hamper tumor metastasis seem reasonable [1].The aim of our project is to generate mAbs able to bind and inhibit VEGF-C, the major growth factor for lym-phatic endothelium. We use a phagemid library Tomlin-son I+J and a phage display method to select the set of phages expressing anti-VEGF-C antibodies in a form of scFv (single chain variable fragment). The procedure of phage selection and obtaining of specific mAbs as well as the evaluation of their inhibitory activity will be present-ed. The potential of anti-VEGF-C antibodies to limit tu-mor development and spreading will be discussed.AcknowledgementsThis work was supported by a grant from the Polish-Swiss Research Programme (PSPB-057/2010 to JB).References[1] Antibody-based antiangiogenic and antilymphangiogenic therapies to prevent tumor growth and progression. Bzowska M., Mężyk-Kopeć R., Próchnicki T., Kulesza M., Klaus T., Bereta J., Acta Biochimica Polonica, Paper in Press, No. 2013_481.

35Eurobiotech 2013

L5.4

Bacterial cell wall assembly: new antibiotics and industrial biopolymersDouglas Weibel2, 3, 4, George Auer3, Hannah Tuson4, Nate Cira4, KC Huang5

1Department of Chemistry, University of Wisconsin-Madison; 2Department of Biomedical Engineering, University of Wisconsin-Madison; 3Department of Biochemistry, University of Wisconsin-Madison; 4Department of Bioengineering, Stanford University

This presentation describes how we are using our discov-ery of bacterial cell wall assembly and maintanence to develop new chemotherapeutics and produce new classes of industrial biopolymers. This presentation builds a con-nection between two connected areas of research in our lab that center on the bacterial cell wall: 1) identifying, studying, and exploiting the machinery that bacteria use to synthesize their polymer ‘exoskeleton’ (the peptidogly-can, ‘PG’) as a new feedstock for industrial polymers; and 2) and developing new clinical antibiotics and that target the bacterial cell wall and protecting these valuable bio-medical resources.In the first part of this presentation, I describe a gel-based force clamp technique that we recently developed to mea-sure the mechanical properties of live bacterial cells [1]. We recently used this technique to measure how single gene deletions in bacteria alter cell stiffness [Auer et al. in preparation]. Using this approach we have identified machinery that is essential for regulation of the mechan-ical properties of the PG and are now engineering the biochemistry of these proteins to tailor PG assembly as a source of biopolymers.In the second part of my talk, I introduce the cell wall ma-chinery as the most important targets of clinical antibiot-ics developed to date. Protecting these valuable resources requires providing health care workers with informed data on how to best treat pathogens at the point-of-need. To facilitate this process, we have developed a very sim-ple, inexpensive, and small plastic cartridge, which we re-fer to as QuickChip that uses an isothermal, DNA-based amplification technique to identify antibiotic resistance elements in bacteria in clinical samples and determines antibiotic susceptibility [2–4]. Integrating QuickChip with a smart phone or tablet-powered manifold enables its hands-free operation: the manifold heats the cartridge and measures the fluorescence output from the isother-mal assay, the ‘app’ sends the data to the ‘cloud’ for anal-ysis and diagnosis, and the cloud returns information on the bacterial strain and optimal antibiotic dosing to guide the effective treatment.We envision that the integration of these technologies will have an important impact on biomaterials science and clinical medicine by drawing new connections be-tween the structure, assembly, and mechanical properties of the bacterial cell wall.

AcknowledgementsThis research was supported by the Aspen Center for Physics (NSF grant 1066293), the NIH (grants T32 GM07215, DP2OD006466, and DP2OD008735), DuPont, and the Alfred P. Sloan Foundation.References[1] Tuson H.H., Auer G.K., Renner L.D., Hasebe M., Salick M., Crone W.C., Gopinathan A., Huang K.C., Weibel D.B. (2012) Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity. Mol. Microbiol. 84, 874–891.[2] Cira N.J., Dueck M.E., Weibel D.B. (2012) A self-loading microfluidic device for determining antibiotic toxicity. Lab Chip 12, 1052–1059.[3] Cira N.J., Weibel D.B., Self-loading microfluidic device and methods of use. 13/303,982. Filed: November 23.2011[4] Ho J., Cira N.J., Crooks J., Weibel D.B. (2012) Rapid identification of bacterial pathogens in an autonomous microfluidic device. PLoS One. 7, e41245.

36 Eurobiotech 2013

L5.5

Biocatalytic access tochiral intermediates for β-adrenergic receptorsantagonistsJanina Ewa Kamińska

Faculty of Biotechnology and Food Sciences, Institute of General Food Chemistry, Lodz University of Technology

Numerous β-adrenergic receptors antagonists used in the treatment of hypertension, angina pectoris, other cardiac diseases and glaucoma are derivatives of 1-amino-3-ary-loxypropan-2-ol. Among β-blockers of this structural class (S)-enantiomer exhibits higher affinity for β-adren-ergic receptors than (R)-enantiomer e.g. (S)-propranolol which is 130 times more active than its (R)-enantiomer.In spite of that knowledge, some β-blockers are still man-ufactured and used in therapy as racemates, even if there is evidence that distomers may exhibit side-effects. For instance (R)-propranolol may cause thyroid dysfunction. Due to increased demand for more effective, safe medi-cines, and the recommendations of institutions respon-sible for registration of new drugs, the synthesis of single enantiomers of chiral intermediates has become import-ant for pharmaceutical industry. Kinetic resolution of ra-cemic 3-aryloxy-1-halogenopropan-2-ols is convenient approach to synthesis of β-blockers new analogues with varying aromatic substituent as well as amine part.A significant decrease in prices of commercial enzyme preparations made their industrial application economi-cally attractive. However still prediction of enzyme enan-tioselectivity for a new substrate is doubtful and requires experimental checking. We decided to investigate how substitution pattern of aromatic ring and kind of halogen in substrate affects enzyme activity and enantioselectiv-ity. Therefore we obtained several novel racemic 3-ary-loxy-1-halogenopropan-2-ols in two-step sequence by treating phenols with epichlorohydrin following epoxide opening with hydrochloric or hydrobromic acid.Then resolution of 3-aryloxy-1-bromopropan-2-ols and 3-aryloxy-1-chloropropan-2-ols by lipase-catalyzed acetylation was undertaken. From eleven enzyme prepa-rations screened, two commercial lipases – Lipozyme® TL and Novozym® 435 we found effective for kinetic res-olution of 3-aryloxy-1-halogenopropan-2-ols by acetyl-ation with vinyl acetate in organic solvents. In prepara-tive experiments enantioselectivity ratio E from 64 to 99 was achieved, yielding both enantiomers with 89–99% ee. Then both enantiomers we successfully applied as chiral building blocks for preparation of new 1-alkyl-amino-3-aryloxyprop an-2-ols, by nucleophilic halogen substitution with isopropylamine or tert-butylamine. The activity of products as β-adrenergic receptor antagonists was evaluated in vitro.AcknowledgementsThe research was financially supported by Ministry of Science and Higher Education (grant No. N N405 2514 33) and European Social Fund and Polish State (Z/2.10/II/2.6/04/05/U/2/06).

L5.6

A plant-based oral vaccine against Hepatitis B Virus – from edible vaccine to tablet formulaTomasz Pniewski1, Józef Kapusta1, 2, Marcin Czyż1, Justyna Wojas-Turek3, Magdalena Milczarek3, Elżbieta Pajtasz-Piasecka3, Joanna Wietrzyk3, Piotr Bociąg1, Anna Kostrzak1, 4, Andrzej Płucienniczak2

1Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland; 2Institute of Biotechnology and Antibiotics, Warsaw, Poland; 3Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; 4Institut Pasteur, Paris, France

Hepatitis B is still one of the most common human in-fectious diseases, despite the fact that effective anti-HBV subunit vaccines have been available for 30 years. Orally administered plant-based vaccines have been considered as alternatives or supplements for standard injection vac-cines, due to assumed low-cost production and simplified vaccination. First trials showed prospects of mucoso-in-testinal immunisation. Unfortunately, despite the unques-tionable milestone, edible vaccines were impractical or in-sufficiently effective. Professional anti-HBV plant-derived vaccine would require an appropriate plant producer as well as vaccine composition and administration protocol.Herbicide-resistant lettuce lines expressing native HBV antigens, HBsAg subunits (S, M, L) and HBcAg, at levels 5–200 μg/g FW were engineered. Vegetative propagation of efficient plant lines enabled production scaling-up of an antigen-bearing material for an oral vaccine preparation.The prototype manufacture technology of the vaccine has been developed, based on freeze-drying of plant material. Powdered lyophilised tissue facilitated controlled antigen delivery and enabled conversion into oral formulas, e.g. tablets.Exclusively oral delivery of lyophilised tissue containing S-HBsAg induced systemic humoral response in mice at the minimal protection level (≥ 10 mIU/ml of anti-HBs antibodies) with higher S-IgA response in intestinal mu-cosa and immediate stimulation of Treg lymphocytes.Combination of injection priming and low-dose oral boosting triggered significant immune response in mice, as high as classical three-dose intramuscular vaccination. Titre of anti-HBs antibodies reaction reached hundreds mIU/ml, as well as stimulation of specific lymphocyte subpopulations and production of cytokines were clear-ly visible. In turn, tablets administered to humans pre-viously immunised with the standard pattern, increased anti-HBs level from 0 up to 100 mIU/ml or 2 – 3-fold when pre-immune anti-HBs were detectable.The study provides some basics regarding parenteral-oral immunisation strategy using an efficacious and convenient plant-derived formula which would serve as a booster vac-cine for possible human vaccination against hepatitis B.AcknowledgementsThis study was supported by grants No. 2 P04B 001 27 and No. N N302 157837 from the Polish Ministry of Science and Higher Education and under the auspices of Medana Pharma Inc., Sieradz, Poland.

37Eurobiotech 2013

L5.7

New fluorescence methods of visualisation of extracellular matrix in metabolically active tissuesJerzy Dobrucki

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Laboratory of Cell Biophysics

Only a few microscopy methods of visualizing extracel-lular matrix (ECM) in live tissues in situ and in freshly excised tissues are available. Collagen fibers polymerized under laboratory conditions can be imaged in an opti-cal microscope, using dark field, differential interference (Nomarski), or polarization contrast, however these methods are not ECM specific. Two sophisticated im-aging techniques can image collagen – detecting second harmonic generation (SHG), and a coherent anti-Stokes Raman scattering (CARS). There are no simple, inexpen-sive, or widely available techniques for 3-dimensional imaging of collagen and elastin fibers in live animals, or excised metabolically active tissues.A new low molecular weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in confocal imaging of extracellular matrix in freshly excised animal tissues. The dye readily pene-trates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. AcknowledgementsThis research was supported by a grant 2067/P01/2007/32 from Ministry of Science and Higher Education in Warsaw.

Posters

P5.1

The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selectionEwa Kowalska, Filip Bartnicki, Katarzyna Pels, Wojciech Strzalka

Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Crakow, Poland

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from low-weight molecules, such as ions, vitamins, antibiotics, to high-weight molecules, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive syn-thesis, low immunogenicity and high specificity.The classical DNA aptamer selection method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was described in the early 90-ties of the last century. In the first selection cycle a target molecule is exposed to a synthetic DNA aptamer library composed of 1014–1015 different oligonucleotides. The DNA fragments bound to the target are amplified using PCR and the pool of single-stranded DNA used for the next selection round is prepared. Usually several selection rounds are sufficient to obtain aptamers which are highly specific for the target molecule.There are many ways of immobilizing the target molecule to perform the SELEX procedure. One of the commonly used methods for protein/peptide targets is based on im-mobilized metal affinity chromatography (IMAC). There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types and bead material.In this study we tested the impact of different IMAC res-ins on the DNA aptamer selection process during the first four SELEX cycles. A histidine-tagged twenty-nine amino acid peptide corresponding to the inter-domain connecting loop fragment of human Proliferating Cell Nuclear Antigen was used as a selection target. Agarose resins containing identical linkers, but different metal ions (Co2+, Cu2+, Ni2+, Zn2+) were studied. Simultane-ously, different resin materials (agarose and polysty-rene) containing the same metal ion (Co2+) were tested. The results clearly demonstrated the impact of the metal ion and resin material on the aptamer pool enrichment level. Concluding, these data indicate that for successful IMAC-based SELEX, the determination of the optimal resin might be important.AcknowledgementsThis work was financially supported by The National Centre of Research and Development grant LIDER/28/54/L-2/10/NCBiR/2011 to WS.

38 Eurobiotech 2013

P5.2

Bcl-2 family gene expression on HeLa cells treated with Physalis peruviana ethanol extractsOzgur Çakir1, Bilgin Candar Çakir2, Murat Pekmez1, Kerem Fidan1

1Istanbul University; 2Istanbul Kultur University

As a member of Solanaceae family, Physalis peruviana has great horticultural and economic importance due to its high nutritional value, high vitamin content, minerals and antioxidants. P. peruviana also has anti-inflammato-ry, antimicrobial and anticancer properties, so we aimed to investigate potential effects of Physalis peruviana (goldenberry) ethanol extracts for cytotoxic activity and apoptotic gene expression on human HeLa cells.MTT (Tetrazolium blue) colorimetric assay was used to determine the viability of cells in the presence of the ex-tracts. HeLa cells were treated with 10, 25, 50, 100 and 200 microgram/ml concentrations of Physalis peruviana shoot and leaf ethanol extract for 48 h. Furthermore, ef-fects of extracts on apoptotic gene expression on Bcl-2, Bax, Bak, Bcl-x, Bik, Mcl-1, Bfl-1 were determined by re-verse transcriptase polymerase chain reaction (RT-PCR). As a result of these assays, it was found that the shoot and leaf ethanol extracts of Physalis peruviana had cytotoxic effect on HeLa cell cultures when applied as 100 micro-gram/ml concentration. It was also determined that shoot and leaf extracts caused significant changes on especially antiapoptotic genes expressions.Shoot and leaf extracts of Physalis peruviana is consid-ered as a potential source of anticancer compounds. Fu-ture studies are needed to elucidate chemical characteri-zation of the P. peruviana active compounds as potential anticancer agents.

P5.3

Antibacterial and antioxidant activites of Physalis peruvianaMurat Pekmez1, Fatma Elif Çepni1, Bilgin Candar Çakir2, Özgür Çakir1

1Istanbul University; 2Istanbul Kultur University

Medicinal plants are used in folk medicine for centu-ries due to biological activities of bioactive metabolites. Physalis peruviana L. (Solanaceae) is one of the common medicinal herb used for its properties as anticancer, an-timicrobial, antipyretic, diuretic, and anti-inflammatory immunomodulator. For this reason we aimed to evaluate in vitro antibacterial and antioxidant activites and total phenolic content of P. peruviana ethanol extracts.Disk diffusion assay was used to determine antibacterial effect. In antibacterial activity maximum inhibition zone was determined in Lactococcus lactis. The lowest MIC value was 100 microgram/disc for Staphylococcus aureus A950277, the highest MIC value was 700 microgram/disc for Escherichia coli DH5-alpha. The antioxidant activity was evaluated by Cuprac method using trolox as stan-dard and extracts exhibit total antioxidant capacity. Total phenolic content was measured as gallic acid equivalents. The highest TEAC and total phenolic content values of the leaf and the shoot extracts are 0.291 ± 0.04 and 0.192 ± 0.015 and 40.69 ± 0.21 and 30.21 ± 0.71, respectively.These results indicate that P. peruviana ethanol extracts may include effective compounds to be used as therapeu-tic agents and give a great interest for future reseach.

39Eurobiotech 2013

P5.4

Arbutin production via biotransformation of hydroquinone in in vitro cultures of Aronia melanocarpa (Michx.) ElliottInga Kwiecień, Agnieszka Szopa, Kornelia Madej, Halina Ekiert

Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, 9 Medyczna Street, 30-688 Crakow, Poland

Arbutin (hydroquinone β-D-glucoside) is a compound of plant origin possessing valuable therapeutic (uri-nary tract disinfection) and cosmetic (skin whiten-ing) properties, which can be obtained from in vitro cultures of plants belonging to different taxa via bio-transformation of exogenously supplemented hydro-quinone [1, 2].Preliminary studies demonstrated the ability of in vitro cultures of Aronia melanocarpa established in our labora-tory to perform β-D-glucosylation of hydroquinone [3]. The aim of the present experiments was to optimize hy-droquinone biotransformation, in particular, to establish the effect of the dose and the method of precursor appli-cation on the course of the reaction.The agitating cultures of Aronia melanocarpa were main-tained on the Murashige and Skoog medium [4] con-taining growth regulators: the cytokinin – BAP (6-ben-zylaminopurine), 2 mg/ml and the auxin NAA (α-naph-thaleneacetic acid), 2 mg/ml. The biomass was cultured for 2 weeks and then hydroquinone was supplemented at the following doses: 96, 144, 192, 288 and 384 mg/l either undivided or divided into two or three portions added at 24-hour intervals. The content of the product of the reac-tion – arbutin, was determined using an HPLC method [5] in methanolic extracts from biomass and lyophilized medium samples collected 24 hours after addition of the last precursor dose.Arbutin was accumulated mainly in the cultured bio-mass, but also in the medium when higher precursor doses (288 mg/l and 384 mg/l) were supplemented. The total contents of arbutin were very diverse, from 2.71 to 8.27 g/100 g d.w. Arbutin production rose with increasing hydroquinone concentration. Its maximum content was observed after hydroquinone addition at 384 mg/l divid-ed into two portions.Biotransformation yield also widely differed ranging from 37.04%–73.80%. The identity of the product – arb-utin, after its isolation and purification was confirmed by spectral analysis (1H-NMR spectrum).The obtained maximum content of arbutin was higher than that required by the newest 9th Edition of the Pol-ish Pharmacopoeia for Uvae ursi folium (7.00 g/100 g d.w.) [6], and is interesting from practical point of view.References[1] Skrzypczak-Pietraszek E., Szewczyk A., Piekoszewska A., Ekiert H. (2005) Acta Physiol. Plant. 27: 79–87.[2] Piekoszewska A., Ekiert H., Zubek Sz. (2010) Acta Physiol. Plant. 32: 223–229.

[3] Kwiecień I., Majcher J., Ekiert H. (2013) 7th German-Polish Symposium “Interdisciplinary research for pharmacy”, Gdańsk, Abstracts, 136.[4] Murashige T., Skoog F. (1962) Physiol. Plant. 15: 473–497.[5] Štambergová A., Supčiková M., Leifertová I. (1985) Českoslov Farm. 34: 179–182.[6] Uvae ursi folium. In: 9th Edition of the Polish Pharmacopoeia, Vol. I, 1410-1411.

40 Eurobiotech 2013

P5.5

The accumulation of phenolic acids in agitating cultures of Ruta graveolens LAgnieszka Szewczyk, Marzena Surzyn, Halina Ekiert

Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, 9 Medyczna Street, 30-688 Crakow, Poland

Apart from many valuable secondary metabolites, like alkaloids, flavonoids, coumarin compounds, volatile oil, Ruta graveolens contains also phenolic acids. These compounds exhibit very beneficial therapeutic proper-ties, e.g. antioxidant, anticancer and immunostimulating activities. These compounds occur in plant cells free and bound as, for instance, glucosides or esters [1].Our earlier studies demonstrated that stationary cultures of Ruta graveolens maintained in our laboratory were ca-pable of producing some free phenolic acids in the in-teresting amounts from practical point of view. The main metabolite was protocatechuic acid [2]. The aim of the present investigations was to assess productive capacity of agitating cultures of this species.The agitating cultures were maintained on four variants of the Linsmaier and Skoog (L-S) medium [3] containing growth regulators: the cytokinin – BAP (6-benzylamino-purine) and the auxin – NAA (α-naphthaleneacetic acid) in the concentration range from 0.1 to 3.0 mg/l. Biomass cultured in vitro was collected after 4-week growth cycles and extracted with methanol. These methanolic extracts subjected or not to hydrolysis (2 M HCl, 2 h) were used for determination of thirteen phenolic acids (cinnamic and benzoic acid derivatives) and cinnamic acid using an HPLC method [4].None of the compounds under study was detected in un-hydrolyzed samples. However, hydrolyzed extracts were shown to contain eight phenolic acids: o-coumaric acid, p-coumaric acid, ferulic acid, gallic acid, p-hydroxyben-zoic acid, protocatechuic acid, syringic acid and vanillic acid. Protocatechuic acid was accumulated at the high-est levels in biomass cultured on all tested medium vari-ants (19.6–33.6 mg/100g d.w.). Amounts of o-coumaric acid (3.6–6.3 mg/100g d.w.) and p-hydroxybenzoic acid (3.1–6.3 mg/100g d.w.) were much lower. Only small levels of the remaining phenolic acids were confirmed. The total content of phenolic acids ranged from 35.6 to 48.6 mg/100g d.w. The maximum content was found in biomass extract from the L-S medium supplemented with 2 mg/l BAP and 2 mg/l NAA.The obtained results indicate that the phenolic acids un-der study occur in biomass of Ruta graveolens cultured in agitating cultures only in the bound form. They also demonstrate a significant concentration-dependent effect of growth regulators on the accumulation of phenolic ac-ids.This type of Ruta graveolens in vitro cultures can be a po-tential rich source of protocatechuic acid.

References[1] Ekiert H., Czygan F.Ch. (2007) In: Biotechnology – Secondary metabolites. Plants and Microbes (Ramawat K.G. and Merillon J.M. ed.), Science Publishers, Enfield, New Hampshire, USA, 445–482.[2] Ekiert H., Szewczyk A., Kuś A. (2009) Pharmazie 64: 694–696.[3] Linsmaier E.M., Skoog F. (1965) Physiol. Plant. 18: 100–127.[4] Ellnain-Wojtaszek M., Zgórka G. (1999) J. Liq. Chrom. Tech. 22: 1457–1471.

41Eurobiotech 2013

P5.6

The influence of L-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. shoot cultureEwa Skrzypczak-Pietraszek, Joanna Słota

Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, 9 Medyczna Street, 30-688 Crakow, Poland

Phenolic acids are an important group of plant sec-ondary metabolites with different, valuable therapeutic properties (e.g. antioxidant, antiphlogistic, immuno-stimulating, antiseptic) [1]. Besides plants growing in the open air, tissue cultures can be an alternative source of secondary metabolites. Yield of their accumula-tion in in vitro cultures can be increased by different methods, including culture medium supplementation with precursors, elicitors and changing the standard amounts of the medium components. The purpose of this study was to investigate the influence of precur-sor (L-phenylalanine), elicitor (methyl jasmonate) and different sucrose concentration on phenolic acids ac-cumulation in agitated shoot cultures of Exacum affine Balf. f. (Gentianaceae).Cultures were maintained in conical flasks with Mu-rashige and Skoog medium [2] supplemented with plant growth regulators: BAP (6-benzylaminopurine), 1 mg/l, NAA (α-naphthaleneacetic acid), 0,5 mg/l and GA3 (gib-berelic acid), 0,25 mg/l. Variant A’ contained 3% of su-crose (standard amount) and the other six variants (A-F) – 6% of sucrose. After two weeks L-phenylalanine (1,6 g/l of medium) and/or methyl jasmonate (two concentra-tions: 100 µM or 800 µM) were added to B-F variants. Variants A’ and A were treated as references. Plant mate-rials were collected after 1, 3 and 7 days after the addition of the precursor and/or the elicitor. Control samples were collected too. Phenolic acids were assayed in the collected biomass before and after acid hydrolysis (2 M HCl, 2 h). Qualitative and quantitative analysis of phenolic acids in methanolic extracts from biomass were conducted by an HPLC method [3].Fourteen phenolic acids (protocatechuic, gallic, gentisic, chlorogenic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, salicylic, o-coumaric, ros-marinic) and cinnamic acid were found in all samples. The total content of free phenolic acids increased from approximately 0,368% to 0,838% (2,3-times) and the total content of the whole phenolic acids (free and bound) – from 0,866% to 1,390% (1,6–times) – depending on the MS medium variants. The studies show that the best vari-ant contained 6% of sucrose (double amount of the stan-dard), L-phenylalanine – 1,6 g/l of medium and methyl jasmonate – 100 µM.Analysis of the results in the described experiment showed that it is possible to increase the accumulation of

phenolic acids in Exacum affine shoot cultures – adding the precursor (L-phenylalanine), the elicitor (methyl jas-monate) and increasing the sucrose concentration.References[1] Khadem S., Marles R., Molecules, 15 (2010) 7985–8005.[2] Murashige T., Skoog F., Physiol. Plant, 15 (1962) 473–497.[3] Ellnain-Wojtaszek M., Zgórka G., J. Liq. Chrom. & Rel. Technol., 22 (1999) 1457–1471.

42 Eurobiotech 2013

P5.7

Biotechnology in cosmetology: Introduction of Argireline® an anti-aging peptide. Study of cellular cytotoxicity of Argireline® solutionMarek Grosicki, Anna Cukier, Katarzyna Kieć-Kononowicz

Jagiellonian University Medical College, Faculty of Pharmacy, Department of Technology and Biotechnology of Drugs, 30-688 Crakow, Poland

Argireline® is an anti-aging peptide synthesized by com-pany: Lipotec S.A. In cosmetology it is used as synthetic cosmetic ingredient in form of a powder or more com-monly 0,05% argireline® solution. This acetyl hexapep-tide-3 is mimicking the N-terminal end of SNAP-25 pro-tein, that plays important role in formation of neuronal SNARE complex. Argireline® prevents formation of skin lines and wrinkles in a very similar way as botulinum toxin (Botox). It competes with SNAP-25 for a position in the SNARE complex, destabilizing it. As a result, the neuronal vesicles cannot release neurotransmitters from the axon endings. In the end that causes attenuation of skin muscle contraction, especially in the forehead and around the eyes. It also inhibits overproduction of cat-echolamines, which can induce formation of wrinkles. Argireline® can be incorporated in cosmetic formulations such as emulsions, gels or sera. Its’ effectiveness has been confirmed in several anti-wrinkle tests performed in vitro using chromaffin cells and in vivo on healthy volunteers [1]. The aim of presented study is to elaborate the prop-er methodology of cytotoxity study of cosmetic’s active ingredients. Tests were performed on argireline® solution in its’ declared by producer concentration. In order to es-timate cytotoxity of the argireline® solution MTS test was used. This test allows to measure the efficiency of mito-chondrial oxidative activity in living cells [2]. In the ex-periments both HEK293 cells and human fibroblasts were treated with argireline® solution. Examined were: short time cytotoxic effect of argireline® as well as its’ influence on cellular proliferation features. Considered methods result in dose dependent argireline® cytotoxicity effect. Argireline® solution, dissolved more than six times from original concentration decreases cellular proliferation rate and metabolic activity. In conclusion it was shown that the MTS test can be used in order to determine cyto-toxic properties of cosmetic’s active ingredients.AcknowledgementsWe would like to thank: Przedsiębiorstwo Produkcyjno-Handlowe Ryszard Kaczmarek i Synowie Sp. z o.o. Spółka Komandytowa company, that is the only official distributor of Lipotec S.A. products in Poland, for providing argireline® solution sample, that was used in the presented experiments.This work was supported by K/ZDS/001915.References[1] Lipotec S.A. (2002) Neuronal exocytosis inhibiting peptides and cosmetic and pharmaceutical compositions containing said peptides, WO 00/64932.[2] Cory J.G., Owen A.H., Barltrop T.C. (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Communications, 3(7), 207–212.

P5.8

Antibacterial activity of essential oils obtained from leaves and fruits of a native plant from South America, Schinus areiraMarta H. Alabrudzinska1, Liliana S. Celaya2, Ana C. Molina2, Carmen I. Viturro2, Silvia Moreno3

1Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, PC 80-822, Poland; 2PRONOA Laboratory, F.I., National University of Jujuy, S. S. de Jujuy PC 4600, Argentina; 3Plant Biochemistry laboratory, Institute Leloir Foundation, IIBBA-CONICET. Buenos Aires, PC 1405. Argentina

Essential oils (EOs) are aromatic components obtained from different plant parts such as leaves, fruits and flower, and are composed of secondary metabolites such as ter-penes and terpenoids. These compounds act as a mech-anism of defense against pathogen attack (microbes, herbivores or competing plants). A great deal of essential oils is used in different industries, mainly in perfumes (fragrances), food (as flavoring and preservatives), phar-maceuticals (therapeutic action) and for centuries in tra-ditional medicine [1]. It is well known that some essential oils exert antimicrobial and antioxidant properties [2, 3].Schinus is a genus belonging to the Anacardiaceae family. This family comprises about 70 genera and 600 species. They are used traditionally as a healing, stomachic and antidiarrheal agent, due to the presence of tannins and oil resins. In this family, S. molle L. (also known as Califor-nia pepper and pink pepper) was introduced from South America to most tropical and subtropical areas of the world, as well as the Mediterranean. Another tree of this genus, S. areira L. commonly called peppertree, molle and aguaribay, is a native plant from South America and nowadays it is distributed through Argentina, south-east-ern of Brazil, Peru, Colombia, Ecuador, Uruguay and widely distributed in Mexico, Central and Southern of California and West Texas, United States. Several parts of this tree are used in traditional medicine as antibacterial, antifungal and antirheumatic [4].Extensively studies regarding the characterization of EOs isolated from the Schinus molle has been reported, how-ever much less research has been done on the biological properties of the oils of S. areira [5]. Moreover, signifi-cant variations in the chemical composition of the EOs of S. areira have been reported in relation to the geographic origin[5], although correlation between the presence and content of specific compounds and the antimicrobial ac-tivity has not been deeply investigated.The aim of this work was to characterize the antibacterial activity of EOs isolated from leaves and fruits of S. areira trees with different chemical profile. EOs were obtained by distillation of dried leaves and fruits and their chemi-cal composition were determined by gas chromatography (GC)/FID and GC/MS as previously described [6]. A mi-croplate bioassay for quick and sensitive determination of antibacterial activity was used [7]. The antibacterial ac-

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tivity was assayed against the human pathogenic bacteria Staphylococcus aureus ATCC 25923 and against a methi-cillin-resistant S. aureus strain (MRSA).Results showed that the EO isolated from fruits and leaves showed high antibacterial effect on S. aureus. The inhibitory effect of both fractions on the bacterial growth decreases with increasing dilutions, demonstrating di-rect proportionality between concentrations of compo-nents and the antibacterial activity. The EO of fruits have a greater inhibitory effect than the leaves. Notably, both the S. areira oil was able to inhibit the bacterial growth of SAMR strain, which caused a large number of deaths in hospitalized patients every year worldwide. The minimal inhibition concentrations obtained for fruits and leaves were from 3.2 µL/mL to 15.0 µL/mL. In conclusion, our findings demonstrated the potential use of the oils against Staphylococcal infections.AcknowledgementsThis research was supported by the National Agency of Scientific and Technological Promotion, Argentina: Grant PICTO 00150 and the National Council for Scientific and Technological Research (CONICET), Argentina. S. M. is a member of the Research Scientific Career from CONICET.References[1] Bakkali et al. (2008) Food Chem Toxicol 46 (2): 446–475.[2] Burt (2004) Int. J. Food Microbiol 94: 223–253.[3] Hosnia et al. (2011) Crops and Products 34: 1622–1628.[4] Mendonça Rocha et al. (2012) Molecules 17: 12023–12036.[5] Viturro C et al. (2010). Proyecto CYTED IV. 20, Edit. Universitária da PUCRS. Chapter 10.[6] Ojeda-Sana et al. (2013). Food Control 31: 189–192.

P5.9

Cell wall proteins of Candida albicans and non-albicans Candida species as the binders for human proteins and potential therapeutic targetsJustyna Karkowska-Kuleta1, Sylwia Kedracka-Krok2, Karolina Seweryn1, Maria Rapala-Kozik1, Andrzej Kozik1

1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Analytical Biochemistry; 2Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Physical Biochemistry

The development of infections caused by pathogenic fun-gi is inseparably connected with the specific properties of pathogen cell surface. In the yeasts of genus Candida, comprising a number of species pathogenic for humans, the cell wall is composed of β-1,3- and β-1,6-glucans, chi-tin, mannoproteins anchored to the polysaccharide scaf-fold through various types of linkages, loosely attached “moonlighting proteins” and small amount of lipids. As is well known, the surface-connected proteins of C. albicans, the most extensively characterized fungal pathogen, are often involved in the adhesion to host pro-teins and cells, contributing to yeast virulence, whereas so far nothing is known about the adhesins of non-albicans Candida species, particularly two emerging pathogens C. tropicalis and C. parapsilosis. In this work, with the use of affinity chromatography and tandem mass spectrometry coupled with high-per-formance liquid chromatography (LC-MS/MS), we were able to isolate and identify fungal proteins responsible for tight binding of a human serum protein, high molecular mass kininogen (HK), a component of kinin-generating system involved in the regulation of many physiological and pathological processes such the coagulation and fi-brinolysis, inflammation, blood pressure control, neovas-cularization and apoptosis. In the case of unicellular, yeast-like forms of C. albicans, the interaction with HK occurred mainly via the “moon-lighting proteins” such as glycoamidase (Png2p), enolase (Eno1p), phosphoglycerate mutase (Gpm1p) and triose-phosphate isomerase (Tpi1p). In C. tropicalis and C. para-psilosis, in their most adhesive hyphal forms, the typical adhesins, agglutinin-like sequence proteins Als3p and Als7p, respectively, were identified as the key elements that strongly bound HK. Because of multifunctionality of HK, HK-binding proteins which occur on the pathogen surface can be considered as potentially useful targets for new therapeutic approaches.AcknowledgementsThis work was supported in part by the National Science Centre, Poland (the grant No. 2012/07/B/NZ1/02867 to A.K.)

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P5.10

Quercetin decreases myelosuppression and oxidative DNA damage induced by etoposide in bone marrow of ratsMonika A. Papież

Department of Cytobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Crakow, Poland

A common side effect of etoposide is myelosuppression, which limits the use of this anticancer drug. A few percent of patients treated with high-dose of etoposide develop treatment-related acute myeloid leukemia (t-AML). The leukemogenic action of etoposide may correspond to ox-idative damage to DNA in bone marrow precursor cells caused by phenoxyl radicals of this drug. The aim of this study was to determine the effect of antioxidant quercetin on myelosupression and oxidative DNA damage induced by etoposide in bone marrow cells of rats.Brow Norway rats were treated with: 1) quercetin (100  mg/kg bw by gavage) for two weak, 2) etoposide (50 mg/kg bw, i.p.) for three consecutive days. 3) querce-tin + etoposide, and 4) vehicle for examined compounds. The rats were decapitated two hours after the last dose of quercetin and/or one hour after administration of etopo-side. Bone marrow smears were prepared and stained with May-Grünwald-Giemsa stain. Oxidative DNA dam-age was analyzed by comet assay using formamidopyrim-idine DNA glycosylase, which cuts oxidised purine bases.Etoposide administration caused a significant decrease in the percentage of myeloid precursors and erythroid nu-cleated cells. Etoposide also induced significant increase in oxidative DNA damage in comparison to the control. Pretreatment with quercetin, followed by the coadminis-tration of etoposide, led to significant increase in the per-centage of myeloid precursors and nucleated erythroid cells compated to group treated with etoposide alone. Quercetin also significantly diminished the level of oxi-dative DNA damage induced by etoposide. The obtained results indicate that quercetin could be a useful object in the complementary treatment.

P5.11

Application of immobilized ethylbenzene dehydrogenase and whole-cell recombinant phenylethanol dehydrogenase system for synthesis of chiral alcoholsMaciej Szaleniec1, Agnieszka Dudzik1, Mateusz Tataruch1, Wojciech Snoch1, Joanna Opalińska-Piskorz1, Jolanta Bryjak2, Małgorzata Witko1, Johann Heider3

1Jerzy Haber Institute of Catalysis and Surface Chemistry PAS, Niezapomianjek 8 St., 30-239 Crakow, Poland; 2Department of Bioinorganic Chemistry, Wroclaw Technical University, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland; 3Laboratory of Microbial Biochemistry, Philipps-University of Marburg, Karl-von-Frisch Strasse 8, D-3504 Marburg, Germany

Chiral building blocks are valuable synthons for the preparation of biologically active compounds. They find its application as precursors of a range of pharmaceuti-cally active compounds (e.g. (S)-fluoxetine, duloxetine). Up to date two biocatalytic strategies found its way into industry: kinetic resolution of alcohol racemates (partic-ularly with use of lipases) and stereoselective reduction of prochiral carbonyl compounds (catalyzed by alcohol de-hydrogenases). However, both strategies have their own weakness such as the maximum 50% yield for the former or a need to recover NADH/NADPH cofactor and prob-lem of reduction process reversibility in case of the latter. The reversibility of ketones reduction process results with establishing of an equilibrium which decreases the final alcohol yield especially for compounds more thermo-dynamically stable in the keto form than in the alcohol form.Fortunately, the synthesis of chiral alcohols can also be achieved with the stereospecific hydroxylation of achiral hydrocarbons. The oxidation reaction is thermodynami-cally irreversible, which removes limitation imposed by reaction reversibility. Such reaction can be catalyzed by ethylbenzene dehydrogenase (EBDH), a molybdenum bacterial enzyme stereoselectively hydroxylating more than 30 different ethylbenzene derivatives [1]. The en-zyme immobilized on a functionalized solid support in the optimal reaction conditions can carry on conversion of alkylaromatic and alkylheterocyclic compounds to their secondary (S) alcohols and is able to achieve sub-stantial substrate conversions. Moreover, the enzyme im-mobilization enables an easy separation of the biocatalyst from the reaction media.The performance of EBDH will be compared with an outstanding alcohol dehydrogenase, phenylethanol de-hydrogenase (PEDH) [2], that is overexpressed in E.co-li. PEDH immobilized in bacterial cells can be direct-ly used in batch reactors as a biocatalysts. It’s amazing tolerance for organic solvents, wide substrate spectrum and resistance to deactivation makes PEDH an ideal in-dustry catalyst.

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The presentation will compare these two complementary approaches to synthesis of chiral alcohols and will discuss strong and weak points of each method in light of their potential for application in fine chemical industry.AcknowledgementsAuthors acknowledge financial support of the project ‘Biotransformations for pharmaceutical and cosmetics industry’ No. POIG.01.03.01-00-158/09-04.References[1] Knack D., Hagel C., Szaleniec M., Dudzik A., Salwinski A., Heider J., Appl. Environ. Microb., 78 (2012) 6475–6482.[2] Höffken H.W., Duong M., Friedrich T., Breuer M., Hauer B., Reinhardt R., Rabus R., Heider J., Biochemistry, 45 (2006) 82.

P5.12

Studies on sintering process of bone-derived hydroxyapatiteDagmara Malina, Kamila Biernat, Agnieszka Sobczak-Kupiec

Institute of Inorganic Chemistry and Technology, Crakow University of Technology, 24 Warszawska St., 31-155 Crakow, Poland

Being the main inorganic constitution of hard tissues (bone and teeth), calcium phosphates have been attrac-tive in medical and dental applications in hard tissue repair. Hydroxyapatite (HAp) ceramics have attracted attention because of their excellent osteoconductive and bioactive properties. Such materials are biocompatible, bioactive, osteoconductive, nontoxic, noninflammatory and have an ability to form strong bonds with the living hard tissue. Although hydroxyapatite exhibits excellent biological properties in tissue environment, possibilities of HAp applications are limited due to its poor mechan-ical properties. Thermal treatment is one of many routes to improve mechanical parameters of hydroxyapatite.The aim of this research was to investigate the sintering process of different hydroxyapatite materials. The HAp derived from animal bones was calcined for 3 h in differ-ent temperatures (700°C, 800°C and 900°C respectively) in order to obtain three different types of hydroxyapatite powder. HAp powders were then uniaxially pressed and sintered at different temperatures ranging from 800°C to 1400°C. The physical properties of as-produced sintered bodies and raw HAp powders were investigated.The results show that there is a difference in sintering behavior of natural hydroxyapatites depending on tem-perature. The main differences refer to the loss of mass, shrinkage, changes in porosity and density of the inves-tigated materials.AcknowledgementsThe research was supported by The National Centre for Science in Poland.

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P5.13

Sugar analogs as a novel therapeutic agents against cancerAnna Czubatka1, Pawel Tokarz2, Zbigniew Witczak3, Tomasz Poplawski1

1Department of Molecular Genetics, University of Lodz, Lodz, 90-236, Poland; 2Department of Organic Chemistry, University of Lodz, Lodz, 91-403, Poland; 3Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes-Barre, PA 18766, USA

Understanding the biological function of sugars that occur naturally in cells resulted in the development of new ways of research aimed at the use of carbohydrates as drugs in the therapy of various human pathologies. Further development of carbohydrate chemistry synthe-sis requires the development of new compounds of this group and analysis of their properties. Small alterations in chemical structures of the sugars functional group(s) or in a sugars ring can change their chemical character or/and their biological activity. For example sugars after the introduction of the sulfur atom into their molecule have a significant smaller susceptibility into chemical and enzymatic degradation. That’s why sugar analogs could be valuable compounds used in the therapeutic strategies for human diseases including cancer.The aim of this study was to investigate the effect of sug-ar analogs: 6-thio-β-D-fructopyranose, 5-thio-D-glucose and 1-epoxy-myo-inositol on two chosen cancer cell lines: LoVo (human colon adenocarcinoma cell line) and A549 (human lung adenocarcinoma epithelial cell line). Potential cytotoxic properties of sugar analoges were tested through incubation specific cell line with a chosen sugar analog and then determining the number of living cells by using colorimetric assay – Cell Counting Kit-8 (CCK-8). This assay showed that all tested sugar ana-logs caused a decrease in cancer cells viability between 20–80% at a concentration of 8mM, depending on the compound. Comet assay in various versions reveal geno-toxic potential of sugar analogs. As we found our tested compounds induced broad spectrum of DNA damage by introduction single and double strand breaks, alkali-la-bile sites and oxidative modifications into DNA. Comet assay also shown increase of the number of DNA damage generated in the cell lines with increasing time of incu-bation with studied sugars. But the cancer cell lines were able to repair more than 50% DNA damages in 120 min.The results suggest that the sugar analogs may act as a cyto- and genotoxic agents on cancer cells. It is possible that sugar derivatives will be a novel therapeutic agents against various types of cancer.AcknowledgementsThis work was supported by the The National Science Centre, decision No. DEC-2011/01/B/NZ4/03391.

P5.14

Fluorometric assay for screening the new multidrug-resistant strains of Pseudomonas aeruginosa with efflux pumps overexpressionGniewomir Latacz, Zuzanna Rowińska, Anna Matys, Katarzyna Kieć-Kononowicz

Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Crakow, Medyczna 9, 30-688 Crakow, Poland

Pseudomonas aeruginosa is a Gram-negative bacteria which is responsible for 10% of all hospital-acquired (nosocomial) infections. It is now well understood that nosocomial infections caused by those organisms are of-ten hard to treat because of the constitutive expression of AmpC β-lactamase and efflux pumps, combined with a low permeability of the outer membrane (intrinsic re-sistance of the species). Additionally, the bacterial muta-tions leading to overexpression of Resistance Nodulation Division (RND) efflux pumps eliminating several classes of antibiotics may cause one of the major antibiotic resis-tance mechanisms in Pseudomonas.The previously reported fluorescence agar-based meth-od was shown to be simple and useful for the detection of efflux activity among multidrug-resistant Gram-neg-ative and Gram-positive clinical isolates [1][2]. Within this study we modified this method by using fluores-cence plate reader to determinate fluorescent dye acri-dine orange (AO) retention in bacterial cells. Control and clinical Pseudomonas aeruginosa isolates were cul-tured overnight in Mueller-Hinton Bulion (MHB) broth with the presence of AO. The OD of the cultures was adjusted to the value 0,6. The fluorescence of cultures as well as media were measured and ∆RFU were calcu-lated. Internal fluorescence of each bacterial strain were also measured in MHB broth without AO to estimate the presence of pyoverdine – the yellow-green and fluo-rescent pigment secreted by some Pseudomonas species. Additionally, the effect of efflux pump inhibitor PAβN on control and clinical strains was examined at the con-centration of 0,1 mM.The method was applied to one control strain ATCC 27853 and two clinical MDR Pseudomonas strains with described resistance on several classes of antibiotics: P4600/01/09 (named P1) and P3768/03/08 (named P2). We observed that the clinical strains P1 and P2 showed generally lower ∆RFU value after incubation with AO in compare to control ATCC 27853. It sug-gests lower retention of fluorescent dye AO and high-er efflux activity of P1 and P2 strains. The increased ∆RFU value of P1 (increased AO retention) was also observed after incubation with efflux pomp inhibitor PAβN. The high effect of PAβN on ATCC 27853 strain was also confirmed.AcknowledgementsThe work was partly supported by UJ CM grant: K/DSC/001407.

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References[1] Martins A., Amaral L. (2012) Screening for Efflux Pump Systems of Bacteria by the New Acridine Orange Agar Method, In vivo 26: 203–206.[2] Martins M., Viveiros M., Couto I., Costa S.S., Pacheco T., Fanning S., Pagès J.-M., Amaral L. (2011) Identification of Efflux Pump-mediated Multidrug-resistant Bacteria by the Ethidium Bromide-agar Cartwheel Method, In vivo 25: 171–178.

P5.15

Production of triterpenoids with cell and tissue culturesMagdalena Malinowska, Elżbieta Sikora, Jan Ogonowski

Crakow Univeristy of Technology

Triterpenes are group of biologically active compounds which can be found mainly in resins or oils of various plants. The compounds structure is based on isoprene units and there are about 30,000 identified compounds. The biological activity of triterpens are very diverse and many studies have already confirmed the following therapeutic effects: anti-inflammatory, analgesic, anti-microbial, antiviral, antimycotic, immunomodulatory and hepatoprotective [1]. Synthesis of triterpenes is of-ten quite problematic and it requires many complicated stages and hard reaction conditions. These compounds can be also extracted from plant material, however the process very often does not meet all the pharmaceutical requirements.As an alternative, triterpens biosynthesis can be per-formed by enzymatic and chemical reactions, whereas biotransformation is generally more specific and elimi-nates unwanted side products and the molecule alter-ations. Furthermore, modification of the triterpenes structure to enhance their pharmaceutical application can be efficiently carried out using biotransformation processes. These kind of synthesis processes use micro-organisms or isolated enzymes. Apart from that, plant cell and tissue cultures as methods of the triterpen com-pounds production are also popular.. The biotechno-logical methods eliminate some disadvantages of classic methods, such as extract variability and instability of the obtained compound, moreover they ensure high re-producibility, optimal regio- and enantioselectivity and the environmentally friendly reaction conditions [2–4]. Sometime the microbial transformation is the only way to obtain desired product. During the last years, in the lit-erature there are several studies describing the microbial transformation as a useful tool to improve the structural diversity of triterpenoids [5, 6]. The attention is mainly paid on looking for an optimal organism. The most wide-ly used technique is still classical screening of a series of microbial strains. The studies on triterpene biotrans-formation give various information of new synthesized compounds and let predict the metabolism mechanism of the compounds [7]. It should be pointed out that plant in vitro production of biologically active metabolites are cultivated in bioreactors under sterile conditions, due to the substances are completely insulated from adverse en-vironmental factors, both biotic (e.g., diseases and her-bivores) and abiotic [8, 9]. The disadvantage of the men-tioned method is that the amounts of desired metabolites are often lower than the contents in plants [10]. The strat-egies to enhance the yields of the metabolites in plant in vitro cultures include media and hormone optimization,

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improvement of bioreactor designs and operation modes and the use of various techniques such as immobilization and genetic modification [11–13].References[1] Dzubak P., Hajduch M., Vydra D., Hustova A., Kvasnica M., Biedermann D., Nat Prod Rep, 2006, 23, 394.[2] Mufflera K., Leipolda D., Schellera M.C., Haasb C., Steingroewerb J., Bleyb T., Neuhausc H.E., Miratad M.A., Schraderd J., Ulbera R., Process Biochem., 2011, 46, 11.[3] Cheng Z.-H., Yu B.-Y., Cordell G.A., Qiu S.-X., Org Lett, 2004, 6, 3163.[4] Chen Q.-H., Liu J., Zhang H.-F., He G.-Q., Fu M.-L., Enzyme Microb. Tech., 2009, 45, 175.[5] Qian L.-W., Zhang J., Liu J.-H., Yu B.-Y., Tetrahedron Lett, 2009, 50, 2193.[6] Parra A., Rivas F., Garcia-Granados A., Martinez A., Mini-Rev. Org. Chem., 2009, 6, 307.[7] Chatterjee P., Kouzi S.A., Pezzuto J.M., Hamann M.T., Appl Environ Microb, 2000, 66, 3850.[8] Vanisree M., Lee C.Y., Lo S.F., Nalawade S.M., Lin C.Y., Tsay H.S., Bot Bull Acad Sin, 2004, 42, 1.[9] Ramachandra Rao S., Ravishankar G.A., Biotechnol Adv., 2002, 20, 101.[10] Wink M., Charlwood B.V., Rhodes M.J.C. (Eds.), Clarendon Press, Oxford, 1990.[11] Pavlov A., Georgiev M., Bley T., Z Naturforsch C, 2007, 62, 439.[12] Dörnenburg H., Process Biochem, 2004, 39, 1369.[13] Lee M., Jeong J., Seo J., Shin C., Kim Y., J. In, Plant Cell Physiol, 2004, 45, 976.

P5.16

Determination of antioxidant activity of black tea extractMagdalena Malinowska, Kamil Kurleto, Grzegorz Kurowski, Barbara Laskowska, Elżbieta Sikora, Otmar Vogt

Crakow Univeristy of Technology

Tea has been consumed all over the World for over two thousand years and now it is the most popular caf-feine-containing beverage. The first written records of the tea can be found in VIII century BC, where a brews of the tea leaves were treated as a medicine. Over the centuries, tea plantations have been established and the methods of tea collection and processing have been improved [1–4]. The tea is not only important because of its popularity but also due to its beneficial influence on human health. Therapeutic effects of tea have been extensively examined in many in vitro and in vivo studies. It was confirmed that tea leaves ingredients has antibacterial, antifungial, antiviral properties, they also prevent cell mutations and they inhibit progress of heart diseases. Moreover, tea can stimulate neural system and regulate its functions. Pos-itive effect of the tea drinking is associated mainly with high content of polyphenols, mainly catechins that act as an antioxidants. Their amount in tea infusion depends not only on the type of tea, but also on brewing process [5–19].The subject of this work was to investigate the influence of the brewing conditions: temperature, time and degree of leaf fragmentation, on antioxidant activity of black tea. The total antioxidant activity (TAA) of the tea infusions was studied using three different test methods: DPPH free radical reduction, Folin-Ciocalteu and thiocyanate. UV-VIS spectrophotometer was applied to measure the absorbance of the samples.The obtained results showed that brewing conditions have a significant effect on the TAA of the black tea infu-sions. The brewing temperature is the most important pa-rameter influencing on polyphenols content in black tea infusions.. Results of all three applied methods showed that the highest antioxidant activity was characterized the infusions obtained at the 90 and 95°C. Brewing time also determines TAA of the tea infusions and the optimal brewing time was 5 to 10 minutes. Recommended 2–3 minutes of brewing time is not sufficient to obtain the maximum antioxidant activity of the tea extracts. Also the leaf fragmentation has a positive impact on TAA of the tea infusions.References [1] Łuczaj W., Skrzydlewska E., Prev. Med., 2005, 40, 910.[2] Büyükbalci A., Sedef Nehir E., Plant Foods Hum Nutr., 2008, 63, 27.[3] Caprari M., Herbata, Warszawa 2009.[4] Sharangi A.B., Food Res. Intern., 2009, 42, 529.[5] Gramza A., Korczak J., Amarowicz R., Pol. J. Food Nutr. Sci., 2005, 3, 219.[6] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food Chem., 2011, 125, 44.[7] Leung L.K., Su Y., Chen R., Zhang Z., Huang Y., Chen Z.Y., J. Nutr., 2001, 131, 2248.

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[8] Wright L.P., Biochemical analysis for identification of quality in black tea, University of Pretoria, Pretoria, 2002.[9] Friedman M., Mol. Nutr. Food Res., 2007, 51, 116.[10] Davies M.J., Judd J.T., Baer D.J., Clevidence B.A., Paul D.R., Edwards A.J., Wiseman S.A., Muesing R.A., Chen S.C., J. Nutr., 2003, 133, 3298.[11] Ferrazzano G.F., Amato I., Ingenito A., de Natale A., Pollio A., Fitoterapia, 2009, 80, 255.[12] Robak J., Gryglewski R.J., Pol. J. Pharmacol., 1996, 48, 555.[13] Saha P., Das S., Asian Pac. J. Cancer Prev., 2002, 3, 225.[14] Cichoń Z., Miśniakiewicz M., Zesz. Nauk. AE Krak., 2005, 678, 103.[15] Yang D.J., Hwang L.S., Lin J.T., J. Chromatogr. A, 2007, 1156, 312.[16] Gramaza A., Korczak J., Trends Food Sci. Tech., 2005, 16, 351.[17] Thanaraj S.N., Seshardi R., J. Sci. Food Agric., 1990, 51, 57.[18] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food Chem., 2011, 125, 44.[19] Obanda M., Owuor P.O., Mang’oka R., Food Chem., 2004, 85, 163.

P5.17

Homology Modeling of Steroid C25 Dehydrogenase Agnieszka Rugor1, Stefan Mordarski2, Jakub Staroń2, Andrzej Bojarski2, Maciej Szaleniec1

1Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30-239 Crakow, Poland; 2Instytute of Pharmacology, PAS, Smetna 12, 31-343 Crakow, Poland

Steroid C-25 dehydrogenase (S25DH), a new EBDH-like molybdenum enzyme isolated from denitrifying Steroli-bacterium denitrificans Chol-1ST [1], catalyzes regiose-lective hydroxylation at the C-25 tertiary carbon atom of the aliphatic side chain in cholesterol and its deriva-tives [2]. Up to date the structure of the enzyme is still unknown. However, the S25DH amino acids sequence is highly similar to other enzymes from DMSO reductase family such as ethylbenzene dehydrogenase (EBDH, PDB code 2IVF) from Aromaticum aromatoleum EbN1 [3] (identity 40%, similarity 96%).Based on the sequence and topological similarities of the active site, the homology model of S25DH was generated with MODELLER 9v7. The best homology structure was selected based on the induced fit docking results of the known S25DH substrates (cholest-4-en-3-one, cholest-4,6-dien-3-one, choles1,4-dien-3one, cholest-5-en-3-ol, cholesteryl hemisuccinate and cholesteryl sulphate). The docking results allowed detailed analysis of interactions between amino acid residues and substrates. It also pro-vided an insight into the active site structural features which can be used in the elucidation of S25DH reaction mechanism. The results suggest a strong vdW stabiliza-tion of the sterane ring system of the substrate as well as indicated involvement of the Asn531 residue in electro-static interaction with C3 oxo/hydroxyl substituent of the substrate.Moreover, the structural analysis of the MGD binding mode showed strong electrostatic and van der Waals interactions of the cofactor with a range of highly con-served residues, both in case of S25DH homology model and the EBDH. This results suggests that the structural features surrounding the molybdenum cofactor were cor-rectly modelled by our protocol.AcknowledgementsThe authors acknowledge the financial support of the National Center of Science under the SONATA grant UMO-2012/05/D/ST4/00277 “The mechanism of regioselective oxidation of cholesterol derivatives by a novel molybdenum enzyme, steroid 25-OH dehydrogenase from Stereolibacterium denitrificans”.References[1] Dermer J., Fuchs G., J. Biol. Chem. (2012) 287, 36905–36916.[2] Chiang Y. et al., J. Bacteriology (2008) 190, 905–914.[3] Szaleniec M. et al., Biochemistry (2007) 46, 7637–764.

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P5.18

Bacterial steroid C-25 dehydrogenase – a novel biocatalyst for regioselective hydroxylation of steroid compoundsNatalia Zawada, Agnieszka Rugor, Mateusz Tataruch, Maciej Szaleniec, Daniel Knack

Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30-239 Crakow, Poland

Steroid C25 dehydrogenase (S25DH) from Sterolibacte-rium denitrificans is a member of the DMSO reductase family of molybdenum enzymes with a bis-MGD cofac-tor in its active site. It catalyzes the oxygen independent hydroxylation of the tertiary C25 atom of the side chain of cholesterol and other steroid compounds to the respec-tive tertiary alcohols [1].The ability of S25DH to intro-duce hydroxyl groups into the C25 atom of cholesterol and its derivatives has the potential for application in the production of pharmaceuticals such as activated vitamin D3 or 25-hydroxycholesterol [2].Up to date, S25DH was purified by a multi-step anaerobic protocol developed by Dermer and Fuchs. However, it has been shown that EBDH-like enzymes can be stabilized in aerobic conditions by addition of a suitable electron ac-ceptor (e.g. ferrocenium (III) ions) [3]. In the poster we present the shortened anaerobic and aerobic enzyme pu-rification procedures that delivers an enzymatic formula-tion with defined biological activity.To confirm the application potential of S25DH, it was used as a catalyst in the synthesis of 25-hydroxy-cholest-4-en-3-one. The synthesis was carried out in a batch reac-tor system with homogenous and immobilized enzyme.Admittedly, the S25DH purification from S. denitrificans is still not efficient enough for industrial purposes. There-fore, the experiments aiming at the development of a het-erologous expression system were undertaken. An artifi-cial operon containing all the three S25DH subunits and the chaperone gene was generated and the genes overex-pressed in E.coli. The poster will discuss the method used for construction of the artificial operon and its efficiency in production of the enzyme.AcknowledgementsThe authors acknowledge the Polish National Center of Research and Development under grant LIDER/33/147/L-3/11/NCBR/2012.References[1] Dermer J., Fuchs G. (2012) J. Biol. Chem. 287: 36905–36916.[2] Chiang Y.R., Ismail W., Müller M., Fuchs G. (2007) J. Biol. Chem. 282: 13240–13249.[3] Szaleniec M. et al. (2007) Biochemistry, 46 7637-764653: 1085–1091.

P5.19

Screening of porous carriers for covalent immobilization of pepsin and laccaseKatarzyna Szałapata1, Monika Osińska-Jaroszuk1, Kamila Wlizło1, Jolanta Bryjak2, Anna Jarosz-Wilkołazka1

1Maria Curie-Sklodowska University, Department of Biochemistry, Lublin, Poland; 2Wroclaw University of Technology, Department of Bioorganic Chemistry, Wroclaw, Poland

Pepsin (EC 3.4.23.1) is one of the principal enzymes in the digestive system and it belongs to the acidic prote-ase family. Laccase (EC 1.10.3.2), a copper-containing oxidase produced mainly by fungal strains, requires an oxygen as one of the substrates during oxidation of broad spectrum of substrates about different chemical structure. Thanks to catalytic activity of these enzymes they are widely used in many industries. Their potential applications, for example in food, textile or biomedical industries, become more common and replace many of the traditional methods. This situation makes it necessary to increase the stability of biocatalysts in order to higher profitability of their use, which is possible thanks to en-zyme immobilization.In this work pepsin from hog stomach (Fluka) and lac-case (Cerrena unicolor) were covalent immobilized onto typical enzyme carriers. Controlled pore glass, acrylic beads, silica-gel and cellulose-based carriers were func-tionalized by the presence of various chemical groups (–NH2, –COOH and –OH). We used three different cross-linkers for their activation: glutaraldehyde, carbo-diimide and divinyl sulphone. After the immobilization we tested the storage stability for each carrier. For the im-mobilized pepsin the stability under different conditions of pH and temperature were also measured. The results obtained in this experiment allowed to select the optimal carrier for each enzyme.

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P5.20

Poly(AA-co-MMA) micro- and nanoparticles for controlled drug deliveryKatarzyna Bialik-Wąs, Bożena Tyliszczak, Krzysztof Pielichowski

Department of Chemistry and Technology of Polymers, Crakow University of Technology, Warszawska 24 St., 31-155 Crakow, Poland

The modern medicine and pharmacy require more and improved drug delivery systems targeting only on the areas covered by the diseases. Such conditions can fulfill the micro-and nanoparticles based on polymers, pH-sen-sitive hydrogels, biopolymer – based microgels/nanogels (biomicrogels/bionanogels). Furthermore, polymer and copolymer nanoparticles are characterized by high stabil-ity in contact with biological fluids [1–3]. The controlled drug delivery systems are truly important in treatment of human diseases due to their therapeutic advantages in improving bioavailability and minimizing systemic side effects [4].In this study, poly(AA-co-MMA) micro- and nanoparti-cles with feed molar ratios AA:MMA = 7:3 were used for metronidazole loading. Metronidazole (MET) 2-(5-ni-tro-2-methylimidazol-1-yl)-ethanol is very well known antimicrobial agent and commonly used in clinical med-icine for > 45 years. Mainly it is applied for anaerobic infections and for treatment of giardiasis, trichomonia-sis and amoebiasis [5,6]. The reaction for preparation of poly(AA-co-MMA) micro- and nanoparticles was car-ried out at 80°C under nitrogen for 8h, whereas MET was dispersed in distilled water and mixed with micro- and nanoparticles as an aqueous suspensions. The complex-ation was conducted for 24h at room temperature.The drug release behavior of MET-loaded poly(AA-co-MMA) micro- and nanoparticles was evaluated in wa-ter and phosphate buffered saline (PBS, 0,9% NaCl) at 37°C. Introduction of MET into poly (AA-co-MMA) micro-and nanoparticles allow to gradual and controlled release of the active substance. Furthermore, structural analysis using FT-IR (ATR) and 1H NMR, as well as sur-face morphology assessment by SEM, were performed.AcknowledgementsThis research was supported by Ministry of Science and Higher Education – project C-4/257/2013/DS-M.References[1] Bajpai A.K., Shukla S.K., Bhanu S., Kankane S., Prog Polym Sci 33 (2008) 1088–1118.[2] Liu Z., Jiao Y., Wang Y., Zhou C., Zhang Z., Adv Drug Deliv Rev 60 (2008) 1650–62.[3] Agnihotri S.A., Mallikarjuna N.N., Aminabhavi T.M., J Control Release 100 (2004) 5–28.[4] Nafea E.H., Marson A., Poole-Warren L.A., Martens P.J., J Control Release 154 (2011) 110–122.[5] Lofmark S., Edlund Ch., Nord C. E., Clin Infect Dis 50 (2010) 16–23.[6] Herculano R.D., Alencar de Queiroz A. A., Kinoshita A., Oliveira O.N. Jr., Graeff C.F.O., Mat Sci Eng C 31 (2011) 272–275.

P5.21

Effect of conjugated linoleic acid (CLA) on serum lipid profile and markers of liver function in rats fed different dietary fat sourcesMagdalena Franczyk-Żarów1, Edyta Maślak2, Renata B. Kostogrys1

1Department of Human Nutrition, Faculty of Food Technology, Agricultural University of Crakow, Balicka 122, 30-149 Crakow, Poland; 2Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Crakow, Poland

Over the years, various components of dietary fat have been studied to examine their effect on serum lipids. Ac-cording to knowledge about oil, butter and margarine consumption on human health, the current study was de-signed to determine the effects of conjugated linoleic acid (CLA) on serum lipid profile and markers of liver func-tion as well as oxidative stress markers concentrations in rats fed different dietary fat sources. Thirty six male Wistar rats were divided into six groups (n = 6) and fed the following diets: control AIN-93G diet contained soybean oil (O) and diets with modification of fat source: butter (B) and margarine (M). The exper-imental diets were supplemented with 1% of conjugated linoleic acid (O+CLA, B+CLA, M+CLA). After 21 days the blood was collected and lipid profile, glucose, liver enzymes (AST and ALT), malonic dialdehyde (MDA) as well as lipid hydroperoxide (LPO) were analyzed.The dietary treatments had no effect on body weight of animals. Whereas, liver weight was significantly in-creased in B+CLA and M+CLA groups compared to control (O). Total cholesterol (TCh), LDL+VLDL choles-terol, MDA and AST concentrations did not differ sig-nificantly between experimental groups. Concentration of HDL cholesterol was significantly increased only in B and M groups compared to O. However, triacylglycerol (TAG) level significantly increased in animals fed M diet and decreased after CLA supplementation (M+CLA). Concentration of plasma glucose was slightly decreased in all CLA groups, but significant difference was observed in rats fed O+CLA diet compared to O group. Serum ALT levels were increased in all experimental groups, but sig-nificant differences were observed only in B+CLA and M+CLA groups compared to O group. Nethertheless, lip-id hydroperoxide (LPO) level was significantly increased in all CLA groups.In conclusion, CLA supplementation in margarine fed rats shows positive effect on TAG and glucose concentra-tion. Moreover, in rats fed M and B diets, HDL level was increased. AcknowledgementsThis work was supported by the subsidy grant for Young Scientists from the resources of the Agricultural University (grant No. BM-4738/KŻCz/2013).

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P5.22

Novel class of primosomal protein B from Clostridium thermocellumMarta Śpibida, Marta Marszałkowska, Marcin Olszewski

Gdansk University of Technology, Department of Microbiology, Narutowicza 11/12 St., 80-233 Gdansk, Poland

PriB is a primosomal protein that catalyzes DNA repli-cation in Procaryota. The replication pathway starts with PriA protein – the initiator protein that binds to a DNA replication fork, unwinds double-stranded DNA and role of PriB is to stabilize PriA on the DNA. However there are many biochemical differences in replication mechanism in bacteria and only some of them use PriB proteins. A few of PriB proteins were published and only three structures of them were resolved (Escherichia coli, Kleb-siella pneumoniae and Neisseria gonorrhoeae). All up-to-date known PriB proteins have one OB domain per monomer and they are homodimers in solution.Recently, we have published the crystal structure of PriB protein from Thermoanaerobacter tengcongensis that represents new class of PriB with two oligonucleotide/oligosaccharide-binding domain (OB) per monomer that means it exists as monomer in solution.The aim of this study is identification and characterization of the primosomal protein B (PriB) from an anaerobic, thermophilic bacterium Clostridium thermocellum (Cth-PriB). This PriB protein consisting 238 amino acid resi-dues and a calculated molecular mass is 27,5 kDa. What is more CthPriB protein contains two single-stranded DNA binding domain (OB-fold) and functions as monomer like PriB protein from bacterium Thermoanaerobacter tengcongensis.Therefore, our studies suggest that we dis-covered new classes of PriB which weren’t published re-cently.

P5.23

The antioxidant quercetin modifies etoposide acton in HL-60 cells Monika Papież

Department of Cytobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Crakow, Poland

The cells possessing high activity of myelopeoxidase (MPO) are particularly sensitive to the compounds which increase oxidative stress, such as etoposide. The aim of this study was to determine the effect of antioxidant quer-cetin, on the activity of etoposide in HL-60 cell line show-ing constitutively high MPO activity.HL-60 cells were incubated for 24 hours in the presence of etoposide and/or quercetin at concentrations range 1–100 uM. Apoptosis was dectected using annexin V (Becton Dickinson) and propidium iodide staining. The level of free radicals was measured using the CellRox kit (Life Technologies). The cells were analyzed by flow cy-tometry.Etoposide increased apoptosis of HL-60 cells in a dose-de-pendent manner. This anticancer drug at lower concen-trations of 5–20 uM increased the level of ROS, whereas higher concentrations of this drug showed antioxidant activity. Quercetin reduced ROS generated by low con-centrations of etoposide. The investigated polyphenol at concentrations of 1–10 uM protected HL-60 cells against apoptosis induced by etoposide. However, the amount of apoptotic cells was significantly higher after treatment with both quercetin and etoposide in comparison to the control. Higher concentrations of quercetin increased apoptosis induced by etoposide.The observed influence of quercetin and etoposide is dose-depend. Quercetin modifies the activity of etopo-side in myeloid leukemia cells with high MPO activity by antioxidant property.AcknowledgementsThis study was supported by the Ministry of Science and Higher Education (K/ZDS/003318).

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P5.24

Salvia lavandulifolia from spain: aromatic profile by enantioselective gas chromatography-mass spectrometry Ana Belen Cutillas5, Alejandro Carrasco1, Vanessa Ortiz1, Ramiro Martinez-Gutierrez3, Francisco Javier Martinez4, Mariano Sanchez5, Virginia Tomas2, Jose Tudela1

1GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz), Departamento de Bioquimica y Biologia Molecular-A, Campus de Excelencia Internacional Regional Campus Mare Nostrum, Universidad de Murcia, Murcia, Spain; 2Departamento de Quimica Analitica, Universidad de Murcia; 3NOVOZYMES SPAIN S.A. (www.novozymes.com); 4Esencias Martinez-Lozano S.A. (www.esenciaslozano.com); 5Europermanent S.L.

Objectives. The identification and determination of bio-chemicals of the essential oil of Salvia lavandulifolia, grown from organic farming in Murcia (Spain). The oil has been eco-extracted, by steam distillation with a por-table still, next to cultivation, and with a boiler feed with biomass, from plants previously distilled.Methodology (www.um.es/genz/e00605/serv03.htm). Fast Gas Chromatography on a non-polar fast column (SLB-5ms) 15 m × 0.1 mm × 0.1 µm, and Enantioselective Gas Chromatography on a chiral column Chiraldex B-DM 30m x 0.25 mm × 0.12 µm, were carried out on a GC-chromatograph (Agilent 7890), with hydrogen as gas carrier (PDH). The Mass Spectrometry Detector used an electronic impact ionizer (70 eV), and a single quad-rupole analyzer (Agilent 5975 MSD). Sandwich split/splitless injections, with hexadecane as inner standard, and calibration straigths with standards (Gerstel MPS-2XT autosampler).Results. The chromatographic results showed that the S. lavandulifolia oil from Murcia is especially rich in some biomolecules like (mM concentration, % enan-tiomers): Camphor (4068, +76, –24), Eucalyptol (1299), Camphene (811, +26, –74), α-Pinene (483, +52, –48), β-Pinene (365, +42, –58), Limonene (333, +81, –19), Borneol (319, +34, –66), Myrcene (233), Linalool (142, +4, –96), Linalyl acetate (138), α-Terpineol (96, +8, –92), α-Terpinyl acetate (66, +6, –94), Bornyl ac-etate (61, +4, –96), and other minor components.Conclusions. Salvia lavandulifolia oil has a very high concentration of Camphor, Eucalyptol and Camphene, about 10–20 times higher than that of many other biomolecules. Near pure (–)-enantiomers are pres-ent for Linalool, α-Terpineol, α-Terpinyl acetate and Bornyl acetate. There are mainly (+)-Camphor and (+)-Limonene, whereas there are mainly (–)-Camphene and (–)-Borneol. Furthermore, α-Pinene and β-Pinene have similar proportions of both enantiomers. This bio-chemotype is markedly different than that of Salvia la-vandulifolia oil, with similar cultivation and extraction procedures in Castilla-La Mancha (Spain). The essential oil from Murcia has higher concentrations than that stated in the corresponding ISO standard for Camphor, Eucalyptol and Limonene. This oil is a good source of

biomolecules for pharmaceuticals, cosmetics, fragrances and food industries.AcknowledgementsThis work has been partially supported by grants from several Spanish organizations. Projects BIO2009-12956 (MINECO, Madrid) and 08856/PI/08 (Fundación Seneca, CARM, Murcia). AC has a fellowship from Esencias Martinez Lozano S.A. (Murcia). VO has a FPU fellowship (AP2010-4300).

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P5.25

Permeation of iodine from iodine-enriched yeast through porcine intestineFlorian Ryszka1, Barbara Dolińska1, 2, Michał Zieliński1, 2, Dagmara Chyra1, Zbigniew Dobrzański3

1Pharmaceutical Research and Production Plant “Biochefa”, Sosnowiec, Poland; 2Department of Applied Pharmacy and Drug Technology, Medical University of Silesia, Sosnowiec, Poland; 3Department of Animal Hygiene and Animal Welfare, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland

Iodine deficiency is a common phenomenon, threatening the whole global human population. Recommended daily intake of iodine is 150 μg for adults and 250 μg for preg-nant and breastfeeding women. About 50% of human population can be at risk of moderate iodine deficiency. Due to this fact increased iodine supplementation is rec-ommended, through intake of iodized mineral water and salt iodization. The aim of this study was to investigate permeation and absorption of iodine from iodine bioplex (experimental group) in comparison with potassium iodide (controls). Permeation and absorption processes were investigated in vitro using a porcine intestine. The experimental mod-el was based on a standard Franz Diffusion Cell(FD-Cell).The iodine bioplex was produced using Saccharomy-ces cerevisiae yeast and whey powder: iodine content – 388 μg/g, total protein – 28,5%, total fat – 0,9%., glutamic acid – 41,2%, asparaginic acid – 29,4%, lysine – 24,8%; purchased from: F.Z.N.P. Biochefa, Sosnowiec, Poland. Potassium iodide was used as controls, at 388 μg iodine concentration, which was the same as in iodine-enriched yeast bioplex.A statistically significant increase in iodine permeation was observed for iodine-enriched yeast bioplex in com-parison with controls – potassium iodide.After 5h the total amount of permeated iodine from io-dine-enriched yeast bioplex was 85%, which is ~ 2-fold higher than controls – 37%. Iodine absorption was by contrast statistically significant-ly higher in controls – 7,3%, in comparison with 4,5% in experimental group with iodine-enriched yeast bioplex.Presented results show that iodine permeation process dominates over absorption in case of iodine-enriched yeast bioplex.AcknowledgementsOriginal article with the same title will be submitted to Acta Biochimica Polonica. Submitted abstract and article are a part of Prof. Florian Ryszka’s contribution.

P5.26

Novel class of primosomal protein B from Clostridium thermocellumMarta Śpibida, Marta Marszałkowska, Marcin Olszewski

Gdansk University of Technology, Department of Microbiology, Narutowicza 11/12 St., 80-233 Gdansk, Poland

PriB is a primosomal protein that catalyzes DNA repli-cation in Procaryota. The replication pathway starts with PriA protein – the initiator protein that binds to a DNA replication fork, unwinds double-stranded DNA and role of PriB is to stabilize PriA on the DNA. However there are many biochemical differences in replication mechanism in bacteria and only some of them use PriB proteins. A few of PriB proteins were published and only three structures of them were resolved (Escherichia coli, Kleb-siella pneumoniae and Neisseria gonorrhoeae). All up-to-date known PriB proteins have one OB domain per monomer and they are homodimers in solution.Recently, we have published the crystal structure of PriB protein from Thermoanaerobacter tengcongensis that represents new class of PriB with two oligonucleotide/oligosaccharide-binding domain (OB) per monomer that means it exists as monomer in solution.The aim of this study is identification and characteriza-tion of the primosomal protein B (PriB) from an anaer-obic, thermophilic bacterium Clostridium thermocellum (CthPriB). This PriB protein consisting 238 amino acid residues and a calculated molecular mass is 27,5  kDa. What is more CthPriB protein contains two single-strand-ed DNA binding domain (OB-fold) and functions as monomer like PriB protein from bacterium Thermoan-aerobacter tengcongensis.Therefore, our studies suggest that we discovered new classes of PriB which weren’t published recently.

55Eurobiotech 2013

P5.27

A novel DNA polymerase from hyperthermophilic bacterium Thermovibrio ammonificans: gene cloning, expression, and characterizationMarta Marszałkowska, Sandra Zakrzewska, Marta Spibida, Marcin Olszewski

Department of Microbiology, Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12 St., 80-233 Gdansk, Poland

Thermostable DNA polymerases are one of the most important bio-tools widely used in molecular biology. They are able to catalyze synthesis of DNA by addition of mononucleotide units derived from deoxynucleoside 5’ triphosphates (dNTP) to the 3’ hydroxyl terminus of the template DNA under extreme conditions [1]. Howev-er, in molecular diagnostics, which is fundamental field of modern medicine, conventional thermostable DNA polymerases are often inhibited by chemical compounds present in biological samples from blood or soil.In order to find thermostable DNA polymerase that shows novel properties we decided to conduct expression of type A polymerase from hyperthermophilic bacterium Thermovibrio ammonificans. This bacterium is one of three discovered to date representants of newly described genus Thermovibrio [2]. Gene coding Thermovibrio DNA polymerase shows significant sequence homology to commonly used polymerase from Thermus aquaticus (TaqPol) [3], however it lacks 3’ to 5’ exonuclease do-main. As a result of cloning and expression we obtained protein with molecular weight of 99,5 kDa, which was purified from Escherichia coli TOP10 strains expressing the cloned genes. Now we are trying to characterize ac-tivity of Thermovibrio DNA polymerase under different conditions (pH, temperature, inhibitors from blood and soil). References[1] Lehman I.R., Discovery of DNA Polymerase, J. Biol. Chem. (2003) 278: 34733–34738.[2] Vetriani C., Speck M.D., Ellor S.V., Lutz R.A., Starovoytov V., Thermovibrio ammonificans sp. nov., a thermophilic, chemolitotrophic, nitrate-ammonifying bacterium from deep-sea hydrothermal vents, Int. J. Syst. Evol. Microbiol. (2004) 54: 175–181.[3] Huang J.-P., Ito J., DNA Polymerase C of the Thermophilic Bacterium Thermus aquaticus: Classification and Phylogenetic Analysis of the Family C DNA Polymerases (1999) J. Mol. Evol. 48: 756–769.

P5.28

Screening of carriers for lipase immobilization suitable for reactions in water, biphasic and pure organic solvent systemsZofia Hrydziuszko1, Agnieszka Dmytryk1, Paulina Majewska1, Katarzyna Szymańska2, Jolanta Liesiene3, Jolanta Bryjak1

1Wroclaw University of Technology, Faculty of Chemistry, Department of Bioorganic Chemistry, Poland; 2Silesian University of Technology, Department of Chemical Engineering, Poland; 3Kaunas University of Technology, Faculty of Chemical Technology, Lithuania

In biotransformations, lipases encompassed a wide range of reactions, such as esterification, transeserification (al-coholysis) and aminolysis (amide synthesis). Trans-ester-ification and amide synthesis are preferably performed in anhydrous organic solvents whereas glycerolysis of com-mercial oils is practiced at a large scale in a solventless system. Third system is composed of water immiscible organic solvent and water. The choice of the system is dic-tated by substrate/product solubility and/or by preferred reaction direction (synthesis over hydrolysis). Regardless reaction system, lipases are used in immobilized form rather to ensure biocatalyst evenly distributed suspension in water restricted media and/or to ensure easy separa-tion after being reused for several times in all reaction systems.The aim of this study was to select an appropriate en-zyme-carrier preparation for carrying out hydrolysis re-action in aqueous medium or biphasic systems and trans-esterfication in organic solvent. For this purpose Candida rugosa lipase (CRL) was bound by adsorption or covalent attachment into array of carriers having different struc-ture, porosity and kind of functional groups. Selection of proper carrier and method of immobilization was based on measured activity, thermal stability and operational stability in successive batch processes.For immobilization studies 15 carriers (silica gel (Z), acrylic copolymer (A), cellulose beads (G), mesoporous cellular foams (MCF)) were used. Obtained 25 en-zyme-carrier preparations were tested for activity in re-action of 4-nitrophenyl palmitate hydrolysis in water. To test thermal stability, 4 h incubation at 60oC was applied and then the best preparations were subjected to oper-ational stability in 20 batch processes. Selected prepara-tions of each carrier group were used for hydrolysis of ethyl (1-butyryloxyethyl)-phenylphosphinate in biphasic system, and after drying (5 days, 30oC) in ethyl (1-hy-droxyethyl)-phenyl-phosphinate transesterification in organic solvent.Activity of obtained enzyme-carrier preparations var-ied from 20 (G) to 5100 U/mL (MCF). The most stable preparations were those bound by adsorption or cova-lent attachment to NH2-Z and COOH-A carriers (activ-ity over 90%). The highest conversion in reaction of hy-drolysis in biphasic system was obtained for NH2-MCF,

56 Eurobiotech 2013

NH2-Z and OH-A carriers. Moreover, lipase immobilized by adsorption and covalent attachment onto NH2-Z and OH-A carriers maintained almost 100% of initial activity after drying and these preparations were used in trans-esterfication reaction. It was found that in biphasic and non-aqueous systems hyperactivation took place. Taking into account hydrolysis, similar substrate conversion at the same reaction time was achieved when several times higher concentration of native CRL was used than im-mobilized preparations. What was more, no progress in transesterfication was observed when native CRL was applied.For industrial applications operational stability is more important than enzyme activity. Taking into account re-action of hydrolysis in water, CRL covalently bound to NH2-Z preserved 50% of initial reaction rate in 23 batch processes. Satisfactory hydrolysis in biphasic system was obtained for preparations with CRL bound to OH-A (90%) or to NH2-Z (76%). In the case of transesterifica-tion, the best results were obtained when CRL was ad-sorbed on NH2-Z carrier. Thus, NH2-Z carrier can be considered as an universal matrix for CRL immobiliza-tion in all reaction systems tested.AcknowledgementsThis study was supported by the project “Biotransformations for pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-158/09-03 partly financed by the European Union within the European Regional Development Fund.

P5.29

The effect of new hydantoin derivatives on the increase of ciprofloxacin efficacy in drug-resistant E. coliAnna Matys, Ewa Otrębska, Jakub Mazurkiewicz, Daria Studnicka, Beata Mastek, Jadwiga Handzlik, Katarzyna Kieć-Kononowicz

Department of Technology and Biotechnology of Drugs, Jagiellonian University, Collegium Medicum, Faculty of Pharmacy, Crakow, Poland

The aim of the study was to assess a series of hydantoin derivatives in terms of their activity against two E.  coli strains: HEMEC 10 (a clinical drug-resistant strain over-expressing efflux pumps) and E. coli ATCC 25922 (a refer-ence strain). The compounds were tested in combination with ciprofloxacin to see whether they enhance its action by exerting an inhibitory effect on efflux pumps for ex-ample. The chemical families which exhibited an interest-ing chemosensitizing effect in previous microbiological assays conducted on drug-resistant E. aerogenes were se-lected for the study [1]. Three groups of compounds were tested: piperazine derivatives of 5-arylidenehydantoin, piperazine derivatives of 5-arylideneimidazolone and de-rivatives of 5-α-naphtylmethylhydantoin. The activity of the compounds in concentrations no higher than 1/2 of their minimum inhibitory concentrations was examined by assessing the effect of the compounds on reduction of minimum inhibitory concentration of ciprofloxacin in the E. coli strains. The compounds did not show high activity – most of them reduced the MIC of ciprofloxa-cin only 2-4-fold. The most active compound was A10 (p-chlorobenzylidene derivative of hydantoin), which re-duced the MIC of ciprofloxacin in the E. coli ATCC 25922 reference strain 32-fold. However, its effect in the E. coli HEMEC10 strain was much lower (2-fold).AcknowledgementsThe authors wish to thank Professor Leonard Amaral and Professor Isabel Couto (Grupo de Micobacterias, Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal) and dr hab. Anna Białecka (Centrum Badań Mikrobiologicznych i Autoszczepionek, Cracow, Poland) for providing the strains used in this study. The study was financed from the programme K/ZDS/001915.References[1] Handzlik J., Szymańska E., Alibert S., Chevalier J., Otrębska E., Pękala E., Pagès J.-M., Kieć-Kononowicz K., Search for new tools to combat Gram-negative resistant bacteria among amine derivatives of 5-arylidenehydantoin. Bioorg. Med. Chem. 21 (2013) 135–145.

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P5.30

Laccase activity and stability in the presence of menthol-based ionic liquidsJoanna Feder-Kubis1, Jolanta Bryjak2

1Wroclaw University of Technology, Faculty of Chemistry, Department of Chemical Engineering; 2Wroclaw University of Technology, Faculty of Chemistry, Department of Bioorganic Chemistry

In the last years, room temperature ionic liquids (ILs) have gained increasing importance in enzymatic cataly-sis and they are claimed as „green” alternative for volatile organic solvents. ILs can be used in monophasic (pure ILs or water-miscible ILs as co-solvent) or biphasic sys-tems. ILs are viscous salts that causes strong reaction rate reduction, thus interest is directed towards biphasic sys-tems. Apart from better preservation of reactivity, these systems provided better conformational stability. In the case of reactions with oxidases, additional advantages are easiness of oxygen dissolution and/or unchanged diffu-sion rates of redox species in water phase. Oxidases are a very important group of enzymes because they use ox-ygen as the only oxidant without need of cofactor (most of oxidoreductases) or hydrogen peroxide (peroxidases). Laccases (EC 1.10.3.2) attract considerable attention due to their potential for manufacturing pharmaceutical in-termediates and specialty chemicals from a wide array of phenolic and non-phenolic substrates that are sparingly soluble in water.The main aim of this work was to find ILs suitable for Cerrena unicolor laccase. Rehmann et al. (Green Chem. 2012, 14, 725) reported that performances of Trametes versicolor laccase depended strongly on the anion na-ture and showed that bis(trifluoromethanesulfonyl)im-ide supported the highest enzyme activity. For that five water-immiscible ILs with this anion were synthesized. The cations described here contained natural alcohol (1R,2S,5R)-(–)-menthol and varied in the structure: (I) 3-butyl-1-[(1R,2S,5R)-(–)-menthoxymethyl] imidazoli-um, (II) 1-[(1R,2S,5R)-(–)-menthoxymethyl]-3-hepty-limidazolium, (III) 1-[(1R,2S,5R)-(–)-menthoxy-meth-yl]-3-methylpyridinium, (IV) heptyl[(1R,2S,5R)-(–)-menth-oxymethyl]dimethylammonium, and (V) decyl [(1R,2S,5R)-(–)-menthoxymethyl]dimethyl-ammonium. Laccase activity was tested in buffer saturated with ILs. Special attention was paid towards stability tests in bipha-sic systems, lasting 5 days. Kinetic parameters of inacti-vation models were estimated using nonlinear regression (OriginPro 8.0).Correctly chosen ILs can provide at least good conditions for an enzyme activity expression and to be concomitant with its stability not lower than in the buffer solution. It was shown that all but one ionic liquids tested did not significantly alter laccase activity (90–123% respective to the buffer. The best laccase-friendly ILs were no. I and IV whereas IL II decreased reactivity in 20%. This, perhaps, depends on weak influence of ions activity on

proteins structure even at their very low concentrations in the water phase. Stability tests showed that no laccase activity was observed in all ILs phases and that ILs I, IV and V, with the highest reactivity, allowed to maintain the enzyme stability even more than in the buffer alone. Exceptional stabilization of laccase was observed in presence of ILs I and IV. In the contrary, ILs II and III caused destabilization of protein structure, probably via defolding and/or changing water molecules organization in the protein micro-environment. Another factor, that has been discussed was the possibility of laccase stabilization by preventing of microorganisms growth by ILs molecules during 5-days incubation. Destabilization caused by enzyme contact with the interface between the aqueous and ILs phases and air – water phases was discussed as well. For mathematical formalization of inactivation courses, isoenzyme model was applied and kinetic parameters were evaluated. It was stated that this model allowed to fit experimental data accurately only for sets obtained in the buffer (control) and in the presence of ILs II. In the other cases, first-order reaction model reflected experiments very well with concomitant good statistical validation. This shows that ILs, even at very low concentrations, influence conformational stability of proteins and that it depends on the cation structure. In general, the imidazolium and ammonium salts with shorter alkyl chain, tested in this study, supported laccase activity and stability.AcknowledgementsThis work was supported by the Polish State Committee for Science and Research under grant N N209 119337, 2009–2012.

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P5.31

Antiradical capacity of fruits of Polish raspberry and blackberry cultivars – an electron paramagnetic resonance spectrometry study Anna Kostecka-Gugała, Aleksandra Mech-Nowak, Paweł Kaszycki

Departament of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

The world production of raspberry fruits increases over the recent years and in 2011 Poland was the second raspberry producer. It is known that Rubus (raspberry and blackberry) fruit antioxidant activity is highly correlated with the presence of phenolic compounds. We examined differences in antiradical capacity of fruits of several Polish raspberry cultivars in relation to the content and composition of main polyphenols in 80%-methanol extracts.Among the tested cultivars were: summer red-fruited raspberry ‘Benefis’, black-fruited ‘Litacz’ and autumn (primocane-fruiting) red-fruited ‘Polka’, yellow-fruited ‘Poranna Rosa’, and a blackberry cultivar ‘Ruczaj’. All the specimens were collected in 2011 and 2012 at The Friut Experiment Station in Brzezna, Southern Poland. Anti-radical activity was measured with the L-band (1.2 GHz) electron paramagnetic resonance spectrometry which en-abled to directly detect the DPPH free-radical quenching reaction by fruit antioxidants. The analyses of phenolic compounds were performed using high performance liq-uid chromatography and supplemented with a standard Folin-Ciocalteau spectrophotometric assay.The results of the study show that black fruits of the rasp-berry ‘Litacz’ were distinguished by the largest antiradical capacity, higher than that observed for blackberry fruits. The typically coloured, red raspberry fruits revealed mod-erate antiradical capacities and the differences in the ob-tained activities were found insignificant for fruits of the summer and autumn (primocane-fruiting) cultivars. Rel-atively smaller antiradical potential was observed for the yellow fruits of raspberry. The great antiradical properties of the black-fruited raspberry ‘Litacz’ were determined by a high amount of polyphenols, mainly flavonoids. ‘Litacz’ was found to be rich in rutin, catechins, anthocyanins (especially cyanidin derivatives) as well as chlorogenic and p-hydroxybenzoic acids. The above characteristics make this Polish black-fruited cultivar a  valuable and recommendable source of active antiradical compounds.AcknowledgementsThe work was supported by a research grant of Polish National Science Centre No. N N310 306139.

P5.32

Development of novel cell line, expressing human histamine H4 receptor and its use in affinity testing of potent receptor ligandsTadeusz Karcz, Katarzyna Kieć-Kononowicz

Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Crakow, Poland

Recombinant receptors systems are pharmacological tools widely utilized in search for novel G-protein cou-pled receptors (GPCRs) ligands. Contrary to historically used native receptors systems, they can be involved in studies on poorly characterized receptors, for which se-lective ligands are not known and low expression levels are observed in native cells.Recombinant proteins can be obtained in various ex-pression systems. In studies on GPCRs, mammalian cell lines are predominantly used. These models guarantee satisfactory grade of posttranslational protein modifica-tions and enable cellular membrane localization of re-combinant proteins. Moreover mammalian cells contain whole range of necessary intracellular signaling pathway elements [1, 2].Human histamine H4 receptor, being a biological target of presented studies is, by far, the last histamine recep-tor discovered. Its gene has been cloned in 2000, inde-pendently by seven groups [3]. Histamine H4 receptor expression on basophils, eosinophils, mast cells and den-dritic T cells suggests its role in immunological answer [4]. Therefore its ligands could provide promising drugs for immuno-based diseases [5], however none of the po-tent ligands was brought to the market so far. Thus, hista-mine H4 receptor remains an important pharmacological target in drug development.The aim of current work was to develop a novel cell line, expressing human histamine H4 receptor, with use of retroviral expression system. Following the molecular biology experiments, which led to obtaining of pQCX-IN-hH4R plasmid, containing human histamine H4 receptor sequence, transduction of CHO cells was per-formed with use of retroviral particles produced in GPen-v+AM12 packaging cells. Resultant cell line was tested for its utility in radioligand binding assays.First, recombinant receptor was characterized in satu-ration binding assay with use of [3H]histamine as a ra-dioligand. Homological histamine competition binding was employed for comparison of obtained cell line’s prop-erties with membranes preparation from commercial source. Competition binding experiment for estimation of unknown Ki values of tested compounds was validated against the insect cells system used for the same purpose by our scientific collaborators, proving the usefulness of developed cell line in testing of novel compounds affini-ties towards histamine H4 receptor.AcknowledgementsWork supported by Polish National Science Center, project Preludium I, No. UMO-2011/01/N/NZ4/01126.

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References[1] Siehler S., Cell-based assays in GPCR drug discovery. J Biotech, 2008, 3: 471–483.[2] Hermans E., Generation of model cell lines expressing recombinant G-protein-coupled receptors. Methods Mol Biol, 2004, 259: 137–153.[3] Oda T. et al., Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes. J Biol Chem, 2000, 275: 36781–36786.[4] Nguyen T. et al., Discovery of a novel member of the histamine receptor family. Mol Pharmacol, 2001, 59(3): 427–33.[5] Jablonowski J., Carruthers N., Thurmond R., The histamine H4 receptor and potential therapeutic uses for H4 ligands. Mini Rev Med Chem, 2004, 4: 993–1000.

P5.33

The effect of egg yolk lipids enriched with CLA on cytotoxicity of human melanoma cell line WM793Dominik Domagała1, Aneta Koronowicz1, Jarosław Oczkowicz1, Elżbieta Sikora1, Piotr Laidler2, Teresa Leszczyńska1

1University of Agriculture, Department of Human Nutrition, Crakow, Poland; 2Jagiellonian University Medical College, Crakow, Poland

Conjugated linoleic acids (CLAs) are a family of at least 28 isomers of linoleic acid found mainly in the meat and dairy products derived from mammals called ruminants. Among these compounds two isomers cis9,trans11 and trans10,cis12 have received considerable attention. In re-cent study we have demonstrated that fatty acids extract obtained from egg yolks enriched naturally with the CLA isomers may exhibit a strong tendency to inhibit prolifer-ation and induce cytotoxicity in melanoma WM 793 cell lines.Cytotoxicity in cells increases with higher concentrations of CLA, however it was still lower than one caused by fat-ty acids without addition of CLA, which correlated with decreased level of cell proliferation, which most proba-bly is caused by cell apoptosis. Apoptotic mechanism was proved by staining cells with annexin and propidium io-dide.These results demonstrated that fatty acids of CLA-enriched egg yolks may induce apoptosis in carcinoma cells. The received data suggests that CLA may be used in future to prevent cancerogenesis, however it still require deeper insight into molecular mechanisms which are in-volved into that process.Material and Methods: Experiments were performed using melanoma cancer cell line WM 793 treated with fatty acids of CLA-enriched egg yolks. Proliferation tests were performed with use of BrdU proliferation kit (Roche). Cytotoxicity was measured by LDH Detection kit (Roche). Apoptosis assay was conducted with use of Annexin-V-FLUOS Staining Kit (Roche).

Plant molecular breeding

Lectures

L6.1

Engineering of a retrotransposon for insertion mutagenesis in plantsNikola Winter, Eneda Xhikaj, Lilian Nehlin, Andrea Tramontano, Andreas Bachmair

Dept. of Biochemistry and Cell Biology, Center for Molecular Biology, Max F. Perutz Laboratories, Univ. of Vienna, Dr. Bohr Gasse 9, A-1030 Vienna, Austria

New methods allow acquisition of sequence data for many plants of agricultural interest. However, insight into gene function by mutant analysis in crop plants has not kept pace with sequence data accumulation, due to the lack of efficient mutagenesis tools. We are studying the plant retrotransposon Tto1 with the goal to generate a “next generation mutagenesis tool“ for plants. Retro-transposons have been used for insertion mutagenesis in plants, but optimization of this process is difficult due to the limited understanding of the retroelement life cycle. In order to facilitate experimental analysis of plant ret-rotransposon Tto1, we replaced the natural promoter of Tto1 by an artificial, inducible one. The engineered ele-ment can transpose in the model plant Arabidopsis thali-ana after induction [1]. Sequences of the long terminal repeat region were investigated for their role in transpo-sition [2], with the goal to reduce the length of sequence repeats as potential inducers of silencing. In order to op-timize insertion frequency, we are currently testing differ-ent inducible promoters and integrase versions for their impact on transgenerational transposition events. Based on insights obtained in the model plant, Tto1 constructs are generated for transposon tagging in the agricultural-ly important plants bean (Phaseolus vulgaris) and barley (Hordeum vulgare). Mutant plants can eventually serve for analysis of gene function, and in breeding programs for crop improvement.AcknowledgementsThe authors’ work is supported by the Austrian Science Foundation FWF (grant TRP14-B12).References[1] Böhmdorfer et al. (2010) Syst Synth Biol 4, 133–138.[2] Tramontano et al. (2011) Virology 412, 75–82.

L6.2

Pea resistance genes to Pea Seed-borne Mosaic Virus (PSbMV), from basic science to applications in breedingPetr Smýkal

Department of Botany, Slechtitelu 11, Palacky University in Olomouc, 783 71 Oolomouc, Czech Republic

Pea seed-borne mosaic virus (PSbMV) belongs among the most frequent viral pathogens causing severe losses in field pea and other legumes. These losses might be prevented by growth of resistant varieties. Resistance to the common P1 strain of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus), while to lentil L1 strain is localized on LGII at sbm-2 locus. We have analyzed variation in the eIF4E genomic sequences from pea lines, reported as donors of resistance. Subsequently, gene-spe-cific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed (Smýkal et al. 2010). All markers were reproducibly am-plified across a broad spectra of pea varieties and breed-ing lines and found to be 100% accurate when compared to symptomology and ELISA testing. Hence, application of these DNA markers substantially speeds-up resistance breeding processes. From the comparison of genotype verses phenotype for selection of resistance/susceptibili-ty in F2 plants, there was a 26% discrepancy between the PCR and ELISA-based assays with potentially susceptible heterozygote plants missed (Smýkal et al. 2010). The nov-el eIF4E alleles were detected in wider pea germplasm and TILLING mutants (Dalmais et al. 2008) are tested for var-ious potyviruses. The series of identified allelesprovide the basis for the testing of various potyviruses and pathotypes to reveal possible co-evolution of potyviruses and its pea host. Durability of host based resistances in relation to ge-netic plasticity of viral pathogens is an issue for practical breeding, however as seen in the case of PSbMV, ancient alleles are still effective in pea resistance. It can be expected that information gain in this study might be extended to other legume species, particularly to economically import-ant species of tribus Vicieae or even to subfamily Papilion-oideae such as chickpea, lentil, faba bean or soybean where Potyviruses cause problems. The implications for marker assistend breeding in legumes will be presented.AcknowledgementsThe work was supported by NAZV QI91A229 project.Reference[1] Smýkal P., Šafářová D., Navrátil M., Dostalová R. (2010) Mol Breeding 26: 425–438.[2] Dalmais M., Schmidt J., Le Signor C. et al. (2008) Genome Biol 9: R43.

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L6.3

High through output phenotyping and genotyping – stepsfor drought tolerance improvement in barleyMarcin Rapacz1, Magdalena Wójcik-Jagła1, Anna Fiust1, Bartłomiej Kozera1, Mirosław Tyrka2

1University of Agriculture in Crakow; 2Rzeszow University of Technology

Water deficit limits crop production all over the world. Also in Poland periodic droughts cause serious decrease in agricultural production. Spring barley production is especially affected, mainly due to short vegetation peri-od and development schedule, in which critical periods of water demand occurs often at the time of periodic droughts. Because of the limitations of classic breeding methods in drought tolerance improvement biotechnolo-gy seems to be the only solution. Either the development of marker systems or genetic modifications are taken into consideration. Despite many attempts made all over the world the progress in this field is, however, limited. The main problems seems to be 1) a very complex drought re-sponse of plants with hundreds of genes involved in, and 2) different drought response mechanisms desired at dif-ferent environments, eg. in Middle East, Mediterranean area or in Poland. We hope that the solution is possible and very simple – we must take the advantage from the above mentioned disadvantages.Two mapping populations based on Polish breeding ma-terials of barley well adapted to local conditions and var-ied in their drought tolerance were used for QTL analysis of the traits describing their short-time drought response at the seedlings stage. High throughput genotyping (DArT markers) and phenotyping techniques, including many physiological characteristics important for high yielding potential of plants affected by periodic droughts were performed. The results showed either a high genetic diversity of studied populations, enabling the creation of high density linkage maps, as well as a high diversity in the physiological response of studied breeding materials. The analysis revealed in total 23 QTLs for 9 physiologi-cal characteristics. Three QTL regions were common for both mapping populations.This analysis was a start point for further work of either applied and basic research character.68 DArT markers from QTLs regions were successful-ly converted into SSR and STS polymorphic markers. During the further analysis of breeding strains differ-ent in their drought tolerance we confirmed that 20 markers were effective in selection for drought toler-ance and these markers are used now for a pilot large scale selection in plant breeding. The same sequences were used for sequence analysis which reveals con-nections between fenotype and certain genes, protein activity or signaling pathways. Results give significant sight into candidate genes which may be involved in different drought responses observed among Polish

breeding materials of barley and may be useful for ge-netic modifications.It seems that the creating of mapping populations from current breeding materials, the use of high throughout-put genotyping and phenotyping methods based on deep physiological knowledge may be a key strategy for im-proving not only drought tolerance but also other com-plex agronomic traits.AcknowledgementsThe research were supported by Polish National Research and Development Center (PBZ-2/3/2006/17 and GENMARK – PBS1/A8/1/2012).

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L6.4

In vitro induced variation in Triticale cv. BogoJoanna Machczyńska1, Piotr Bednarek1, Janusz Zimny2

1Department of Plant Physiology and Biochemistry, Plant Breeding and Acclimatization Institute-National Research Institute, Radzikow, 05-870 Blonie; 2Department of Plant Biotechnology and Cytogenetics, Plant Breeding and Acclimatization Institute-National Research Institute, Radzikow, 05-870 Blonie

In vitro culture plant regeneration is a widespread method used to obtain genetically uniform plants – double hap-loids (DH) that should be identical to their donor plants. However, in vitro culture induces genetic and epigenetic variation that may be observed in regenerants. Genetic changes include translocation and transposition as well as sequence mutations, while epigenetic alterations concern gene silencing, gene regulation and chromatin conden-sation.The metAFLP approach is a valuable tool for the analysis of the quantitative characteristics of (epi)genetic changes affecting restriction sites (Acc65I & KpnI) and their vicin-ity within the whole genome. Moreover, it enables quanti-fication of unaffected sites. MetAFLP assumes utilization of two isoschizomes Acc65I and KpnI. Acc65I and KpnI recognize the same restriction sequence but differ in their sensitivity towards DNA methylation of their recognition sequences; the former is sensitive to methylation of the restriction site and adjacent sequences whereas the latter is not. Comparison of the molecular data obtained with the Acc65I/MseI and KpnI/MseI metAFLP platforms for donor plants and their regenerants allows qualification and quantification to distinct variation types (sequence changes, de novo methylation and demethylation).Plant DNA methylation occurs on CpG sites (symmetric methylation) as well as on CpXpG (X denoting A, C or T) and CpXpX (asymmetric) sites. In our study specially designed primers were used to distinguish between sym-metric and asymmetric methylation types present within restriction sites.Triticale is important for Europe as it uses less good soil, less fertilizer, exhibits better disease tolerance, high yield potential, grain quality, resistance to pathogens, favour-able amino acid composition. Hexaploids gained prom-inence in Poland as feed grains. However triticale is a polyploid species, which demonstrates high genetic and epigenetic instability.In our research 4 DH lines of triticale cv. Bogo were used to evaluate the level of in vitro induced variation. Regen-erants obtained via androgenesis in anther culture and shed-microspore culture and via somatic embryogenesis in immature embryo culture. Our results demonstrate that triticale is not genetically stable during tissue culture manipulations. Average total tissue culture induced vari-ation amounted to 28%, where sequence variation con-stituted of 18%, de novo methylation reached up to 4,5% and demethylation equaled to 5,3%.

L6.5

Conversion of a DArT marker differentiating wild and cultivated carrots to a codominant CAPS markerAlicja Macko-Podgórni, Krzysztof Smółka, Dariusz Grzebelus1Department of Genetics, Plant Breeding and Seed Science, University of Agriculture Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

Carrot is one of the most important vegetables grown in the world, mostly due to high content of β-carotene – vitamin A precursor. Wild carrot (Daucus carota sub-sp. carota) is an ancestor of cultivated carrot (D. carota subsp. sativus), and Central Asia is a likely region where the biennial cultivated carrot was domesticated. Culti-vated and wild carrots spread from their centre of origin to distant geographical regions. Both forms can easily hybridize, thus a considerable increase of genetic varia-tion is exhibited both among and within each subspecies. Even though carrot diversity was extensively studied us-ing a variety of molecular marker techniques, until now no handy markers allowing determination between culti-vated carrots and wild populations was available.Recent results of investigation on genetic diversity of cul-tivated and wild carrots using Diversity Arrays Technol-ogy (DArT) allowed identification of a DArT marker dif-ferentiating wild and cultivated accessions. We sequenced the polymorphic region and identified a 16 bp-long indel containing a PstI restriction site and causing the DArT polymorphism. We designed site-specific primers an-chored in regions flanking the indel and converted the DArT marker into an inexpensive, efficient, codominant cleaved amplified polymorphic site (CAPS) marker for routine detection of wild/cultivated variants present in that locus.The CAPS-based assay was used to screen 72 cultivated and 16 wild accessions of different origin, each accession being represented by three individuals. All wild acces-sions were characterized by presence of the allelic variant associated with the wild phenotype. In all plants from 60 accessions of cultivated carrot the expected variant typi-cal for cultivated carrot was observed. In the remaining 12 cultivated accessions the ‘wild’ variant was present in at least one plant. Most of them were old primitive land-races from Turkey and Iran, likely poorly monitored for cross-pollination with wild carrots. The result suggests that the identified ‘cultivated’ variant, rare in wild germ-plasm, was enriched in the cultivated germplasm in the course of domestication.

63Eurobiotech 2013

Posters

P6.1

New sources of phenolic compounds and anthocyanins for biotechnolgyNijolė Anisimovienė1, Jurga Jankauskienė1, Milda Jodinskienė1, Vidmantas Bendokas2, Vidmantas Stanys2, Tadeušas Šikšnianas2

1Institute of Botany of Nature Research Centre; 2Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry

Introduction. One of the current problems in biotech-nology is a search for plant original bioactive compounds important due to their benefits to human health (He, Guisti, 2010; Oancea, Oprean, 2011). Anthocyanins from berries have received considerable interest as dietary antioxidants among the different bioactive substances in human health (Tsuda, 2012). These compounds are being able to capture reactive oxygen species, delay the initiation or propagation of oxidative chain reactions. The prevention role of anthocyanins in cardiovascular disease, cancer, diabetes and other chronic diseases was indicated (Pascual-Teresa, Sanches-Ballesta, 2008; Shipp, Abdel-Aal, 2010; Tsuda, 2012).The growing interest in impact of antioxidants on human health has triggered our study of berries from different Ribes and Prunus species. Also, complex interspecific hy-brids were created in order to obtain plants with higher phenolic compound and anthocyanin amount, as well as clarification of anthocyanin composition in order to identify cultivars or hybrids with highest antioxidant ac-tivity.Material and Methods. Four different Ribes species: R. nigrum L. ‘Ben Tirran’ (black berries), R. aureum ‘Au Gs–5’ (yellow berries) and ‘Corona’ (black berries), R. petreum ‘Jonkher van Tets’ (red berries), R. uva-cripsa ‘Lūšiai’ (red berries) and ‘Čiornyj negus’ (black berries) and four interspecific Ribes hybrids were studied. Antho-cyanin and phenol compound content, and antioxidative activity were evaluated in P. avium, P. cerasus cultivars, P. machii wild form and 4 their interspecific hybrids, too. All berries were collected at technical maturity phase and immediately frozen at –70°C. Anthocyanins and other phenolics from frozen berries were extracted by 90% aqueous methanol, acidified by HCl to 0.1 N, at ra-tio 1:20 (g/ml). Total phenolics content in extracts was determined by Folin-Ciocelteu method, using galic acid as standard. Antioxidant activity (capacity) was eval-uated spectrophoretically, by radical scavenging assay, according DPPH test (Anisimovienė et al., 2009). The anthocyanin content was determined using a spectropho-tometric differential pH method (Wrolstad et al., 2005) and calculated using a molar extinction coefficient of cy-anidin-3-glucoside 29600 (Cy-3-Glu). The composition of anthocyanins was determined by HPLC procedure (Durst, Wrolstad, 2001; Liobikas et al., 2009).

Results. Large anthocyanins content differences (up to 38 times) were identified in berries of tested Ribes spe-cies. It ranged from 16 mg 100 g–1 FW to 615 mg 100 g–1 fresh weight (FW), and the amount of total phenolic compounds ranged from 172 mg 100 g–1 FW to 672 mg 100 g–1 FW. The highest content of anthocyanins was ob-tained in R. aureum ‘Corona’ and R. nigrum ‘Ben Tirran’ berries. The lowest was identified in R. aureum ‘Au Gs-5’ and gooseberry ‘Lūšiai’. The antioxidative activity of all tested Ribes berry extracts was higher than 65%. All stud-ied interspecific hybrids were characterized as possessing high biological activity – values of tested indexes matched or outfaced antioxidant activity of R. nigrum ‘Ben Tirran’, which berries accumulate high anthocyanin content in Lithuanian climatic conditions. The highest content of anthocyanins was identified in berries of R. nigrum x R. petraeum and R. nigrum x R. aureum hybrids. Del-phinidins were predominant (68.6%) in berries of black-currant ‘Ben Tirran’, cyanidins dominated in gooseberry berries independently on berry colour and amount of anthocyanin and it constituted 75.8–90.8% of total an-thocyanins. Results of quantitative and qualitative analysis of an-thocyanins and phenolic compounds in fruits of tested Prunus species cultivars and interspecific hybrids re-vealed, that anthocyanin and phenol compound contents varied, too. Anthocyanin content in fruits of P. cerasus cultivars ranged from 45 to 147 mg 100 g–1 of FW, and total phenol 241–430 mg 100 g–1 of FW. 126–192 mg 100 g–1 FW of anthocyanins and 251–323 mg 100 g–1 FW of total phenols was identified in P. avium cultivars. The highest phenolic compound content – 568 mg 100 g–1 FW was found in berries of P. padus, while amount of antho-cyanins was similar to P. cerasus and P. avium. Antho-cyanin amount in fruits of interspecific Prunus hybrids ranged from 156 to 333 mg 100 g–1 FW; phenolic com-pounds varied from 302 to 484 mg 100 g–1 FW. Anthocy-anin amount in fruits of interspecific hybrid P. cerasus x P. maximovitchii 2n was 2 times higher than in fruits of P. cerasus cultivar ‛Rechta’ and P. avium cultivar ‛Kita-janka’ with high anthocyanin content, compared to other sour and sweet cherry cultivars. The antioxidative activi-ties of Prunus were 20% lower, in comparison with Ribes. Cyanidins were the most abundant anthocyanin class in fruits of most studied Prunus species and interspecific hybrids, however, in fruits of ‘Irema BS’ and P. cerasus x P. maximovitchii 2n cyanidins amount was significantly lower, and the most abundant anthocyanins were pelar-gonidins.Conclusion. The newly created interspecific hybrids of Ribes and Prunus species are proposed to be perspective new sources of anthocyanins. AcknowledgementsResearch was funded by a grant No. SVE-01/2011 from the Research Council of Lithuania.References[1] Anisimovien, N., Rubinskienė M., Viškelis P., Stackevičienė E., Stanys V., Šikšnianas T., Jankovska, E., Sasnauskas A. (2009) Anthocyanins in currants, cherries, blueberries, and antioxidative activity of berry extracts. Zemdirbyste=Agriculture, 96(3): 158–167.

64 Eurobiotech 2013

[2] Durst R.W., Wrolstad R. (2001) Separation and characterization of anthocyanins by HPLC. p. F1.3.1–F1.3.13. In: R.E. Wrolstad (ed.), Current Protocols in Food Analytical Chemistry. Wiley, New York.[3] He J., Guisti M.M. (2010) Anthocyanins: natural colorants with health-promoting properties. Annual Review Food Sci. Technol., 1: 163–187.[4] Liobikas J., Trumbeckaitė S., Bendokas V., Baniulis D., Majienė D., Kopustinskienė D.M., Šikšnianas T., Anisimovienė N. (2009) Pro-apoptotic effect of black currant berry extracts on rat heart mitochondria. Zemdirbyste=Agriculture, 96(3): 149–157.[5] Pascual-Teresa S., Sanchez-Ballesta M.T. (2008) Anthocyanins: from plant to health. Biochemistry Reviews, 7: 281–299.[6] Oancea S., Oprean L. (2011) Anthocyanins, from biosynthesis in plants to human health benefits. Acta Uninversitatis Cibiniensis. Series E: Food Technology 15: 3–15.[7] Shipp J., Abdel-Aal El-S.M., Food application and physiological effects of anthocyanins as functional food ingredients. The Open Food Science Journal, 4, 2010, 7–22.[8] Tsuda T. (2012) Dietary anthocyanin-rich plants: Biochemical basis and recent progress in health benefits studies. Mol. Nutr. Food Research, 56: 159–170.[9] Wrolstad R.E., Durst R.W., Lee J. (2005) Tracking color and pigment changes in anthocyanin products. Trends in Food Science and Technology, 1: 423–428.

P6.2

Selection efficiency of converted DArT markers in spring barley breeding for drought toleranceMagdalena Wójcik-Jagła1, Anna Fiust1, Marcin Rapacz1, Mirosław Tyrka2

1Department of Plant Physiology, University of Agriculture in Crakow; 2Department of Biochemistry and Biotechnology, Rzeszow University of Technology

21 DArT markers converted into STS and SSR markers were tested on 24 spring barley genotypes that differed in their response to drought and all proved to be polymor-phic. Selection efficiency was determined on the basis of correlation of the presence of a certain allele and values of physiological traits associated with drought tolerance (Rapacz i in. 2010 [1]). Among the markers tested, sev-eral seem to be suitable for discarding of the susceptible to drought genotypes during the selection process. The chosen markers were most efficient in differentiating the genotypes in terms of gas exchange, especially net pho-tosynthesis, which is the most informative parameter for assessment of plant growth ratio in drought. Two of the tested markers appear to be potentially most suitable for marker-assisted selection (MAS) of drought-tolerant Pol-ish spring barley genotypes. They differentiated the tested genotypes in terms of several physiological parameters, including net photosynthesis. Average net photosynthesis stress index (ASI) of the genotypes that did not contain those markers was only ca. 70% of the average ASI for all of the tested genotypesAcknowledgementsThis study was supported by the by Polish National Research and Development Center [PBZ-MNiSW-2/3/2006/17].References[1] Rapacz M., Kościelniak J., Jurczyk B., Adamska A., Wójcik M. (2010) Different Patterns of Physiological and Molecular Response to Drought in Seedlings of Malt- and Feed-type Barleys (Hordeum vulgare). J. Agronomy & Crop Science 196: 48–54.

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P6.3

Studies on micropropagation of Polish garlic (Allium sativum L.) varietiesAlicja Chuda

University of Agriculture in Crakow, Faculty of Horticulture, Institute of Plant Biology and Biotechnology, Department of Genetics, Plant Breeding and Seed Science, al. 29 Listopada 54, 31-425 Crakow, Poland

Garlic is one of the most important Allium vegetable crop widely cultivated throughout the world. Due to sexu-al sterility commercial varieties of garlic are propagated vegetatively. Unfortunately, this system favors the accu-mulation of viruses in plant material and thereby causes significant yield losses. Tissue cultures, especially mer-istem cultures and shoot tip cultures have been applied successfully to generate virus-free garlic plants. More-over, through somaclonal variation in vitro cultures can generate new forms at the phenotypic, cytological and molecular levels. The somaclones can be used in breeding programmes designed for increasing the genetic variabil-ity within Allium sativum species. However, due to the low propagation rates plant tissue cultures require opti-mization associated with the selection of donor varieties, type of explants and regeneration media. This study was conducted to provide an efficient protocol for microprop-agation of polish garlic varieties. In the experiment the influence of variety: Huzar, Jarus, Mega and Ornak and medium: LS [Linsmaier and Skoog 1965], MS [Murashige and Skoog 1962], LIK (LS+0.2 mg/l IAA+ 2 mg/l kine-tin) and MIK (MS+0.2 mg/l IAA+ 2 mg/l kinetin) on the development of garlic plantlets in in vitro cultures were investigated.AcknowledgementsThe research project was funded by the Polish Ministry of Science and Higher Education, decision No. 202914/E/377/M/2013.References[1] Linsmaier E., Skoog F. (1965) Organic growth factor requirements for tobacco tissue cultures. Physiol. Plant., 18: 100–127.[2] Murashige T., Skoog F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473–497.

P6.4

Analysis of mitochondrial transcripts associated with cytoplasmic male-sterility in cauliflower (Brassica oleracea L. var. botrytis)Gabriela Machaj, Marek Szklarczyk

Dapartament of Genetics, Plant Breeding and Seed Science, University of Agriculture in Crakow, al. 29 Listopada 54, Crakow, Poland

When transferred to cauliflower (Brassica oleracea L. var. botrytis) the cytoplasm of black mustard (Brassica nigra L.) causes cytoplasmic male sterility (CMS). In sterile plants stamens are substituted by petal-like organs and as a result pollen is not produced. CMS in plants is de-termined by specific sequences of the mitochondrial ge-nome. The present work was aimed at identification of mitochondrial transcripts which were associated with the sterilizing effect of the nigra cytoplasm in cauliflower plants. The analysis was based on the use of northern hy-bridization, RT-PCR, real-time RT-PCR and long PCR. The obtained results indicate that CMS-nigra is associat-ed with altered length of the high-molecular ccb206 tran-script and lowered accumulation of the low-molecular atp9 and ccb206 transcripts. The lowered accumulation of the ccb206 mRNA was confirmed by RT-PCR and re-al-time RT-PCR analyses. Moreover, the northern data suggest co-transcription of the atp9 and ccb206 sequenc-es. Their physical vicinity in the cauliflower mitochondri-al genome was proved with the use of long PCR. These experiments also indicate altered organization of the atp9 and ccb206 loci in the nigra and oleracea cytoplasm. Fur-thermore, long PCR and hybridization anlyses showed increased expression of the rrn26 gene in male-sterile plants.

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P6.5

The role of nitric oxide in calamine plant species response to lead stress during in vitro cultureEwa Muszyńska, Ewa Hanus-Fajerska

Department of Botany and Plant Physiology, University of Agriculture in Crakow

Abiotic stresses such as increased concentration of heavy metal ions results in reduction of rate plant growth and development. Therefore, the searching of effective meth-ods increasing the probability of plant survival in harsh conditions is very important task. The results of some recent experiments point at protective role of nitric ox-ide in species subjected to the various stress factors. For this reason, the aim of our work was to evaluate the ef-fect of exogenous nitric oxide application on the seed germination ability and further growth and development of plants cultivated in vitro under lead stress. For exper-iment seeds of Dianthus carthusianorum and Gypsphilla fastigiata (Caryophyllaceae) representing calamine pop-ulations from the Olkusz Ore – bearing Region (Southern Poland) were used. Seeds were treated with nitric oxide applied as sodium nitroprusside (SNP) and S-nitro-so-N-acetylpenicillamine (SNAP) in concentration of 50, 100 and 150 µM. Afterwards they were surface sterilized and placed on germination medium. Cultures were estab-lished on modified MS medium using 10 mm fragments of obtained seedlings shoots. Three concentration of lead nitrate were tested: 0,1 mM, 0,5 mM, 1,0 mM. After eight weeks of culture, micropropagation efficiency was eval-uated on the basis of number and length of regenerated organs (shoots and roots) and their fresh and dry mat-ter. Vitality of plants was also determined through the measurements of several physiological parameters. The chlorophyll a fluorescence with using Handy-PEA (Han-satech, UK) spectrofluorometer was provided and photo-synthetic functional parameters (Fv/Fm and PI) were cal-culated. Additionally, the content of photosynthetic pig-ments according to Wellburn (1994) and the content of total phenolic compounds according to the protocol pro-posed by Fukumoto and Mazza (2000) were investigated with using UV-Vis spectrometry. The obtained results will be presented and discussed during poster session.AcknowledgementsSupport of experimental work by the Ministry of Science and Higher Education of Republic of Poland (4562/2013) is gratefully acknowledged.References[1] Fukumoto L.R., Mazza G. (2000) Assesing antioxidant and prooxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 4: 3597–3604.[2] Wellburn A.R. (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol, 144: 301–313.

P6.6

Evaluation of the nucleotide sequences of DNA rearrangements in wheat-rye hybrids using ISSR-PCR, IRAP-PCR, REMAP-PCR and “ITAP-PCR”Izabela Szućko, Stanisława Maria Rogalska

Chair of Cell Biology, University of Szczecin, Waska 13, PL-70415 Szczecin, Poland

Early generations of wheat-rye hybrids are characterized by cytogenetic, genetic and physiological instability. This is manifested by a significant percentage of aneuploids in next generations of the hybrid, the occurrence of new genotypes that are difficult to predict and the diversi-fied development of hybrid plants. Many literature data confirm a significant changes of nucleotide sequences in DNA hybrids in generations F1-F4. These changes were most commonly identified as the elimination of a  se-quence and the emergence of new DNA sequence motifs which were not present in the parental genomes. We con-ducted a study on changes at the DNA sequences level in order to show whether similar changes occur in our hybrids material in the F2 and F3 generation.The aim of this study was to investigate the occurrence of the nucleotide sequences of DNA rearrangements in wheat-rye hybrids based on a combination of four mark-er systems (ISSR, REMAP, IRAP and “ITAP”).The material consisted of F2 and F3 generations of wheat-rye hybrids derived from the Plant Breeding Company Strzelce, agency Małyszyn. They were obtained by cross-ing three hexaploid wheat varieties (Triticum vulgare L cv Zyta, Tonacja, Ostka Strzelecka and two families (STHN 5067, 1002/1003) with the widely cultivated rye variety – Dańkowskie złote (Secale cereale L).Six primers were used for the IRAP analysis. Primers were designed based on sequences of the retrotransposons de-rived from cereals of the Secale genus (Bilby, Cassandra, Sukkula), Triticum (Angela) and Hordeum (Nikita, Sabri-na). For the ISSR analysis, 16 arbitrary primers were se-lected. For REMAP analysis, 15 combinations of primers were used. Each pair consisted of outward-facing retro-transposon primer and a second primer from a micro-satellite sequence. Five primers were used to the “ITAP” method, primers were designed on the basis of the trans-posons sequences from the Secale cereale (Rev) and Trit-icum (Sher, Mut, Jude, Caspar) genomes.The results obtained from the IRAP, REMAP, ISSR and “ITAP” methods enabled evaluation of the nucleotide sequence rearrangements. In addition, the value of each marker system was assessed. Marked efficiency index method (EMR), diversity index (DI) as the average value of the PIC system and marker index (MI) as the product of EMR and DI were counted. In the analysis, the pres-ence or absence of band was treated as a single feature and assigned as 1 or 0. Results of ISSR, IRAP, REMAP and “ITAP” methods showed changes in the structure

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of specific DNA nucleotide sequences. Disappearance of certain types of rye bands was mainly observed, and the percentage of wheat-specific bands which disappeared was lower. A new sequence types, absent in the parent species were observed less frequently. This indicates pos-sible rearrangements or elimination of sequences of wheat and rye. These results suggest that the early generations of wheat-rye hybrids undergo many genetic changes. It is worth noticing, that the DNA elimination (5-14% of the genome) increases the differences between the diploid genomes contained in the same nucleus in allopoliploid which is triticale. Through such differentiation the pos-sibility of homoelogous chromosome pairing is reduced, and the result may be a diploid-like meiosis behaviour, affecting polyploidy plant growth and fertility. Disomic inheritance leads to stabilization of progeny plants what makes them potentially useful for crop breeding.The values of the coefficients showing the quality of the individual marker systems were similar, indicating that each of these markers could be used separately to evaluate changes in the genomes of wheat-rye hybrids, however, using the whole set makes the analysis much deeper and more precisely shows the degree of rearrangement of the DNA nucleotide sequence variations in wheat-rye hy-brids.AcknowledgementsThis work is co-financed by the European Union within the European Social Fund, “Investment in knowledge as a driving force for development of innovation in the region – II edition” – implemented within the framework of Subaction 8.2.2 Regional Strategies of Innovation SOP HRD 2007–2013.

P6.7

Intergenic Spacer length variability in cultivated, weedy and wild rye speciesLidia Skuza, Ewa Filip, Izabela Szućko

Chair of Cell Biology, University of Szczecin, Waska 13, PL-70415 Szczecin, Poland

Ribosomal DNA (rDNA) in plants is organized in tandem arrays with a high copy number (Arnheim 1983). Each re-peating unit contains sequences coding for 18S, 5.8S and 25/26/28S rRNA genes (25S is found in most of the plants; 26S is found in wheat and 28S in mammals) and three spacer regions, i.e. internal transcribed spacers (ITS1and ITS2) and the intergenic spacer (IGS). Because there are highly conserved portions (18S, 26S, 5.8S)(Gerbi 1985; Gerbi et al. 1987) as well as less conserved (ITS) and more variable (IGS) regions (Appels et al. 1980; Beech and Stro-beck 1993; Borisjuk et al. 1994), this area has been used for both phylogenetic and genetic diversity analyses. Sequenc-ing of this region has also been used to detect and quantify variability in some species (Chou et al. 1999).The non-coding rDNA spacers (IGS) and the internal transcribed spacer (ITS) could be substantially variable in size due to differences in the number of repetitive el-ements among the closely related species. The repetitive and highly variable nature as well as the fast evolution rate allowed the rDNA spacers to be considered valuable alternative molecular marker systems. Three pair of uni-versal primers were used for amplification of non-cod-ing regions of ribosomal (rRNA) IGS. The IGS amplified products obtained from 19 Secale accessions, consisting of cultivated and non-cultivated rye, and represent three species and four subspecies of rye genus, showed a high level of polymorphism. The material was obtained from several world collections (Center for Biological Diversity Conservation in Powsin Warsaw, Poland, Nordic Genetic Resource Center, Sweden and National Small Grains Ger-mplasm Research Facility, National Small Grains Collec-tion, Plant Germplasm Inspection Stadion Agricultural Research Sernice United States Department of Agricul-ture, USA).The primers for IGS PCR resulted in multiple bands (2 – 13), different size (59.9bp-3249.7bp) and polymor-phism average 81,3%. The genetic similarity of the 19 Se-cale accessions ranged from 8.4 to 72.2. Cluster analysis by the Neighbor-Joining method in the Molecular image Gel DocTM XR (Bio-Rad) program on the basis of the Dice’s coefficient of genetic similarity indicated a divi-sion of the species studied into three groups of similarity. S. vavilovii (Hungary) and S. vavilovii (Russia) made a separate group. The second group includes nine species: S. strictum (Turkey), S. strictum (Australia), S. ancestrale (Japan), S. afganicum (Armenia), S. segetale (Azerbaijan), S. anatolicum (Canada), S. ancestrale (Turkey), S. cereal (Macedonia), S. cereal (USA). The third – the remaining species of rye.

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The variation in the total size of the IGS among the spe-cies detected in this work could be due to dissimilarity in the sequence of the repetitive elements or in their tandem repeat number (Saghai-Maroof et al. 1984; Barker et al. 1988; May and Zhiyong 1996). Highly inter specific poly-morphisms for rDNA IGS region suggesting the IGS will be a useful molecular marker for studies of Secale species.AcknowledgementsThis work is supported by the State Committee for Scientific Research grant No. N N310 435498 “Degree of relatedness within the genus Secale using non-coding chloroplast and mitochondrial sequences, and nuclear rDNA IGS regions”.

P6.8

Intra-population genetic diversity of cultivated carrot (Daucus carota L.) assessed by analysis of microsatellite markersAnna Maksylewicz, Rafal Baranski

University of Agriculture in Crakow, Institute of Plant Biology and Biotechnology, Unit of Genetics, Plant Breeding and Seed Science, al. 29 Listopada 54, 31-425 Crakow, Poland

Cultivated carrot (Daucus carota L. ssp. sativus) is one of the most important vegetable crops grown worldwide. It has spread from the centers of origin to remote geograph-ic regions that contributed to large diversity of accessions at the genetic level. The use of molecular markers, includ-ing microsatellites (SSRs), allowed for differentiation of Asiatic and European gene pools (Baranski et al. 2012), yet the molecular evidence for intra-population diversity has not been evaluated.The aim of the present study was to assess intra-popu-lation variation of cultivated carrot using SSR markers. The study was performed on a collection of 18 carrot ac-cessions originating from Europe, Asia, Japan and USA. Seeds were obtained from gene bank collection, breeding companies and research institutes. The accessions includ-ed hybrids (F1), open pollinating varieties (OP) and land-races (LR) that differed in root color. Diversity of carrot accessions represented by 9 to 20 single plants was eval-uated using 27 codominant SSR markers detected after separation in a capillary sequencer.In total 253 alleles were identified with a mean 9.4 and range from 2 to 18 alleles per locus. Most of the alleles (73.5%) had frequencies below 0.1 and only 3.95% alleles occurred with frequencies above 0.6. Rare alleles (fre-quency < 0.05) were detected in all except two loci and they represented 60.5% of all alleles identified. In 23 loci 64 unique alleles were detected (25,3%). The observed heterozygosity (Ho = 0.39) was lower than the expected heterozygosity (He = 0.6), which is consistent with pre-vious results on polymorphism of SSR markers in carrot (Baranski et al. 2012), and the fixation index was high-er than zero indicating excess of alleles in the homozy-gous state, probably due to the use of advanced cultivars in the analysis. AMOVA results showed that 29% of the observed variation was due to differences among popula-tions and 71% within the populations that is often noticed for cross-pollinated species having greater diversity with-in population than between populations. The first three PCoA coordinates visualized 61% of total variability. Two OP cultivars (‘Yellow Belgian’ and ‘Shima Ninjin’) of the lowest intra-population variation clearly differed from the remaining accessions. Accessions originating from continental Asia and Europe had more allelic variants (allelic richness 1.47 and 1.42, respectively) than those from Japan and USA (1.30 and 1.32, respectively). Also allelic richness in landraces (1.47) was higher than in F1

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hybrids (1.40) and OP cultivars (1.39). In consequence, accessions from Asia and Europe were more diverse (He = 0.47 and 0.42, respectively) than those from Ja-pan and USA (He = 0.26 and 0.33, respectively). Land-races were more diverse (He = 0.47) than F1 hybrids (He = 0.40) and both types were more diverse than OP cultivars (He = 0.30). It should be however noted that all accessions from Asia were landraces and those from Eu-rope were either OP or F1 cultivars so greater variability in the Asian gene pool can be partially explained by the fact that it was represented by landraces only.AcknowledgementsThe research was supported by the Ministry of Science and Higher Education grant No. NN310782440.References[1] Baranski R., Maksylewicz-Kaul A., Nothnagel T., Cavagnaro P.F., Simon P.W., Grzebelus D. (2012) Genetic diversity of carrot (Daucus carota L.) cultivars revealed by analysis of SSR loci. Genet Resour Crop Evol 59: 163–170.

P6.9

Detection of Plasmodiophora brassicae in soil by PCR methodAnna Czubatka, Jozef Robak, Agnieszka Czajka, Wojciech Szczechura, Miroslawa Staniaszek

Research Institute of Horticulture, Konstytucji 3 Maja 1/3 St., 96-100 Skierniewice, Poland

Plasmodiophora brassicae Wor. fungus is the cause of clubroot – one of the most dangerous and most frequent diseases of brassicas. Resting spores of this pathogen are able to remain viable in soil even up to 8 years. The most effective way to protect cultivations against this disease is introduction resistant plants varieties and analysis of fields in the presence of this pathogen before planting brassicas. Detection of P. brassicae relied on bioassays are long and require large amount of infected soil and space in greenhouse. Molecular methods use in the detection of this pathogen should be more sensitive and faster than traditional plant bioassays. Furthermore, applica-tion real-time PCR method could allow detect spores of P. brassicae and specify their amount in soil samples. De-velopment of reliable and sensitive method to detect this pathogen is necessary to protect cruciferous crops culti-vation against clubroot.The aim of this work was compare traditional bioassays and PCR methods as a tools for the detection of P. bras-sicae in soil. Soil samples containing series of different concentrations of studied pathogen between 0 to 108 spores per 1 g–1 were analysed using bioassay on Chinese cabbage cv. Granaat, nested PCR and real-time PCR. Ge-nomic DNA for PCR reactions were isolated from soil samples using commercial kit.In a biological test P. brassicae was detected in the soil when the resting spores concentration was 106 of spores per 1 g–1. In nested PCR the specific DNA fragment was amplified in soil in which the concentration of spores was higher than 104 spores per 1 g–1. Preliminary results indi-cate that real-time PCR detects 103 spores per 1 g–1, which gives a chance to use it as a sensitive method for the de-tection and quantification of P. brassicae in soil. However, this method still requires further work to become a rou-tine diagnostic test replaces the biological assays in detec-tion of P. brassicae in soil.AcknowledgementsThe study was performed in the frame of Multi-annual Programme financed by Polish Ministry of Agriculture and Rural Development.

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P6.10

Chromosomal distribution of repetitive DNA sequences and karyotyping in Cucumis metuliferusKohei Yagi, Ewa Siedlecka, Zbigniew Przybecki, Stefan Malepszy, Pląder Wojciech1Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences – SGGW, Nowoursynowska, Warsaw, Poland

Cucumis metuliferus (African cucumber, African horned cucumber, African horned melon, Jelly melon, kiwa-no) is cultivated species in Cucumis genus like C. sati-vus. Cucumis metuliferus has become economically and medically important species in Cucumis genus. It has attracted attention as a genetic resources for cucumber and melon. Genomic base information about C. metu-liferus will be necessary for grifting or gene introduction to the major Cucumis crops in the future. Cytological and molecular cytogenetic studies of C. metuliferus have been performed by some researcher (Dane and Tsuchiya 1976, Yadava et al. 1984, Nijs 1985, Ramachandran and Narayan 1990). Most of the these studies reported only chromosome number (2n = 24) by basic staining (Dane and Tsuchiya 1976, Yadava et al. 1984, Nijs 1985). Giem-sa C-banding has been reported by Ramachandran and Narayan (1990). They also isolated repetitive DNA, and indicated that it hybridized to 12 sets of inter phase and 6 heterochromatin knob of pachytene chromosomes. But distribution patterns and chromosomal locations are still unclear due to small chromosome size. In this study, we analyzed the early-metaphase chromosomes of C. metuliferus by means of 4’-6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA) fluorescence stain-ing and found the location of the 45S rDNA (18S rDNA), 5S rDNA genes, telomere sequence and species-specific tandem repeats using fluorescent in situ hybridization (FISH).

P6.11

The effect of temperature on growth, and several parameters of Pheodactylum tricornutum in batch cultureMonika Bojko1, Klaudia Brzostowska1, Paulina Kuczyńska1, Dariusz Latowski1, Monika Olchawa-Pajor1, Weronika Krzeszowiec2, Kazimierz Strzałka1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland; 2Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland

The effect of different temperatures on the kinetics of diatoms growth in batch cultures was examined. The experiments were performed using Pheodactylum tri-cornutum CCAP/1055/1 strain as a model diatom with known genome sequence. The elucidation of significance of temperature as an important factor for growing of di-atoms is crucial for planning all experiments with these algae in in vitro systems. The studies compared the effect of temperature on several parameters important for cul-tivation of the diatoms in culture medium. These param-eters were: the concentration of photosynthetic pigments, proteins and Fv/Fm, cells size, RNA and DNA content as well as the optical density (OD405, OD600) of the culture. The diatoms were grown at two optimal temperatures: 15°C and 20°C and under temperature stress conditions at: 12°C and 23°C with photoperiod 10/14h and light in-tensity of 40 µEm–1s–1. The above-mentioned parameters were registered for 14 days, individually for each culture at different temperatures.The analysis of the obtained results demonstrated that temperature had significant effects on the growth kinet-ics of Pheodactylum tricornutum. Two phases of culture growth were observed. The faster phase was detected between first and 5th day after inoculation, The second one was slower and was recorded from 6th till 14th day, In cultures growing at 20˚C and 23°C the highest chlo-rophylls and proteins concentration as well as OD were measured at the last day of observation. The analysis of the relation between investigated parameters shows that in first 5 days after inoculation there are significant positive correlation between OD increase, chlorophyll a, chlorophyll c and protein concentration, and Fv/Fm at different temperatures. The pattern and strength of correlations varied with temperatures. Several days after inoculation (from 6th till 14th day) chlorophylls and pro-teins concentration as well as OD were increasing con-tinually at all growth temperature. On the other hand the Fv/Fm parameter achieved maximum level at 6th or 7th day and then decreased to the values registered on the first day of observation. At the highest temperature (23°C) the values of this parameter were the lowest. To-tal level of RNA and DNA as well as cell size of Ph. tri-cornutum were additional parameters tested during two weeks culture of diatoms. Genetic material undergoes

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gradual degradation 10 days after inoculation. Size of the cells of Ph. tricornutum was invariable and indepen-dent of the time and growth temperature.All analyzed parameters demonstrated that the optimal time of the diatoms culture is the period between 4th and 7th day after inoculation.AcknowledgementsThis work was supported by project No. 2011/01/M/NZ1/01170.

P6.12

DNA markers as a tool for selection of tomato plants with resistance to Fusarium oxysporum f.sp. radicis-lycopersiciMirosława Staniaszek, Wojciech Szczechura, Elżbieta Kozik, Marzena Nowakowska, Hanna Habdas

Research Institute of Horticulture, Department of Genetic Breeding and Biotechnology Vegetable Plants, 96-100 Skierniewice, Poland

Tomato is one of the most important and popular vegeta-ble grown in many world regions. In modern breeding of this species more frequently are using molecular markers, especially based on the polymerase chain reaction (PCR). These methods allowing to selection of plants with specif-ic trait such as resistance to fungal pathogens. Fusarium oxysporum f.sp. radicis-lycopersici (Forl) is an important tomato pathogen. This pathogen causes fusarium crown and root rot, one of the most destructive disease of toma-to in crops under cover. Resistance to Forl is determined by single dominant gen, named Frl, located on the long arm of chromosome 9 tomato genome. In studies under-taken in Research Institute of Horticulture, Skierniewice, CAPS C2-251100 marker was identified which is linked to Frl gene. The amplification products was digested by XapI restriction enzyme to obtain a DNA polymorphism. This marker can be useful in marker assisted selection in to-mato breeding programs.This work was performed in the frame of Multi-annual Programme “Development of sustainable methods of horticultural production to ensure high biological and nutritional quality of horticultural products and to pre-serve the biodiversity of the environment and to protect its resources”, finance by Polish Ministry of Agriculture and Rural Development; Task 6.6.

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P6.13

Effect of different conditions for cell immobilization and phytosulfokine supplementation on somatic embryogenesis in protoplast cultures of Daucus speciesKatarzyna Maćkowska, Ewa Grzebelus

University of Agriculture in Crakow, Dept. of Genetics, Plant Breeding and Seed Science

Somatic embryogenesis more or less follow the pattern of zygotic embryo development and therefore is an im-portant pathway of plant regeneration from cell culture systems. Additionally, in comparison to often applied or-ganogenesis, plant production through embryogenesis is much faster and effective. Embedding of the cells in solid matrices such as alginate may significantly enhance effi-ciency of that process by inducing asymmetrical divisions of the cells promoting further polar organization of the developing structures and in consequence giving rise to compact embryos. Phytosulphokine (PSK), a peptidyl plant growth factor found lately in both monocotyledon-ous and dicotyledonous plants, also has been shown to stimulate somatic embryogenesis. The aim of the present study was to evaluate influence of different conditions for protoplasts immobilization in alginate gel and PSK appli-cation on somatic embryogenesis in Daucus species.Protoplasts were isolated from shoot cultures of seven Daucus accessions including D. carota subsp. sativus, D. carota subsp. azoricus, D. carota subsp. drepanensis, D. carota subsp. maritimus, D. aureus, D. montevidensis, and D. pusillus. Tissue digestion and protoplast cultures were performed as described by Grzebelus et al. (2012) with some modifications: isolated cells were embedded in filter- or autoclave-sterilized alginate solution as thin alginate layer (TAL) or extra thin alginate film (ETAF) and cultured in the presence of 100 nM PSK. Culture time points both for first cell division and microcolonies up to 70 µm and 150 µm in size were recorded. Moreover frequency of protoplasts undergoing asymmetric cell di-vision, number of macrocolonies (above 500 µm in size), presence of somatic embryos, and number of regenerat-ing plants were estimated.First cell divisions were observed between fourth and fifth day of culture for D. carota subsp. sativus, D. caro-ta subsp. drepanensis, D. carota subsp. maritimus, and D. aureus while for the remaining accessions around 7-8th day of culture. Cell colonies up to 70 µm and 150 µm in size were microscopically visible in 8-16-day-old cultures and 14-20-day-old cultures, respectively. Both method of sodium alginate sterilization, culture technique and ad-dition of PSK to the culture medium did not influenced on the time of the first cell division and cell colonies formation. Percentage of asymmetric cell divisions was accession-dependent and varied from 91% to 98%. Pro-toplasts of D. c. subsp. maritimus and D. montevidensis

most frequently in comparison to the other accesisons underwent this type of division. After 2 months of cul-ture macrocolonies above 500 µm in size were found and their number varied from 13 (D. c. subsp. azoricus) to 574 (D. c. subsp. sativus). For D. montevidensis and D. pusillus such macrocolonies were not observed. Additionally, at that time in the cultures of all D. carota subspecies and D. aureus somatic embryos in torpedo-stage were found. After transferring of macrocolonies and embryos to a sol-id hormone-free medium regeneration into plants were occurred. The number of morphologically normal plants varied from 8 for D. c. subsp. maritimus to 73 for D. au-reus. Application of the TAL technique for protoplasts immobilization and supplementation of the cultures with 100 nM PSK increased frequency of plant regeneration.AcknowledgementsThis work was supported by the Polish Ministry of Science and Higher Education, grant No. N N310 440 238.References[1] Grzebelus E., Szklarczyk M., Baranski R. (2012) An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot. Plant Cell, Tissue and Organ Culture, 109: 101–109.

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P6.14

Characterization of wheat mRNAs expressed during grain developmentMagdalena Simlat, Michał Nowak, Patrycja Wieczorek, Maria Moś

Department of Plant Breeding and Seed Science, University of Agriculture, Lobzowska 24, 31-140 Crakow, Poland

The study was performed using wheat grains harvested at different developmental stages (from 10 to 45 days after pollination, DAP) from cultivars Piko and Sława – sus-ceptible and resistant to pre-harvest sprouting (PHS), respectively. The RNAs isolated from these grains were used for differential display (DDRT-PCR) analysis of gene expression. Transcript derived fragments (TDFs) which were specific for one or more grain developmental stages were cut out from the gel, re-amplified, T/A-cloned and sequenced. BLAST analysis revealed homology for 22 from the 25 analyzed fragments. The identified homol-ogies included sequences coding for VP1 transcription factor, SNF/SWI2 family transcription factor, protein kinase PK3, flowering locus C (FLC) protein, starch syn-thase I and germin-like proteins (GLP). Seven fragments showed high similarity to transposon sequences e.g. the gene coding for the putative gag-pol poliprotein. Differ-ential expression of the identified sequences will be veri-fied using real-time PCR.

P6.15

Molecular and cytogenetic verification of the F1 and F2 hybrids between Betula nana and Betula utilis ‘Doorenbos’Małgorzata Czernicka1, Jarosław Pławiak1, Piotr Muras2

1Department of Genetics, Plant Breeding and Seed Science, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland; 2Department of Dendrology and Landscape Architecture, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

The birch (Betula sp.) is very popular as garden and park tree. Most of birch species and varieties grow rap-idly and for this reason they are not appropriate enough to cultivate in gardens. In the research, the breeding program was conducted in order to obtain the interspe-cific hybrid (‘Nanabos’) which could be characterized by slow growth rate and interesting shape of purple leaves. Crossing and selection were done in Tomaszkowice near Krakow in 2001 while the female inflorescenc-es of Betula nana were manually pollinated by Betula utilis ‘Doorenbos’ pollen. In 2002 the total number of seedlings was about one hundred and only several ones have survived up to date. The research purpose was the verification of the hybid character of F1 and F2 puta-tive progeny derived from the interspecific cross i.e. B. nana x B. utilis ‘Doorenbos’. The verification was based on the RAPD-PCR method and the ploidy level analysis which were supported by morphological results. Plant material were: parental taxa, 8 putative F1 hybrids, 8 pu-tative F2 hybrids, 3 cultivars of B. pendula (‘Doopurea’, ‘Purpurea’, ‘Dalecarlica’). For molecular research, DNA was extracted from young leaf tissue using DNeasy Plant Mini Kit (Qiagen). RAPD analysis was performed using twenty 10-mer random primers and RAPD-PCR products were analyzed using agarose gel electrophore-sis. For karyotype, mitotic chromosomes were isolated from young leaf buds using protoplast dropping method of Anmthawat-Jónsson (2003). The phenotypic analysis suggested that most hybids had leaves similar to mater-nal species and they were characterized by unbranched, columniform growth pattern with tall straight trunk, that could be inherited from the paternal form. How-ever, the main branches showed spiral growth what was typical for B. nana.One specimen from the hybrid F1 population was intermediate in phenotype (relative to parental genotypes) i.e. columniform but low growth and size of leaves was higher in comparison with the rest of hybids. Molecular markers were obtained and a den-drogram was worked out, which illustrated the level of the genetic diversity among the parental taxa and F1 and F2 hybrid plants. Classification of the hybrid plants by ploidy level proved that most of the putative hybrids in-dicated that they were diploid (2n = 28) and no triploid as expected.AcknowledgementsThe research was supported by Polish Ministry of Science and Higher Education grant for Young scientists (Project No. 4554/2013).

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References[1] Anamthawat-Jónsson (2004) Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization. Methods in Cell Science 25(3-4): 91–95.

Environmental biotechnology

Lectures

L7.1

Metallophytes: a biodiversity and phytotechnological resource for soil clean-up, phytomining and mine site restoration Alan J M Baker

School of Botany, The University of Melbourne, Australia; Centre for Mined Land Rehabilitation, The University of Queensland, Australia; Department of Animal and Plant Sciences, The University of Sheffield, UK

Metallophytes – plants that have evolved on metal-en-riched soils – have key ‘values’ that must drive research on their unique properties, and ultimately their conserva-tion. The ability of metallophytes to tolerate extreme metal concentrations commends them as the optimal choice for phytostabilization and ecological restoration of mineral wastes and metal-contaminated soils. Metallophytes, and in particular metal-hyperaccumulating plants, have also spawned several novel phytotechnologies, including phy-toremediation, phytoextraction and phytomining. Other new potentials for exploiting their unique properties are emerging. The last decade has seen an ever-increasing interest in metal-tolerant and metal-accumulating plants both from an academic standpoint and in developing their potentials in phytotechnologies. Few studies have high-lighted the need to conserve these species. This presenta-tion identifies future research needs for the conservation and utilization of the global metallophyte biodiversity.

L7.2

Bioaugmentation with engineered endophytic bacteria improves phytoremediation in the fieldJaco Vangronsveld, Panagiotis Gkorezis, Sofie Thijs, Nele Weyens

Hasselt University, Environmental Biology, Centre for Environmental Sciences, Agoralaan Building D, B-3590 Diepenbeek, BelgiumL 7.5L.7.6

Phytoremediation is a promising technology: driven by solar energy, plants are able to pump contaminations to their rhizosphere and even take them up. In the rhizo-sphere and during its transport throughout the plant, the present plant-associated micro-organisms can take care of the organic contaminant’s degradation.Successful application of phytoremediation was demon-strated in 2 field cases (BTEX and oil contamination). On these sites, poplar trees were planted in the contamina-tion plume and groundwater concentrations and possible evapotranspiration to the atmosphere were monitored.Despite the above mentioned and other successful field ap-plications, phytoremediation is not yet applied on a large scale due to some constraints. At first, plants should tol-erate the occurring contaminant levels. Further, the deg-radation capacity of the plant-associated micro-organ-isms must be high enough to prevent phytotoxicity and evapotranspiration. To solve these constraints, a diversity of interesting characteristics of plant-associated bacteria can be exploited. The availability/uptake of many organics can be stimu-lated by bacteria producing e.g. surfactants, siderophores and organic acids. Bacteria with the appropriate degra-dation pathway(s) can strongly improve the degradation efficiency. In a third field case, this concept of endophyte-en-hanced phytoremediation was successfully applied on a TCE-contaminated site. Poplar trees were planted and in situ inoculated with TCE-degrading endophytic bacte-ria. Three months after inoculation, a 90%-reduced TCE evapotranspiration was observed. Further investigations revealed that this reduction was not only achieved by an enrichment of the inoculated strain, but also by of trans-fer of the degradation genes from the inoculated strain to bacteria from the natural abundant population.

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L7.3

Support of Plant Growth by Selected Soil Microbes and Manipulation of Soil Texture in Metal Contaminated SoilI. Lichtscheidl1, S. Wernitznig1, 2, W. Adlassnig1, A. R. Sprocati3, K. Turnau4, A. Neagoe5, C. Alisi3, S. Sassmann1, A. Nicoara5, V. Pinto3, C. Cremisini3

1University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Althanstraße 14, A-1090 Vienna, Austria; 2Medical University of Graz, Institute of Cell Biology, Histology and Embryology. Harrachgasse 21, A-8010 Graz, Austria; 3Italian National Agency for New Technologies, Energy and Sustainable Economic Development. Via Anguillarese, I-301-00123, Rome, Italy; 4Jagiellonian University, Institute of Environmental Studies, Gronostajowa 7, Pl-30387 Crakow, Poland; 5University of Bucharest, Research Center for Ecological Services. Splaiul Independentei 91–95, Sector S, Ro-050089 Bucureşti, Romania

Establishment of a vegetation cover on mine waste sub-strate is one of the crucial but also one of the most dif-ficult parts of the remediation process. This study tests two different approaches to facilitate plant growth on toxic and oligotrophic substrates, i.e., the addition of coarse mineral particles and the inoculation with putative growth supporting bacteria.Extremely fine grained mine waste from Ingortosu/Italy containing high amounts of Zn, Cu, Cd and other tox-ic elements was supplemented with a mixture of sand and volcanic clay, with a bacterial consortium (Bacillus cereus, Curtobacterium flaccumfaciens and eight other strands) or with a combination of both, a control was left untreated. On these substrates, Euphorbia pithyusa, Heli-anthus annuus, Agrostis capillaris, Deschampsia flexuosa and Festuca rubra were grown in pots under greenhouse conditions.The bacteria established well and reduced the extractabil-ity of most metals. Their effect on plant growth, howev-er, was ambiguous and usually negative. The addition of sand and volcanic clay, on the other hand, had a positive effect on all plant species except E. pithyusa. The effects of a double treatment with both bacteria and sand and volcanic clay were insignificant or negative.It is concluded that manipulations of the soil texture of mine substrates may play a key role in the process of bioremediation.AcknowledgementThis study was supported by funding of the the EU project Umbrella (EU 226870), of the Austrian Ministery for Science and Research, and the Appear project Biorem. We acknowledge the use of the green house facility of the University of Vienna and we are thankful for the help of the gardeners, Thomas Joch and Andreas Schröfl. The soil from Ingurtosu/Sardinia was kindly provided by Giovanni de Giudici from the University of Cagliari/I.

L7.4

Highland plants under extreme conditions and their phytoremediation potentialEdwin J.P. Cadenas

UNASAM, Huaraz, Peru

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L7.5

X-Ray Microanalysis of Copper and Zinc Uptake in the Moss Physcomitrella patens Hedw

S. Sassmann, M. Weidinger, W. Adlassnig, I. Lichtscheidl, I. Lang

University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Althanstraße 14, A-1090 Vienna, Austria

Mosses are frequently regarded as tolerant to toxic met-als and as accumulators of such metals regardless of their original habitats. The reasons for this tolerance are poorly understood. Unlike vascular plants, mosses absorb nu-trients via the entire surface, and do not possess exclu-sion mechanisms like an exodermis or an endodermis. In preliminary experiments, we confirmed an exceptionally high zinc tolerance of moss gametophytes from heavy metal contaminated mining sites, and also for Physcom-itrella patens Hedw. (Funariaceae) which avoids metal enriched habitats in nature.Gametophytes of P. patens were cultivated on sterile agar plates. We offered zinc (as Na2Zn EDTA, ZnCl2 and ZnSO4) and copper (as Na2Cu EDTA, CuCl2 and CuSO4). After five weeks of cultivation the moss samples were washed, air-dried, mounted and carbon-coated. During sample preparation, great attention was paid to avoid contamination with the metal rich growth medium. Cop-per and zinc uptake of P. patens leaves were semi-quan-titatively analysed by X-ray microanalysis in a scanning electron microscope.We observed differences in tolerance and uptake of cop-per and zinc. A gradient elevation of the heavy metal con-tent of leaf tissues corresponding to the added amount of zinc and copper in the growth media could be observed. Furthermore, our results indicate strong differences in uptake and resistance depending on the anion. E. g., met-als coordinated with EDTA proved to be significantly less toxic for P. patens. The relevance of coordinating agents for metal toxicity is discussed.AcknowledgementsThis research was supported by the Gesellschaft zur Förderung der Pflanzenwissenschaften, the Hochschuljubiläumsstiftung der Stadt Wien (grant H-1939/2008), the University of Vienna (Forschungsstipendium 2010 to S.S.) and Appear (Biorem)

L7.6

Bioremediation by Phormidium Biofilms a Case Study from a Historic Mine SiteW. Adlassnig1, N. Pérez2, S. Sassmann1, S. Reipert1, F. Hofhansl3, I. K. Lichtscheidl1

1University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Althanstraße 14, A-1090 Vienna, Austria; 2Luleå Tekniska Universitet, Institutionen för Samhällsbygnad och Natururser, Campus Luleå, F Huset, SE-97187 Luleå, Sveden; 3University of Vienna, Department of Microbiology and Ecosystem Science, Althanstraße 14, A-1090 Vienna, Austria

Mining and ore processing frequently results in the for-mation of metal rich drainage water, representing a seri-ous threat for people and environment. The remediation of such waters is difficult and expensive, and alternatives to conventional water treatment are intensively investi-gated.At a historic mining site in the Austrian Alps, a Cu con-taminated creek is remediating via constant binding of metals to microbial mats. This study deals with the lo-calisation of the absorbed copper, as well as the isotopic signature and species composition of the microbial com-munity.The biofilm is almost exclusively dominated by the Cy-anobacterium Phormidum sp. and contains 3.9  ±  1.8% Cu. The constant absorption from the water reduces the Cu content of the creek from 0.6  ±  0.1  mg  ∙  l–1 to 0.2 ± 0.2 mg  ∙ kg–1 after only 300 m, thereby exceeding abiotic Cu precipitation by far. Ancient dead layers of bio-film covered by humus but still rich in Cu indicate that the immobilisation is permanent. In spite of the variable Cu content of the biofilm, it exhibits a remarkably constant δ65Cu of 0.5 ± 0.1‰ which is significantly higher than in the contaminated water (–0.9 ± 0.2‰) making uptake of the Cu into the living cell improbable. This is confirmed by the ubiquitous occurrence of electron dense mineral particles in the gelatinous sheath of Phormidium, partly identified as the secondary Cu mineral sampleite.Fixed mats of Phormidium have been frequently used for metal absorption under laboratory conditions. The spontaneous self-remediation of a creek by Phormidium, however, is a novel feature and suggests new strategies for the application of Cyanobacteria in the field of bioreme-diation.AcknowledgementsWe are grateful to Prof. Dr. A. Beran (University of Vienna) for the identification of sampleite. Thanks are due to Forstmeister R. Schilcher, who made scientific research in the Schwarzwand possible. This study was supported by the EU project Umbrella (EU 226870), the Appear project Biorem and the OEAD project Promote.

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L7.7

FISH detection of autochthonous bacteria of ceramic clayey raw materialsPaulina Supel1, Paweł Kaszycki1, Joanna Brzeszcz2, Piotr Kapusta2, Piotr Wyszomirski3

1Departament of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Crakow; 2Department of Microbiology, Oil and Gas Institute, Crakow, 3AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Crakow

Contamination of clay raw materials with organic com-pounds causes severe problems in ceramic industry. Oc-currence of organic fraction leads to lowered aesthetic value, profoundly reduced mechanical strength, and thus decreases the overall industrial suitability. There-fore, quality requirements along with increasingly strict environmental standards give strong reason to elaborate innovative methods of beneficiation of ceramic clays. Among these, biotechnological methods based on micro-biological enzymatic activities seem to be the most chal-lenging and efficient. The main principle of these meth-ods is to degrade undesirable organic admixtures by het-erotrophic microorganisms capable of bioremediation of an organic fraction as well as to remove iron compounds by bioleaching.Autochthonous microorganisms appear as the best nat-ural candidates for biological improvement of ceramic industry clay materials. If present, they are best fitted and adapted to the clayey microenvironment and can possibly utilize material organic impurities as a source of carbon.The aim of the study was to prove the occurrence of autochthonous bacteria in three selected ceramic clay materials, obtained from different areas and differing in the content and form of an organic fraction: (1) Lower Jurassic (Liassic) clay from Żarnów, with a low organ-ic substance content, (2) Tertiary material form Turów, characterized by a moderate level of carbon com-pounds, especially the so called fine-dispersed brown coal substances, obtained from a site adjacent to brown coal deposits, and (3) a clay from the Radzymin deposit, belonging to the relatively young, Quaternary materials, used mainly in building ceramics due to the high con-tent of organic compounds.Standard microbiological plating techniques showed the occurrence of autochthons in materials (2) and (3). Iden-tification of several colony morphotypes indicated high biological diversity of the strains. The FISH (fluorescence in situ hybridization) technique enabled to reveal even greater biodiversity, including clay (1). The visualisation method was based on several specific molecular probes directed at conservative genes 16S rRNA (EUB338I, II, III-Cy3, ALF968-Cy3) and 23S rRNA (BET42a-Cy3, GAM42a-Cy3, HGC69a-Fluos). The above sets of oligo-nucleotide probes were constructed to identify bacteria belonging to the following systematic groups: α-Prote-

obacteria (ALF 968), β-Proteobacteria (BET42a), γ-Pro-teobacteria (GAM42a), Actinobacteria (HGC69a), and Eubacteria (a mix of three probes EUB 338I, II, III.)The above data bring for the first time the information of bacterial consortia inhabiting unique environments of clayey minerals. We claim the research that we have just launched will bring interesting and valuable applications in terms of treatment of ceramic industry materials prior to their processing.AcknowledgementsThe work was financially supported by the grant for scientific research No. 3500, approved by the Polish Ministry of Science and Higher Education.

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L7.8

Phytoremediation crops for sustainable biofuels – scope and limitationsPrasad M.N.V.

Department of Plant Sciences, University of Hyderabad, Hyderabad 500 046, India

Energy and environmental security are the crux of the sustainable development. Nature’s cure using plant re-sources (= Phytoremediation) is a sustainable solution for environmental decontamination. As of now about 18000 articles have been published on various aspects of using biological resources for environmental cleanup starting with only 11 in 1989. Often policy and decision makers, academia and civic governance think that phytoremedi-ation is a temporary solution of transferring the pollut-ants and contaminant are transferred from one place to another. Scientists and academia are no exception to this feeling. Regulators have expressed apprehensions about phytoremediation due to lack of knowledge of contem-porary knowledge of environmental sustainability. Often it is generally believed, that this kind of environmental decontamination is a temporary solution. How to dispose of the contaminated phytomass is a puzzling question by environmental managers and regulators.Plants are assisted by soil microbes and mediate wide array pollutants and are able to decontaminate the tox-ic substances, thus contribute significantly to pollution abatement. Phytoremediation is the use of plants to ac-cumulate, remove or render harmless toxic compounds contaminating the environment. Plants absorb/adsorb/exclude, translocation, store or detoxify inorganic and or-ganic contaminants. Thereby they contribute significantly to the fate of these contaminants of the biosphere. Biofuel production is other side of the coin of the “Phytoremedi-ation crops”.Keywords: bioremediation, contaminants, energy and environment security, geoenvironment, inorganics, nature’s cure, natural resources, organics, phytoremediaiton, pollutants, rhizoremediationAcknowledgementsThe author is grateful to the Ministry of Environment and Forests, GOI, New Delhi For the award of Pitamber Pant National Environment Fellowship (MoEF Ref. No. 17/3/2010-RE Dt 29-2-2012).

Posters

P7.1

The influence of phytase addition during the enzymatic starch hydrolysis process with the use of basic amylolytic enzymes on yield and the course of alcoholic fermentation processDawid Mikulski, Grzegorz Kłosowski

Department of Biotechnology, Institute of Experimental Biology, Kazimierz Wielki University, 85-667 Bydgoszcz, Chodkiewicza 51 St., Poland

Phytic acid (myo-inositol hexa-phosphoric acid, IP6) is the major phosphorus storage compound of most cereal grains. Phosphorylated form of inositol due to ionized phosphates groups has strong chelating proper-ties. Phytic acid creates complexes, the so-called phytin complexes with divalent cations (Mg2+, Ca2+, Fe2+, Zn2+), with proteins and polysaccharides, i.e. starch [1]. These complexes limit the availability of the divalent cations, proteins and starch for organisms which do not pro-duce the phosphohydrolases enzymes. The applications of phytase, the enzyme hydrolysing phytin complexes during the preparation of the fermentation medium, should result in the increase of availability of biogenic compounds [2]. The aim of the study was to determine the influence of phytin hydrolysis before and after starch hydrolysis process present in high gravity maize mashes (HG mashes) on yield and the course of alcoholic fer-mentation process. The raw material used in prepara-tion of HG mashes according to pressureless liberation of starch technology (PLS technology) was milled maize grain (starch concentration – 53.34%, phytic acid con-centration – 2.76 mg/g, total phosphorus concentration – 2.93 mg/g). The process of enzymatic starch hydrolysis was conducted with heat-stable α-amylase (Termamyl SC DS preparation) and glucoamylase (Saczyme prepa-ration) produced by Novozymes® company. Phytin com-plexes hydrolysis was conducted with the use of Phytase 10000L preparation produced by Erbsloh® company. During the alcoholic fermentation the basic technologi-cal indicators (alcohol concentration, productivity, yield and fermentation energy) and indicators of the physio-logical condition of yeast (number, viability, phosphorus concentration in yeast cells) were determined. Alcoholic fermentation volatile by-products were determined with capillary gas chromatography (GC method). The analy-sis of variance (significant level α3/100kg of starch was observed in the variant with phytin hydrolysis before starch hydrolysis process. In the initial fermentation phase irrespectively of the research variants the viability of yeast was ca. 98%. In subsequent phases the increase of ethanol concentration caused the decrease of viability and number of yeast cells in variants with addition of phytase in relations to the control variant. The highest

80 Eurobiotech 2013

concentration of acetaldehyde (524.1±25.6mg/L) and higher alcohols (4144.2±154.0mg/L) was observed in the distillate obtained in the fermentation medium with phytin hydrolysis before hydrolysis of starch. AcknowledgementsThe study financed from the science resources in the years 2013–2014, as an experimental study of the National Science Centre (No. 2012/05/N/NZ9/02436). References[1] Garcia-Estepa R.M., Guerra-Hernández E., Garcia-Villanova B. (1999) Phytic acid content in milled cereal products and breads. Food Research International 32, 217–221.[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and biotechnology. Biotechnology Letters, 25, 1787–1794.

P7.2

The Kinetic reduction of Cr(VI) by yeast cells of Saccharomyces cerevisiae, Phaffia rhodozyma and their protoplastsJarosław Chwastowski, Henryk Kołoczek

Crakow University of Technology, Department of Inorganic Technology and Environmental Biotechnology

Contamination with heavy metals is a serious environ-mental problem. Pollution by chromium originating from several industrial processes such as leather tan-ning, electroplating, textile industries, nuclear power plant, water cooling, pulp production, ore and petro-leum refining processes is a reason for deterioration of midland water quality. In trace amounts, chromium is beneficial to living organisms (M. Pas et al., 2004). How-ever, at higher concentrations it is extremely toxic, caus-ing allergies, egzema, and respiratory track disorders. It is also a strongly mutagenic and cancerogenic agent, es-pecially in its oxidized form, Cr(VI) (Barceloux, 1999). Once inside the cell Cr(VI) is being reduced to the Cr(V) and can react with H2O2 what leads to the pro-duction of hydroxyl radicals (•OH) via the Fenton-like reaction. Cr(V) causes DNA breaks and various muta-tions in chromosomes.Chromium on the sixth oxidation state can enter easily into the living cells because Cr(VI) ion has tetrahedral symmetry (same as SO4

2– and HPO42–, unlike Cr(III)

which has octahedral symmetry) and can enter the cell membrane through non-specific sulfate transporters by facilitated diffusion.The adverse health effects and diverse cellular and mo-lecular reactions make the studies on chromium toxi-cology and metabolism very crucial in terms of en-vironmental protection and clinical medicine. Such studies are performed using eukaryotic organisms, mainly yeast, plants, mammalian cells and transgenic mice (Cervantes et al., 2000). Among these, yeast has proved to be a very suitable model for the research of eukaryotic cell response to chromium stress and biore-mediation pathways (Ksheminska, Koloczek et al., 2001, 2005). Microbiological bioremediation of chromium contamination is performed by means of the reduction of chromate to the less toxic Cr(III) form which can be easily removed by precipitation. The mechanisms of chromate reduction and its transport to the yeast cells are the subject of the studies.The kinetic reduction of Cr(VI) presented in this study is determined in intact yeast cells and their protoplasts. The study evaluates the behavior of yeasts: Saccharomyces cerevisiae and Phaffia rhodozyma cells and their isolated protoplasts to reduce Cr(VI) to lower oxidation states. The viability of yeast cells was carried out using the meth-ylene blue staining method and the plate method (after the incubation with different concentration of Cr(VI), which varied from 0,5 mM to 10 mM).

81Eurobiotech 2013

Chromate-reducing activities are monitored by means of Cr(V) free radical form determination using the L-band (1.2 GHz) EPR (electron paramagnetic resonance) spec-trometer.Each reduction process was carried out in 120 minutes and it was measured in vivo in the EPR resonator. Both strains of yeast displayed substantial reduction of 4 mM Cr(VI). However, the cells of P.rhodozyma showed about 3.5 times higher reduction than S.cerevisiae. The Cr (VI) concentration equal to 4 mM was the sub-lethal concen-tration for S.cerevisiae while for P. rhodozyma didn’t have any negative effect on the viability. The data showed that Phaffia rhodozyma is far more resistant to higher concen-trations of chromium than Saccharomyces cerevisiae. The obtained result is confirmed by others (H.I Nechay et al., 2009).The protoplasts of the above mentioned strains were iso-lated using the method developed and optimized by the authors with the use of the Zymolyase enzyme produced by a culture of Arthrobacter luteus. Protoplasts of both strains showed couple times faster reduction of chro-mium compared to intact cells which suggests the high influence of the cell wall on uptake and/or inhibition of the reduction process of Cr(VI). Moreover, P. rhodozyma protoplasts have demonstrated greater Cr(VI) reduction than S. cerevisiae protoplasts. The observed effect of chro-mium reduction could be connected with more sufficient production of metabolites like glutathione and cysteine which also can be the factors responsible for its resistance for high concentration of toxic chromium. AcknowledgementsThis work was supported by grant of Ministry of Science and Higher Education, Poland, No. NN 304326136.

P7.3

The citricacid-modified enzyme-resistant dextrin from potato starch as potentialprebioticKatarzyna Śliżewska1, Renata Barczyńska2, Janusz Kapuśniak2

1Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, 171173 Wolczanska Street, 90-924 Lodz, Poland; 2Institute of Chemistry, Environmental Protection and Biotechnology, Jan Dlugosz University, 13/15 Armii Krajowej Avenue, 42-200 Czestochowa, Poland

The objective of the study was to determine whether dextrin obtained as a result of heating starch with citric acid (patent claim no. 392895) is a substance with pre-biotic properties. The enzyme-resistant dextrin, prepared by heating of potato starch in the presence of hydro-chloric (0.1% dsb) and citric (0.1% dsb) acid at 13°C for 3 h (CA-dextrin), was tested as the source of carbon for probiotic lactobacilli and bifidobacteria cultured with intestinal bacteria isolated from faeces of three heathy 70-year old volunteers. The dynamics of growth of bac-terial monocultures in broth containing citric acid (CA)-modified dextrin was estimated. It was also inves-tigated whether lactobacilli and bifidobacteria cultured with intestinal bacteria in the presence of resistant dex-trin would be able to dominate the intestinal isolates. Prebiotic fermentation of resistant dextrin was analyzed using prebiotic index (PI). Fermentation products were determined by HPLC. It was shown that all of the test-ed bacteria were able to grow and utilize CA-modified dextrin as a source of carbon, albeit to varying degrees. In co-cultures of intestinal and probiotic bacteria, the environment was found to be dominated by the probiot-ic strains of Bifidobacterium and Lactobacillus, which is a beneficial effect.AcknowledgementsThe study was supported by the Polish Ministry of Science and Higher Education, Grant No. N N312 3353 39.

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P7.4

Comparison of ARDRA and ARISA fingerprinting patterns analysis as a methods in early bulking activated sludge symptoms detectionDagna Sołtysik, Daria Matczyńska, Ilona Bednarek

Department of Biotechnology and Genetic Engineering, Medical University of Silesia in Katowice, Narcyzow 1, 41-200 Sosnowiec, Poland

Microbial community very often is characterized on the basis of ribosomal DNA analysis. Presence in this part of genome highly variable and conserved regions allow, not only to differentiation and classification microorganism species, but also, to observe permanent transformation of bacterial population. Appropriate balance between microorganisms from different taxonomy group is crit-ical for proper wastewater treatment process. Using fin-gerprinting pattern method gives the possibility to detect those changes and to analyze activated sludge microbial community. In this study two molecular methods were used: amplified ribosomal DNA restriction analysis (AR-DRA) and automated ribosomal intergenic spacer region analysis (ARISA) to differentiate activated sludge samples concerning microbial qualitative fluctuation between bulking and non-bulking activated sludge.Active sludge samples (collected from September 2007 to February 2009, twice per month) were derived from waste water treatment plant in Łaziska Górne. Each sample was physicochemical and microscopic analysis conducted. As a template to molecular investigation ge-nomic DNA was used. To obtain sludge’s DNA finger-prints by ARDRA technique, 16S rRNA genes fragments (amplify by use universal primers 27F and 1492R) was digested by chosen endonucleases. To receive samples genetic profile by ARISA primers complementary to conservative regions of 16S and 23S rDNA were used. Numbers and position of bands in generated genetic populations profiles (poliacrylamide electrophoresis gel for ARDRA, capillary electrophoresis results for ARISA) were comprised basis to created dendrograms using the Ward’s algorithm.As a results fingerprinting patterns for each sample were generated. Results from ARISA as well as ARDRA allow to clustered all samples into two groups. Arrangement of sludge samples in a tree diagrams were similar for both ARISA and ARDRA method (differences in distribution concerning 11 sludge samples). Most normal sludge sam-ples were grouped together suggesting alike microbial composition assurance correct activated sludge condi-tion. Detailed analysis of dendrograms (especially normal sludge samples assigned to bulking sludge samples clus-ter) and comparison obtained results with physicochem-ical and microscopic observations allow to detect subtle transformations in activated sludge bacterial population leading to bulking process. It is very important, especial-

ly in the fact, that recognition of bulking process early symptoms plays important role in prevention processes which are less expensive and easier than fully developed bulking syndrome elimination.AcknowledgementsThis project was financially supported by research grant KNW-1-110/P/2/0 and KNW-1-095/N/3/0, subsidized by Medical University of Silesia in Katowice.

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P7.5

Phytofiltration laboratory prototype of uranium-contaminated watersPaulo Favas1, 3, João Pratas2, 3

1School of Life Sciences and the Environment, University of Trás-os-Montes e Alto Douro, Portugal; 2Department of Earth Sciences, Faculty of Sciences and Technology, University of Coimbra, Portugal; 3IMAR-CMA Marine and Environmental Research Centre, University of Coimbra, Portugal

This study presents the results of a laboratory experi-ments, including a phytofiltration prototype system, to reduce the uranium (U) concentration in contaminated waters. The species Callitriche stagnalis Scop., Potamoge-ton natans L. and Potamogeton pectinatus L. of the na-tive plant community of the uraniferous region of Beiras (Central Portugal) were selected because they are autoch-thonous and they show high accumulation levels and/or high biomass production. Fluorometry was adopted for the determination of the U content in the water and plant samples using a “Fluorat-02–2M analyzer” (Lumex, Rus-sia). The installed prototype consists of a closed circuit of channels. The system was initially contaminated with 500 µg/L of U as uranyl. The performance of this sys-tem was very effective. The U concentration in the water dropped to 220 µg/L in 24 hours and after two weeks it had decreased to 72.3 µg/L, thus representing an efficien-cy of 85.5%. The U concentration in C. stagnalis increased from 0.98 to 1567 mg/kg, in P. natans increased from 3.46 to 271 mg/kg and in P. pectinatus increased from 2.63 to 1588 mg/kg. The results show the effectiveness of these plants to remove U from the water.

P7.6

Biological activity of lipopolysaccharide from cyanobacterium Synechococcus PCC 7002Agnieszka Rombel-Bryzek1, Anna Krop-Wątorek1, 2

1Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland; 2Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

Lipopolysaccharide (LPS, endotoxin) is an integral com-ponent of the outer membrane of the cell envelope of Gram-negative bacteria as well as some cyanobacteria. LPS is the major virulence factor of bacteria and a strong stimulator of innate immunity in diverse of eukaryotic species. LPS massively released by pathogenic bacteria in-fecting the organism, induces overproduction of inflam-matory mediators, leading to the systemic inflammatory response (sepsis) and potentially death. Cyanobacterial lipopolysaccharides, compared with pathogenic bacte-rial LPS such as Vibrio cholerae, Neisseria meningitidis, Yersinia pestis, Plesiomonas shigelloides, have been less studied [1, 2].The purpose of this study was to investigate biological ac-tivities of lipopolysaccharide isolated from cyanobacteri-um Synechococcus PCC 7002. LPS was tested by the chro-mogenic kinetic Limulus amebocyte lysate assay (LAL test) and by cell culture assay – the induction of proin-flammatory cytokines, tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6) by mouse macrophage cell line J774A.1. Lipopolysaccharide Synechococcus PCC 7002, activated of the clotting factors released by amebocytes Limulus and has the ability to induction of the inflamma-tory cytokines in immunocompetent cells. However LPS Synechococcus PCC 7002 was less active than this from the pathogenic Escherichia coli O55.References[1] Łukasiewicz J., Ługowski C. (2003) Post. Hig. Med. Dosw. 57: 33–53.[2] Bernardova K. et al. (2008) J. Appl. Toxicol. 28: 72–77.

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P7.7

Influence of imidazolium ionic liquids on activated sludge processDorota Gendaszewska1, Ewa Liwarska-Bizukojć1, Cedric Maton2, Chris V. Stevens2

1Technical University of Lodz, Institute of Fermentation Technology and Microbiology, Lodz, Poland; 2Ghent University, Department of Sustainable Chemistry and Technology, Ghent, Belgium

Ionic liquids (ILs) have attracted great interest in scien-tists and industrial centers during the last decade. This compounds due to their specific features could be a green alternative for organic solvents. Unfortunately, their properties like high chemical and thermal stability and solubility in water can affect on the quality and quanti-tative composition of the population of activated sludge. As a consequence, biological wastewater treatment can be difficult, not fully effective and cause a threat for aquatic environment. Therefore, it is important to carry out research on the impact of these compounds on bio-logical wastewater treatment. The aim of the study is to investigate the effect of the ILs on activated sludge pro-cess. To achieve it, the selected imidazolium liquids were tested with respect to their influence on morphology of activated sludge flocs, biomass concentration, degrees of ILs and COD removal. Four different ionic liquids were tested:1-ethyl-2H-3-methyl-4,5-dimethylimidazi-olium iodide (IL 1), 1-ethyl-2-methyl-3-methyl-4,5-di-methylimidazolium iodide (IL 2), 1-ethyl-2-isopro-pyl-3-methyl-4,5-dimethylimidazolium iodide (IL 4), l-hexyl-2H-3-methyl-4,5-dimethylimidazolium iodide (IL 6). The activated sludge was taken from the aeration chamber at the Combined Wastewater Treatment in Lodz (Poland). All experiments were conducted in the shake flasks (300 ml) under aerobic conditions. The following concentrations of ILs were applied: 1, 5, 25 and 50 mg/l. The flasks were incubated at 20±0,5°C in a thermostat-ed rotary shaker at 130 min–1 for 24 hours. For each ILs test was performed in triplicate. In addition, control tests without ionic liquid were conducted. The concentration of ILs, Chemical Oxygen Demand (COD) and volatile suspended solids (VSS) were determined at the beginning and at the end of the tests. Moreover, three independent slides from each sample were prepared and approximate-ly 40 images were taken. They were processed and anal-ysed using the automated image analysis procedure. As a result the basic morphological parameters of the flocs were measured. Ionic liquids influenced on activated sludge flocs morphology, particularly with regard to flocs size. The mean projected area of flocs for each sample was lower than the control, especially at the high initial con-centrations of ILs in wastewater (25 and 50 mg/l). The smallest flocs were found in the tests with IL 6 (50 mg/l). Also, the presence of ILs in wastewater at higher concen-trations caused to the decrease of the concentrations of activated sludge biomass. The above mentioned observa-

tions can be explained by the fact that ILs having higher number of longer substituents are more hydrophobic. As a consequence, ILs at higher concentrations can inhibit the growth of biomass and cause the formation of small flocs. Moreover, the degree of removal of ionic liquids decreased with the increase of the initial concentration of ILs in wastewater. The highest degree of removal was observed for IL 6 with the longest alkyl chain. Also, at the higher initial concentrations of ILs, the degree of COD removal decreased. This probably resulted from poor biodegradability of ILs. The obtained results shown that the influence of imidazolium ionic liquids on activated sludge depends on their concentration in wastewater and associates with their chemical structure, particularly the chain length of alkyl substituents and number of substit-uents.

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P7.8

Treatment of fibreboard wastewater by combined ozone, membrane bioreactor and activated carbon processDorota Krzemińska, Magdalena Madeła, Ewa Neczaj

Czestochowa University of Technology, Institute of Environmental Engineering, Brzeznicka 60a, 42-200 Czestochowa, Poland

The aim of this work was to estimate the influence of ozo-nation pretreatment of fibreaboard wastewater (FBM) on anaerobic treatment efficiency in membrane bioreactor (MBR). Moreover, granular activated carbons (GAC) were used as a tertiary treatment step.The influent fed into the anaerobic bioreactor was collected from the raw wastewater generated in fi-breboard company (Silesian Region, Poland). Ozone gas was generated from air using an ozone generator (CH-KTB-3G with a  power on 0,075 KW). The gas mixture of air and ozone was fed into the glass tank containing wastewater via a porous diffuser. Batch ex-periments were conducted with the use of 1 L wastewa-ter with natural pH and temperature room (20–23°C). A 2–3,5 mg/L of ozone dose in different time was ap-plied for ozonation experiments with an air flow rate of 0,18 m3/h. Anaerobic treatment experiment was con-ducted by means of 12 L capacity laboratory scale sub-merged membrane reactor with the capillary ultrafil-tration module. The reactor was filled up with granular sludge from industrial anaerobic wastewater treatment plant (UASB) from brewing industry. Experiments were performed under temperature of 37oC with gran-ular sludge concentration of 10 g/l. After acclimatiza-tion period reactor was fed with mixture of synthetic wastewater (80%, v/v) and FBM wastewater (20%, v/v). Membrane bioreactor was operated at organic loading rates (OLR) of 1.51 and 1.24  kg COD/m3·d and hy-draulic retention time (HRT) of 2,4 d. In the next step of this investigations a two types of activated carbons: WG-12 and Picabiol with different characteristic were used as a post-treatment step.The results of our investigations indicated that ozona-tion irradiation improves the treatability of fibreaboard wastewater resulting in a higher percent COD removals and biogas yield. Under the optimal condition, the in-vestigated dose of 19,5 mgO3/L/h and reaction time of 10 min, anaerobic biodegradability of FBM wastewater in MBR reactor was increased by 19%. Moreover the higher biogas production (0.75÷0.78 m3/kgCODrem.·d) was also observed under ozonation pretreatment. Therefore ozone pretreatment of fibreboard wastewater is a promising method to enhance fermentation efficiency. Moreover, the results obtained showed that GAC can be used in the removal of COD from fibreboard wastewater. The higher reduction of COD (67%) was obtained for carbon WG-12 with adsorbent dose of 6 g/l.

Keywords: fibreboard wastewater, ozonation, activated carbon, anaerobic membrane bioreactorAcknowledgementsThis work was supported by the Faculty of Environmental Protection and Engineering (Czestochowa University of Technology) BS/MN-401-315/11 and BS/PB-401-303/11.

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P7.9

Treatment of fibreboard wastewater by combined fenton’s process, membrane bioreactor and activated carbon processDorota Krzemińska, Magdalena Madeła, Ewa Neczaj

Czestochowa University of Technology, Institute of Environmental Engineering, Brzeznicka 60a, 42-200 Czestochowa, Poland

The present study was aimed to treat the fibreaboard wastewater (FBM) by Fenton’s process on anaerobic treatment efficiency in membrane bioreactor (MBR). Moreover, granular activated carbons (GAC) were used as a tertiary treatment step.The first part of this study examined the effect of operating conditions on Fenton’s process pretreatment of industri-al wastewater. The effectiveness of the AOP pretreatment was assessed by evaluating wastewater biodegradability enhancement (BOD5/COD), as well as monitoring major pollutant concentrations (COD) with reaction time. An-aerobic treatment experiment was conducted by means of 12 L capacity laboratory scale submerged membrane re-actor with the capillary ultrafiltration module. The reac-tor was filled up with granular sludge from industrial an-aerobic wastewater treatment plant (UASB) from brewing industry. Experiments were performed under tempera-ture of 37°C with granular sludge concentration of 10 g/l. After acclimatization period reactor was fed with mixture of synthetic wastewater (80%, v/v) and FBM wastewater (20%, v/v). Membrane bioreactor was operated at organic loading rates (OLR) of 1.54 and 1.25 kg COD/m3·d and hydraulic retention time (HRT) of 2,4 d.In the next step of this investigations a two types of acti-vated carbons: WG-12 and Picabiol with different charac-teristic were used as a post-treatment step. The sorption process was carried out by the static method. The main difference between those carbons is bulk density. The car-bon WG-12 has a reported density of 26,1 lb/cu ft. The carbon Picabiol is lighter and has reported density of 14,54 lb/cu ft. Taking into consideration capillary disin-tegration, it was possible to affirm, that WG-12 is typical agent of microporous carbons, however carbon Picabilo macroporous carbons.The results of our investigations indicated that the opti-mum dose Fe2+ and H2O2 was found to be 1,0 and 1,0 g/L, respectively. In a single biological treatment the average removal efficiencies of COD, and BOD were 69%, and 86%, respectively. Integration of Fenton’s process and bi-ological treatment resulted in 91% removal of COD and 96% BOD from the fibreaboard wastewater. However, the biogas production was on the same level in both process-es (0.54÷0.56 m3/kgCODrem.•d). The results indicated that the combined process would be a promising alter-native for the treatment of fibreaboard wastewater. More-over, the results obtained showed that granular activated carbons can be useful in the removal of COD from fi-breboard wastewater after MBR treatment process. It was

found that carbon WG-12 has better adsorption capacity as compared with carbon Picabiol. The higher reduction of COD (54%) was obtained for carbon WG-12 with ad-sorbent dose of 6 g/l.Keywords: fibreboard wastewater, Fenton’s process, activated carbon, anaerobic membraneAcknowledgementsThis work was supported by the Faculty of Environmental Protection and Engineering (Czestochowa University of Technology) BS/MN-401-315/11 and BS/PB-401-303/11.

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P7.10

Alga Spirogyra sp. – biosensor of surface water contamination with heavy metalsMałgorzata Rajfur1, Małgorzata Anna Jóźwiak2, Andrzej Kłos1

1Chair of Biotechnology and Molecular Biology, Opole University, kard. B. Kominka 6 St., 45-032 Opole, Poland, phone +48 77 401 60 42, fax: +48 77 401 60 50, 2Department of Environment Protection and Modelling of the Jan Kochanowski University, Swietokrzyska 15 St., 25-406 Kielce, Poland, phone +48 41 349 64 34, fax +48 41 349 64 18

Algae, due to their presence in various conditions and high resistance to physicochemical factors, are the pio-neers which inhabit new environments, and their sorp-tion properties are used in water biomonitoring. The ca-pacity of heavy metal sorption by algae used for in situ study depends on the abiotic factors, eg. water pH and the presence of other cations naturally present in water ecosystems.In order to assess the algae sorption properties and the effect of other factors on the sorption equilibria, the sorption properties od freshwater alga Spirogyra sp. were studied in the laboratory conditions. Saline solutions of the following heavy metals: Mn, Cu, Zn and Cd were used for the study. The heavy metals affinity, determined on the basis of the conduced analyses, with the thallus of alga Spirogyra sp. increases in the following sequence: Cd2+ < Mn2+ ≈ Zn2+ < Cu2+. It was reported that the sorp-tion competitiveness of the cations naturally present in the algae habitat in relation to Mn2+ ions changes in the following series: Na+ < Ca2+ < H+ determined for concen-trations referred to ion unit charge.The study results were compared with the results of the analyses carried out on dried marine algae Palmaria pal-mata.The conducted study is a part of the scientific project, the aim of which is to develop classification method of surface waters on the basis of the chemical composition analysis of algae thalli.Keywords: alga Spirogyra sp., sorption, ions, heavy metals, metals affinity seriesAcknowledgementsThe Project received financial assistance from the funds of the National Science Centre granted by force of the decision No. DEC-2011/03/D/NZ9/00051.

P7.11

Kinetics and equilibria of heavy metal sorption by aqueous plants Elodea canadensis L. and Myriophyllum spicatum L. Paweł Krems, Dominik Jerz, Małgorzata Rajfur, Andrzej Kłos

Chair of Biotechnology and Molecular Biology, Opole University, kard. B. Kominka 6 St., 45-032 Opole, Poland, phone +48 77 401 60 42, fax: +48 77 401 60 50

In the laboratory conditions, the sorption of the following ions: copper, cadmium, zinc and lead was carried out by the chosen species of aqueous plants: Elodea canadensis L. and Myriophyllum spicatum L. Sorption of the stud-ied heavy metals in solutions was conducted under static conditions and under the conditions of a dynamic solu-tion flow. The process kinetics, equilibrium parameters, the effect of pH and the manner of preparing the chosen species on the heavy metal ions sorption were studied. Process kinetics was described with the pseudo-first and pseudo-second order reaction models, while the equilib-rium was described with the Langmuir isotherm model.The affinity series of the studied heavy metals was deter-mined for Elodea canadensis L. and Myriophyllum spi-catum L. It was found out that the equilibrium between biosorbent and solution, with which it was in contact, is attained after approximately 20–30 min. It was also stated that heavy metal sorption processes restrict the presence of hydrogen cations in solution and that preparation of the studied plants has effect of their sorption properties.Keywords: heavy metals, aqueous plants, sorption kinetics and equilibrium, Langmuir isotherm

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P7.12

Heavy metal sorption by moss Pleurozium shreberi and lichen Hypogymnia physodesDominik Jerz, Małgorzata Rajfur, Andrzej Kłos

Chair of Biotechnology and Molecular Biology, Opole University, kard. B. Kominka 6 St., 45-032 Opole, Poland, phone +48 77 401 60 42, fax: +48 77 401 60 50,

The conducted study regarded the assessment of sorp-tion properties of moss Pleurozium shreberi and lichen Hypogymnia physodes in reference to heavy metal cati-ons: Mn2+, Ni2+, Cu2+, Zn2+, Cd2+ and Pb2+. The aim of the study was to confirm the thesis that moss reveals better sorption properties regardless of the type of sorbed cat-ions. The sorption process of the studied analytes was conducted under static conditions (at changing analytes concentration in solution) and under dynamic condi-tions (at constant analytes concentration in solution). To describe the equilibrium, the Langmuir isotherm model was applied. On the basis of the conducted study, it was found out that the equilibrium is attained after approximately 30 min. In the experiment conditions, 90–95% of metals are sorbed within the first 10 min. It was also stated that moss Pleurozium shreberi is characterised by better sorption properties as compared to lichen Hypogymnia physodes, and that they sorb heavy metals proportionally to their amount in the solution, in which they were immersed. On the basis of the conducted study, the affinity of the studied cations with moss and lichen sorption structures was determined. Keywords: lichen Hypogymnia physodes, moss Pleurozium shreberi, heavy metals, sorption kinetics and equilibrium, Langmuir isotherm

P7.13

Preparative scale production of immobilized lipase preparations for continuous bioconversion processesLukasz Stanczyk, Katarzyna Struszczyk-Swita, Miroslawa Szczesna-Antczak, Tadeusz Antczak

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland

Currently, bioconversion and biotransformations are important processes in pharmaceutical and chemical in-dustries. Evidence of their significance is provided by the growing tendency to replace classical chemical manufac-turing technologies by purely biotechnological processes or chemical operations coupled with bioconversion and biotransformations that are named chemo-enzymatic processes. Applications of biocatalysis are becoming fully competitive compared to chemical catalysis and consti-tute one of fundamental elements of “green chemistry”.Among approximately 4000 various enzymes which have hitherto been known only around 200 ones have been used in practice. This group has been dominated by hy-drolases including lipases.Lipases are versatile enzymes of industrial importance. They can catalyze various reactions thereby giving di-verse products that are used in many branches of indus-try: cosmetic, pharmaceutical, production of fuels, food processing etc.The potential of lipase has been only partially exploit-ed, mainly because of high costs of lipase preparations caused by their purification. New and inexpensive lipo-lytic preparations with excellent catalytic properties un-der technological conditions, high stability, catalytic ef-ficiency, and molecular and kinetic characteristics, have been still prospected for. The cheapest biocatalysts were found to be whole-cell biocatalysts.The main objective of this project is the development of a method of preparative scale production procedure of microbial lipase preparations, e.g. from lipolytic Mucor circinelloides and Mucor racemosus strains from pure culture collection at ITB TUL, in a large laboratory scale.1. Mycelium of Mucor filamentous fungi immobilized in a porous carrier in the form of uniform thin foams with open porosity and the large internal surface.2. Whole-cell preparations Mucor, dehydrated, ground (particles with a diameter close to 3 µm) and additionally stabilized.Processes of production of immobilized preparations of Mucor lipases was optimized in terms of: culture me-dium composition, method of agitation in a fermentor, type of porous carrier (pore size, shape and dimensions of the porous carrier), conditions of de-fatting and de-hy-dration of mycelium (including pH of lipase microenvi-ronment and water activity (aw) of preparation). It is also planned to develop a quick method enabling analysis of the degree of colonization of the porous material by

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filamentous fungi based on digital techniques of image analysis. One of objectives of this task is also the develop-ment of a method of long-term storage and activation of immobilized preparations of Mucor lipases.AcknowledgementsProject Nr POIG.01.03.01-00-158/09 “Biotransformations useful in pharmaceutical and cosmetic industries”.Project partially financed by the European Regional Funds within the scope of Operation Program Innovative Economy.

P7.14

Changes in dynamics of ascorbate-glutathione metabolism in response to trace metal stress in Pisum sativumAneta Piechalak1, Arleta Malecka1, Agnieszka Kutrowska1, Anetta Hanc2, Danuta Baralkiewicz2, Barbara Tomaszewska1

1Department of Biochemistry, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland; 2Department of Trace Element Analysis by Spectroscopy Method, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 90, 61-614 Poznan, Poland

Heavy metals are common pollutants in ecosystems and due to anthropogenic activities its concentration steadily increase each year. They are not biodegradable and are thus persistent in the environment. Many heavy metals (such as cadmium, lead but also high amount of copper and zinc) are toxic for plants. They can reduce photosyn-thesis, cause water imbalance, disturb nutrient accumu-lation and induce oxidative stress. The toxicity of heavy metals is generally thought to be due to inactivation of enzymes and/or functional proteins by directly binding to them or caused their oxidation by reactive oxygen species (ROS) generated in the presence of heavy met-als, such as hydrogen peroxide (H2O2), hydroxyl radicals (OH˙) and superoxide radicals (O2

–). These can react very rapidly with DNA, lipids, pigments and proteins and cause lipid peroxidation, membrane damage and inacti-vation of enzymes (Singh et al. 2006). To minimize the harmful effects of ROS, plants activate defense system in-cluding both enzymatic antioxidants and non-enzymatic antioxidants that participate in scavenging ROS (Malecka et al. 2009, 2011). Among the antioxidant defense system, ascorbate (AsA), glutathione (GSH) and related enzymes (such as ascorbate peroxidase, glutathione reductase and glutathione peroxidase) play a pivotal role in direct or indirect scavenging of ROS from plant cells (Noctor and Foyer 1998, Aravind and Prasad 2005).In order to elucidate the response of the ascorbate-gluta-thione (ASC-GSH) cycle to trace metal stress, the activ-ities of antioxidant enzymes and the levels of molecules involved in the ASC-GSH metabolism were studied in Pisum sativum L. plants subjected to copper, zinc, cad-mium or lead. Compared to the control, the contents of H2O2, ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) increased in metal-treated plants, whereas the reduce glutathione content decrease due to GSH role as antioxidant and substrate to phyto-chelatin biosynthesis. Further more, the ASC/DHA and GSH/GSSG ratios decreased in the metal ions presence. The activities of ascorbate peroxidase (APX), glutathione reductase (GR) and gamma-glutamylcysteine synthetase (ECS) were up-regulated compared to control. These data indicate that fluctuations in the ASC-GSH metabolism observed during treatment may have a positive effect on metal stress mitigation in P. sativum.AcknowledgementsThis research was supported by grant MNiSWN N310 107840.

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P7.15

Biological denitrification of wastewater: Metabolic acticity in a microbial consortiumAgnieszka Piotrowska-Cyplik1, Wojciech Juzwa2, Zuzanna Szczepaniak1, Justyna Staninska2, Jakub Czarny3, Roman Marecik2

1Institute of Food Technology of Plant Origin, Poznan University of Life Sciences, Wojska Polskiego 31, 60-624 Poznan, Poland; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Poznan, Poland; 3Institute of Forensic Genetics, al. Mickiewicza 3/4, 85-071 Bydgoszcz, Poland

The wastewater originating from explosives manufactur-ing plants are characterized by a high concentration of nitrates, sulphates and low pH. The determined compo-sition of treated wastes was: 3200 mg N L–1, 48 mg L–1 chlorides, 1470 mg L–1 sulfates. The DNC6 consortium bacterial cell samples collected during denitrification of industrial wastewater in the bioreactor were subjected to separation with the use of the FACS Aria III flow cy-tometer (Becton Dickinson, USA). Cytometric analysis of the metabolic activity, expressed as the red-ox poten-tial, allowed for the evaluation of specific single microbial cells. Separation of cells allowed for the isolation of pure monocultures, which were used for sequencing and sub-sequent genetic identification. The microorganisms pres-ent in the bacterial population used for the denitrification were identified at the first, second, fourth and sixth day of the process. The combination of flow cytometry and genetic identification allowed for determination of bac-terial species present in both areas of metabolic activity. The low metabolic activity area accounted for 32% of the total cell count, while the high metabolic activity area constituted for 19% of the total cell count. In the area of highest activity P. stutzeri accounted for 43% of total cell count, while P. putida accounted for 39%. The remain-ing 18% included freundi, P. alcaligenes and A. xylosox-idans. The composition of cells present low activity area was more diverse, with 79% accounting for four main species – O. intermedium, R. rubber, S. multivorum and S. maltophila). The highest difference in metabolic ac-tivity between these two areas was observed during the second day of the denitrification process. The microor-ganisms in the high activity area exhibited a fluorescence intensity at 12500, while the value for microbes in the low activity area was at 960.AcknowledgementsThis work was financially supported by the Ministry of Science and Higher Education, Poland (Grant No. N N523 419 837).

P7.16

Influenceof radical reactions on the kinetics of PAH biodegradation in aqueous mediaJustyna Staninska1, Zuzanna Szczepaniak2, Paweł Cyplik1

1Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Poznan, Poland; 2Institute of Food Technology of Plant Origin, Poznan University of Life Sciences, Wojska Polskiego 31, 60-624 Poznan, Poland

The industrial development and expansion of urban ag-glomerations, which have been constantly progressing since the 18th century, contribute to the accumulation of compounds from the group of polycyclic aromatic hydrocarbons (PAH) in different areas of environment and to their subsequent deposition into surface waters. The scale of the phenomenon is evidenced by numerous exceeding of standards recorded during the diagnostic monitoring of priority substances conducted under the State Environmental Monitoring. Since it was found that the prolonged effects of PAH on living organisms are highly unfavorable, there has been an increasing need to develop technologies of their effective degradation. This study aimed to determine the effect of ozonation-induced radical reactions on the biodegradation kinetics of select-ed PAH compounds by a microbial consortium isolated from soils contaminated with organic substances. The degradation was carried out under aerobic conditions at 25°C for 240 h in aqueous mineral solutions containing naphthalene, phenanthrene or pyrene at concentrations of 50 mg/dm3. The ozonation was performed before mi-crobial inoculation and lasted 60 minutes (at an efficien-cy of 2–5 g ozone/h). Measurement of PAH removal was performed using the photoluminescence method with prior hexane extraction. It was found that ozonation had no lethal effects on the microbial consortium, reflecting the lack of toxicity of radical reactions products. In addi-tion, the biodegradability of particular PAH in ozonated systems showed similarity to the reference tests. The in-crease in the number of aromatic rings was related with a decrease in the degradation rate, which was associated with a decrease in water solubility and lower bioavailabil-ity of the studied compounds. Assessment of biodegra-dation kinetics (based on the comparison of PAH degra-dation half-times) showed that ozonation increased the biodegradation efficiency by approx. 25%. These results demonstrate the possible application of combined tech-niques: ozonation and biodegradation in environment protection.

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P7.17

Different patterns of SOD generation and scavenging in barley SSD lines during droughtRenata Bączek-Kwinta1, Małgorzata Borek2, Janusz Kościelniak3, Katarzyna Żmuda4

1University of Agriculture in Crakow, Faculty of Agriculture and Economics, Plant Physiology Department; 2University of Agriculture in Crakow, Faculty of Agriculture and Economics, Plant Physiology Department; 3University of Agriculture in Crakow, Faculty of Agriculture and Economics, Plant Physiology Department; 4University of Agriculture in Crakow, Faculty of Agriculture and Economics, Plant Physiology Department

Breeding lines obtained via SSD technology provide the benefits of using 5–6 rounds of crossing-over during the breeding process [1]. The aim of the POLAPGEN project is to supply breeding companies with some tools useful in generation of new cultivars of barley resistant to water scarcity, new methods for assessing plants’ resilience, and to develop plant ideotype for drought resistance. One of the elements of such resistance is the activity of antiox-idant system [2]. Therefore, the intensity of superoxide anion generation (O2

–; histochemical method) and su-peroxide dismutase activity (SOD, spectrophotometric determination by cytochrome c) in leaves of plants of barley SSD lines subjected to drought during their seed-ling stage was studied. For the measurement and analy-ses the 3rd leaf (counting from the base) was collected. For a thorough evaluation of the results of histochemical analyses, an image analysis software (WinDIAS 3, Delta T, United Kingdom) was used.It was found that the intensity of the O2

– production var-ied widely in both the control plants and these subjected to drought. Drought caused an increased generation of O2

– in leaves of 69, a reduced in 20 lines, and 12 lines showed no changes when compared to their controls. SOD activity revealed an increase in 43, and a drop of 18 lines. The direction of changes in O2

– generation and SOD activity was consistent in 43 per 101 studied cases.While analysing the pattern of staining the tissue against O2

–, which can provide information about small injuries and/or leaf structure, in 5 cases an extremely weak O2

– generation, resulting from tissue drying, was noticed. The type of the staining was also very differentiated, from only marginal (leaf blade not dyed), mottled and/or localised along the veins, through the patchy one to the complete coverage.Protein content was genotypically differentiated, but a strong stimulating impact of drought was noticed, as in the most of the lines an increase was triggered (even 7-fold in relation to the control). In 4 cases no effect was observed, but also a drop in 10 cases, including a 3-fold in leaves of two lines, was established.The results reveal a very weak antioxidant activity of plants of some studied lines, and suggest the high met-abolic activity of these genotypes, in which both intense

generation and scavenging of O2– was accompanied by

an increase in protein level during drought. The compari-son of the results with these obtained in the other tasks of the project will improve the phenotyping of barley lines tested for antioxidant efficiency and signalling processes.AcknowledgementsProject Biotechnological tools for breeding cereals with increased resistance to drought (POLAPGEN-BD) is co-financed by European Union from European Regional Development Fund in a framework of the Innovative Economy Operational Programme 2007–2013.References[1] Surma M., Adamski T., Kaczmarek Z., Czajka S. (2003) Zmienność cech ilościowych w populacjach linii DH i SSD jęczmienia. Biuletyn IHAR 226/227/1, 277–283.[2] Bączek-Kwinta R., Borek M., Kościelniak J., Żmuda K. (2013) Does the increased SOD activity reflects drought resistance of barley? BioTechnologia 94(2), 163.

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P7.18

Influence of additional carbon sources on pentachlorophenol transformationMagdalena Kurek, Edyta Burdzik, Joanna Surmacz-Górska

Faculty of Energy and Environmental Engineering, The Silesian University of Technology, Konarskiego 18A, 44-100 Gliwice, Poland

The influence of additional carbon and energy sources (phenol and glucose) on pentachlorophenol transfor-mation by microorganisms of activated sludge, in batch reactors, was investigated. Pentachlorophenol is a high-ly toxic, cancerogenic and resistant to biodegradation compound, which has been widely used as a wood pre-servative and biocide. Obtained results showed, that the degradation of 15 mg/l of pentachlorophenol as a only carbon and energy source, with unacclimated activated sludge is inefficient. The presence of phenol, glucose or both, phenol and glucose, brought about the increase of pentachlorophenol degradation rate. The highest removal of pentachlorophenol (62%) was gained for both growth substrates (phenol and glucose) together. Furthermore, the presence of readily growth substrates, resulted in in-crease of biomass concentration, correlated with penta-chlorophenol and growth substrates degradation. When pentachlorophenol was only carbon and energy source the decrease in biomass concentration was observed.AcknowledgementsThis work was financially supported by National Science Centre (NCN), PRELUDIUM project No. 2012/05/N/ST8/03804.

P7.19

The influence of bioaugumentation and biosurfactant addition on bioremediation efficiency of diesel-oil contaminated soil: feasibility during field studiesAlicja Szulc, Daria Pęziak, Aleksandra Piotrowska, Marta Woźniak, Mateusz Sydow, Łukasz Ławniczak, Anna Parus, Łukasz Chrzanowski

Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland

The study focused on assessing the influence of bioaug-mentation and addition of rhamnolipids on diesel oil bio-degradation efficiency under environmental conditions. Initial laboratory studies (measurement of emitted CO2 and dehydrogenase activity) were carried out in order to select the consortium for biougmentation as well as to evaluate the most appropriate concentration of rham-nolipids. The selected consortium consisted of following bacterial taxa: Aeromonas sp., Alcaligenes sp., Gordo-nia sp., Pseudomonas fluorescens, Pseudomonas putida, Rhodococcus sp., Stenotrophomonas maltophilia, Xan-thomonas sp. It was established that the application of the rhamnolipids at 150 mg/kg of soil was most appropriate in terms of dehydrogenase activity. Based on the obtained results, four treatment methods were designed and tested during long-term field studies: I) natural attenuation; II) addition of biosurfactants III) bioaugmentation; IV) bio-augmentation and addition of biosurfactants. It was ob-served that bioaugmentation contributed to the highest diesel oil biodegradation efficiency, whereas the addition of rhamnolipids did not notably influence the treatment process.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.20

Contributions of rhamnolipids to natural or induced bioremediationAlicja Szulc1, Marta Woźniak1, Mateusz Sydow1, Piotr Lisiecki1, Grzegorz Framski2, Roman Marecik3, Kamila Myszka3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 3Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland

Surface-active compounds of biological origin have at-tracted much attention and their popularity seems to steadily increase during recent years. Like every other natural substance, biosurfatants have numerous advan-tages compared to their synthetic counterparts. They are equally effective as synthethic surfactants in terms of solubilization and emulsification, however they are read-ily biodegradable, less toxic and more environmentally friendly. Moreover, biosurfactants can be obtained from waste materials, which renders their production favor-able on account of economics. All these relevant traits contribute to a high applicability of biosurfactants, which currently stems to several branches of industry as well as remediation. This review summarizes the recent rev-elations in the field of biosurfactant amended bioreme-diation, focusing mainly on a critical approach towards potential limitations and causes of failure, while studying the effects of biosurfactants on the efficiency of biodegra-dation and phytoextraction processes.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.21

Biodegradation of ionic liquids by microbial consortia in aqueous mediaJoanna Wojtera-Kwiczor3, Anna Gielnik3, Łukasz Ławniczak1, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland; 3Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland

Ionic liquids are specialized chemical compounds which have gained much attention due to their broad range of properties. Since the applicability of ionic liquids is rapidly expanding, it is imperative to evaluate their in-fluence on the environment. Due to their high biologi-cal activity and potential toxicity, these compounds have been considered as major xenobiotics. In particular, the biodegradability of ionic liquids has been recognized as a topic of high environmental relevance. In order to as-sess their recalcitrance and develop a potentially efficient removal method, a set of biodegradation studies was car-ried out with the use of a defined microbial consortium which displayed a high catabolic potential towards nu-merous pollutants. The studies focused on investigation of the biodegradation efficiency of selected ionic liquids by the microbial consortium as well as on the determi-nation of the qualitative and quantitative changes in the microbial consortium during the biodegradation of ionic liquids. The following compounds were selected for bio-degradation studies: trihexyl(tetradecyl)phosphonium chloride, trihexyl(tetradecyl)phosphonium bromide, trihexyl(tetradecyl)phosphonium benzotriazolate, tri-hexyl(tetradecyl)phosphonium 3-amino-1,2,4-triazolate, trihexyl(tetradecyl)phosphonium 1,2,4-triazolate, tet-radecyl(trihexyl)phosphonium (dicyano)imide, tetra-decyl(trihexyl)phosphonium bis(2,4,4-trimethylpentyl)phosphinate, tetradecyl(trihexyl)phosphonium bis(tri-fluoromethylsulfonyl)imide, Benzalkonium chloride, benzalkonium 3-amino-1,2,4-triazolate, 1-Butyl-1-meth-ylpiperidinium bromide, 1-Butyl-1-methylpyrrolidini-um bromide, 1-dodecyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium bromide, Didecyldime-thylammonium bromide, Didecyldimethylammonium 3-amino-1,2,4-triazolate. The biodegradation rates were assessed via HPLC-MS as well as the DBAS (disulphine blue active substances) method while the quantitative and qualitative changes in bacterial community dynam-ics were determined by employing real-time quantitative PCR with the ddCt approximation method. The obtained results suggest that phosphonium-based ionic liquids were much more biodegradable, whereas the ammoni-um-based compounds were toxic, recalcitrant and con-tributed to distinguishable changes in the abundance of specific consortium members.AcknowledgementsThis work was supported by the grant number 2011/03/B/NZ9/00731 from The National Science Centre.

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P7.22

Influence of 1-alkoxymethyl-2-methyl- 5-hydroxypyridinium chloride homologues on biodegradation of diesel fuel by a microbial consortiumJakub Idkowiak2, Bogdan Wyrwas2, Agnieszka Zgoła- -Grześkowiak2, Anna Syguda1, Joanna Wojtera-Kwiczor3, Anna Gielnik 3, Łukasz Ławniczak 1, Łukasz Chrzanowski 1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland; 3Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland

Fast development of ionic liquids is gaining more and more attention. New formulations and application of ion-ic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothesize that in the presence of diesel fuel, the low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxy-pyridinium chloride homologues (side chain length from C3 to C18) on biodegradation of diesel fuel by a bacte-rial consortium was investigated. The tests performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids de-creased with increase in side-chain length, only higher homologues (C8–C18) caused a decrease in diesel fuel biodegradation. Additionally, the modification in cell surface hydrophobicity was observed as a result of ex-posure to toxic compounds (MATH). Disulphine blue active substances method was employed to determine the partitioning index of ionic liquids between the water and diesel fuel phases, which varied from 1.1 to 51% for C3 and C18 homologues, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel.AcknowledgementsThis work was supported by the grant number 2011/03/B/NZ9/00731 from The National Science Centre.

P7.23

Biodegradation of diesel/biodiesel blends in sandy soilPiotr Lisiecki1, Daria Pęziak1, Aleksandra Piotrowska1, Łukasz Ławniczak1, Anna Parus1, Grzegorz Framski2, Jacek Staniewski1, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

This study was aimed at evaluation of the biodegradation rates of aromatic and aliphatic hydrocarbon fractions in saturated sandy soil spiked with diesel/biodiesel blends (D, B10, B20, B30, B40, B50, B60, B70, B80, B90 and B100, where D is commercially available petroleum diesel fuel and B is commercial rapeseed biodiesel). The bac-terial consortium of petroleum degraders previously iso-lated from crude oil contaminated site in the Carpathian mountains was applied as a biological agent. The biodeg-radation kinetics were evaluated using measurement of the amount of CO2 evolved within 578 days. The residual aromatic and aliphatic fractions of diesel fuel were de-termined by GC-FID and GC×GC-TOF-MS techniques. Furthermore, the influence of biodiesel amendment on the community dynamics was assessed using Real-Time PCR. The obtained results suggest that there was no pref-erential degradation of aliphatic fraction of diesel fuel due to amendment with biodiesel since the ratio of aliphatic/aromatic hydrocarbons remained constant regardless of the biodiesel concentration. Our observation leads to the conclusion that biodiesel does not present many of the previously described environmental advantages.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.24

The influence of biodiesel on the biodegradation of hydrocarbons in aqueous microcosmsPiotr Lisiecki1, Marta Woźniak1, Mateusz Sydow1, Anna Parus1, Joanna Wojtera-Kwiczor2, Roman Marecik3, Kamila Myszka3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland; 3Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland

Biodiesel is considered as an attractive alternative to pe-troleum derived diesel fuel. However its’ environmen-tal impact has not been fully investigated yet. Many re-searches concerning the emission of green-house gases and exhaust emission have been conducted, but little at-tention has been paid towards the fate of biodiesel fuels in case of an environmental spill. As the production and consumption of biodiesel grows, the threat of its’ spills into the environment increases as well. In fact, biodiesel is mainly used in the form of blends with petro-diesel. Therefore simultaneous presence of fatty acids alkyl esters and petroleum hydrocarbons in the fuel contaminated site is highly probable. Although it has been confirmed by several authors that biodiesel is more readily biode-graded than petroleum diesel in general, the impact of biodiesel on the biodegradation of hydrocarbons remains unclear. Some authors suggest that the presence of a read-ily available carbon source may stimulate the growth of microorganisms and hence increase the dissipation of hydrocarbons. On the other hand, other researchers do not confirm this hypothesis. The aim of this work was to investigate the influence of biodiesel on the biodeg-radation of hydrocarbons in the water environment. For this purpose, a set of aqueous microcosms was prepared. Mixtures of model hydrocarbons – paraffins, aromatics and naphthenes – served as a sole carbon source in one half of the microcosms. The other half was supplement-ed with 20% addition of biodiesel. During the 3 weeks lasting experiment, sets of samples were sacrificed peri-odically for chromatographic determination of carbon source residues. The results of the research show that the applied bacterial consortium isolated from crude oil polluted site exhibits great abilities to decompose various chemical groups of hydrocarbons and biodiesel, although branched alkanes and naphthenes were degraded in less-er extent. On the other hand, the most readily degraded compounds were fatty acids methyl esters and straight chain alkanes, which were dissipated in a similar rate. There was no significant change of hydrocarbons biodeg-radation rate in the presence of biodiesel. This suggests that biodiesel addition to diesel fuel will not affect the biodegradation of the petroleum fraction of such fuels.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.25

Preferential degradation of Triton X-100 and polyethylene glycols during biodegradation of diesel fuelŁukasz Ławniczak1, Daria Pęziak1, Aleksandra Piotrowska1, Marta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Bogdan Wyrwas2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland

Biological methods based on biodegradation processes mediated by anionic or nonionic surfactants have recent-ly received much scientific attention. Surfactants may intensify the bioprocesses through solubilization and mobilization of water-immiscible hydrocarbons. In the concept, hydrophobic droplets are enclosed in micelles and transported to the aqueous phase. On the other hand, the negative influence of addition surfactants, especially synthetic, on biodegradation process was also frequent-ly reported. These compounds can have a toxic effect on microorganisms able to degrade petroleum compounds. It was also described that molecules of surfactants may interact with cell surface and decrease the adhesion to organic phase. Moreover, it is possible that hydrocarbon droplets entrapped in micelles are not available for bacte-ria. It is also noted, that some surfactants can be preferen-tial carbon sources for degrading bacterial species. Triton X-100 is an intensively studied nonionic surfactant with application in bioremediation processes. Therefore, the hypothesis regarding preferential biodegradation of Tri-ton X-100 applied for enhancement of microbial removal of hydrocarbons was studied. The microbial degradation of Triton X-100 as a sole carbon source as well as with co-degradation of surfactant and diesel hydrocarbons by a soil-isolated bacterial consortium was confirmed under both full and limited aeration with nitrate as an electron acceptor.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.26

Biological treatment of spent metalworking fluids by non-autochthonous bacterial consortiaŁukasz Ławniczak1, Daria Pęziak1, Aleksandra Piotrowska1, Marta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Roman Marecik2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland

Metalworking fluids (MWF) are commonly used in the metalworking industry as coolants and shear reducing media. The metalworking fluids which are currently in use are mostly oil-in-water emulsions, which contain several chemical additives (surfactants, stabilizing and anticorrosive agents, biocides etc.). Due to rapidly pro-gressing industrialization, the MWF market is constantly developing. The most popular types of MWF (emulsified oil, semi-synthetic and synthetic fluids) are usually used as 5% aqueous solutions. Constant dilution the metal-working plants contributes to the generation of immense wastewater volumes, which must be subjected to treat-ment.A fundamental shift in the interest regarding the micro-biology of metalworking fluids could be observed over the recent years. While the previous approach was mainly focused on suppressing microbial growth in operational media, presently, a need to explore the principals of colo-nization and biodegradation of waste MWF has been rec-ognized. This effect is clearly associated with the growing popularity of biological remediation methods.The results of previously conducted studies suggest that populations which inhabit metalworking fluids are char-acterized by low biodiversity and high similarity in terms of prevalent taxa, even when comparing samples of fun-damentally different origin. Additionally, several scientif-ic reports indicate, that the native microorganisms often display virulence, pathogenic traits and the tendency to resort to antagonistic interactions (i.e. secretion of toxins).With this in mind, the aim of this study was to evaluate the ability of non-autochthonic bacterial consortia origi-nating from petroleum-contaminated soil to colonize and biodegrade spent metalworking fluids. Several crucial process factors were studied (temperature, pH, carriers for cell immobilization, addition of nitrogen and phos-phorous) in order to evaluate the optimal biodegradation conditions. The biodegradation efficiency was assessed by determining the decrease of chemical and biochemi-cal oxygen demand. Additionally, the biodegradability of selected MWF components was also studied by employ-ing GC-MS and HPLC-MS. The obtained studies confirm that the use of non-autochthonic bacterial consortia may contribute to several advantages for biological treatment of metalworking fluids.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P 7.27

Bioavailability of hydrocarbons to bacterial consortia during Triton X-100-mediated biodegradation in aqueous mediaDaria Pęziak1, Aleksandra Piotrowska1, Marta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Jakub Idkowiak2, Bogdan Wyrwas2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland

The aim of our study was to investigate the effect of Tri-ton X-100 on the biodegradation efficiency of hexadec-ane and phenanthrene carried out by two bacterial con-sortia isolated from soil. It was established that the tested consortia were not able to directly uptake compounds entrapped in micelles, since biodegradation extents of both hydrocarbons were limited by the presence of Triton X-100. In micellar systems, the nonionic synthetic sur-factant was preferentially degraded (the degradation effi-ciency of Triton X-100 after 21 days was 70% of the initial concentration – 500 mg/l), followed by a lesser decompo-sition of hydrocarbon released from the micelles (30% for hexadecane and 20% for phenanthrene). In comparison, when hydrocarbons were used as the sole carbon source, 70% of hexadecane and 30% of phenanthrene were de-graded. Those results confirmed limited bioavailability of hydrocarbons entrapped in micelles. In conclusion, one can observe that Triton X-100 was the preferred carbon source compared to hydrocarbons. The degradation of the surfactant did not contribute to notable shifts in bac-terial community dynamics, as determined by Real-Time PCR. The obtained results suggest that if surfactant-sup-plementation is to be used as an integral part of a biore-mediation process, then possible bioavailability decrease due to entrapment of the contaminant into surfactant micelles should also be taken into consideration, as this phenomenon may have a negative impact on the biodeg-radation efficiency. Surfactant-induced mobilization of otherwise recalcitrant hydrocarbons may contribute to the spreading of contaminants in the environment and prevent their biodegradation.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.28

Rhamnolipids increase biodegradation of diesel oil in the presence of chlorophenolsDaria Pęziak1, Aleksandra Piotrowska1, Grzegorz Framski2, Bogdan Wyrwas3, Roman Marecik4, Kamila Myszka4, Agnieszka Piotrowska-Cyplik5, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 3Institute of Chemistry, Poznan University of Technology, Poznan, Poland; 4Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland; 5Institute of Food Technology of Plant Origin, University of Life Sciences in Poznan, Poznan, Polan

Surfactant-mediated treatment increases hydrocarbon solubilization and is widely considered as being re-sponsible for potentially facilitated biodegradation. This changes dramatically when toxic co-contaminants inhib-iting microbial activity are present in the hydrocarbon mixture. This study was aimed at assessing the effect of rhamnolipids on the performance of a bacterial consor-tium degrading diesel fuel employed as a model hydro-carbon-rich effluent, co-contaminated with toxic phenol, 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP). This approach led to the unexpected finding that rham-nolipids reduced the toxicity of 4-CP and 2,4-DCP to the hydrocarbon-degrading cells. The facts that rhamnolipids decreased diesel fuel/water partition coefficient (KFW) of both 4-CP and 2,4-DCP and modified the aggregate size distribution profiles of the dispersed diesel fuel/chlori-nated phenols solutions, suggest the existence of specific interactions between rhamnolipids and the co-contami-nants. Due to the polar nature of both 4-CP and 2,4-DCP, possible explanations involve adsorption of 4-CP and 2,4-DCP on the surface of biosurfactant aggregates. This property of rhamnolipids might potentially be of interest to all those using biosurfactants for microbial treatment of hydrocarbon-rich wastewaters co-contaminated with toxic compounds.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.29

Simultaneous degradation of phenol and alkanes by unconventional yeast strainsAleksandra Piotrowska1, Daria Pęziak1, Marta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Jakub Idkowiak2, Bogdan Wyrwas2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland

This study was focused on assessing the modification of the yeast cell surface hydrophobicity in the presence of two diametrically different carbon sources, n-alkanes (represented by a mixture of dodecane–hexadecane) and phenol. Degradation efficiency of either sole hydrocar-bons or hydrocarbons in the mixture with phenol as well as phenol itself by unconventional yeast species Candi-da maltosa, Yarrowia lipolytica and Pichia guilliermondii were investigated. Simultaneously, the formation of bio-mass was also monitored. The cell surface hydrophobicity was assessed by microbial adhesion to hydrocarbons test (MATH). Phenol degradation was assessed using GC-SPE technique, whereas hydrocarbons were extracted prior to gravimetric determination. The obtained results suggest that the hydrophobic/hydrophilic nature of the carbon source had a significant influence on the yeast cell surface hydrophobicity. The observed dependence be-tween hydrophobicity and biodegradation efficiency may lead to a conclusion, that the more hydrophobic cells are, the better the biodegradation efficiency of hydrocarbons they express. A similar relation is observed for hydrophil-ic compounds like phenol. Higher biomass formation during biodegradation of both contaminants present in a  mixture, followed by changes in hydrophobicity and possible correlation between them, attracts our attention and this phenomenon would become an interesting sub-ject of further research in the future.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.30

Biodegradation of Triton X-100 by a bacterial community isolated from activated sludgeAleksandra Piotrowska1, Daria Pęziak1, Marta Woźniak1, Mateusz Sydow1, Grzegorz Framski2, Bogdan Wyrwas3, Agnieszka Zgoła-Grześkowiak3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 3Institute of Chemistry, Poznan University of Technology, Poznan, Poland

Triton X-100 is a commonly used non-ionic surfactant which has a branched hydrocarbon chain with an aro-matic ring and a hydrophilic part consisting of ethylene oxide chain. Through the past few years it has found sev-eral industrial, commercial and household applications, such as paper manufacturing, metal processing and pro-duction of detergents. However, with increasing produc-tion of Triton X-100 the environmental contamination with this surfactant has been rising continuously. The result of primary biodegradation of this compound is the shortening of the ethoxyl chain and formation of stable metabolites, which can be toxic and capable of bioaccu-mulation in aquatic organisms. Therefore, it is important to create a  strategy focusing on biological reduction of alkylophenoletoxylates from the environment. Most of the previous biodegradation studies concerning Triton X-100 were carried out with the use of pure bacterial cul-tures. However, the aim of this study was to isolate a mi-crobial community capable of utilizing this surfactant from activated sludge biomass, employed to treat plant wastewaters of different origin. In this study the complex structure and biodiversity of the activated sludge allowed for selection of a bacterial community, which could be potentially applied as a biosorbent for treatment of waste-water with a high surfactant concentration.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.31

Biodegradation of diesel and biodiesel fuels under various aeration conditionsMateusz Sydow1, Daria Pęziak1, Aleksandra Piotrowska1, Marta Woźniak1, Piotr Lisiecki1, Łukasz Ławniczak1, Roman Marecik2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland

Biodiesel, as renewable fuel, is becoming the alternative to commonly used petroleum diesel. It has a document-ed advantage over diesel associated with a more suitable environmental behavior. Biodiesel easily undergoes aero-bic biodegradation and it was observed that its addition facilitated the utilization of petroleum diesel. Therefore, supplementation with biodiesel has been offered as the remediation technic to accelerate the biodegradation of diesel. However, the microbiological consortium capable of utilizing diesel with the addition of biodiesel is charac-terized by considerable diversity and complex structure of mutual interactions between the degrading species, which may change during the biodegradation process. The aim of this study was to investigate the degradation rates of diesel fuel, pure biodiesel and B20 blend under different aeration conditions by a seven-member bacte-rial consortium. It allowed to demonstrate the changes in bacterial community structure depending on the pres-ence of biodiesel in petroleum diesel or changes in aera-tion during biodegradation process.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.32

Biodegradation of rhamnolipids and its effect on bacterial community composition during diesel oil degradationMarta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Piotr Lisiecki1, Anna Parus1, Katarzyna Mucha2, Roman Marecik3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Faculty of Materials Science, Technology and Design, Kazimierz Pulaski University of Technology and Humanities in Radom, Radom, Poland; 3Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland

Susceptibility of rhamnolipids towards microbial degra-dation during biosurfactant-enhanced biodegradation of diesel and B20 (20% biodiesel and 80% diesel v/v) fuels was evaluated under both aerobic and anaerobic condi-tions. Rhamnolipids were supplied at 150 mg/l, which is approx. double the value of critical micelle concentration (CMC). Rhamnolipid-induced changes in communi-ty dynamics were assessed by employing real-time PCR and the ddCt method for relative quantification. The ob-tained results confirmed that rhamnolipids were readily degraded by a soil-isolated consortium of hydrocarbon degraders in all samples. Mathematical modelling of the GC results allowed us to present the following order of microbial preference regarding carbon sources: biodies-el>rhamnolipids>diesel. The presence of rhamnolipids proved to be effective only for B20 however it did not in-fluence the biodegradation rate of pure diesel. No effect associated with rhamnolipids supplementation was ob-served under anaerobic conditions. The biodegradation of rhamnolipids did not favor the growth of any specific consortium taxa, which proved that the employed biosur-factant did not interfere with the microbial equilibrium during diesel/biodiesel biodegradation.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.33

Environmental fate of selected ionic liquids introduced into terrestrial microcosmsŁukasz Chrzanowski1, Daria Pęziak1, Aleksandra Piotrowska1, Bogdan Wyrwas2, Agnieszka Zgoła-Grześkowiak2, Anna Syguda1, Joanna Wojtera-Kwiczor3, Łukasz Ławniczak1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznan, Poland; 3Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland

Ionic liquids (IL) are considered as a novel and prom-ising group of chemical compounds which exhibits vast structural diversity as well as high applicability in several branches of the industry (i.e. organic synthesis, phase transfer catalysis, electrochemistry, formation of functional polymers, textile softening and production of hygiene-related products). These peculiar chemicals are composed of an ionic pair, in which both the cation and the anion may exhibit certain properties, enabling the synthesis of highly specific agents. Ionic liquids have gained immense popularity over the past decades, most-ly due to their abundant applications and high efficien-cy, hence their high-scale production and introduction into commonly used products is becoming a fact. While there are certainly many advantages of employing ion-ic liquids for every-day use, there are also numerous environmental concerns regarding these compounds. Certain IL are characterized by a high biological activ-ity, which is why the presence of such xenobiotics may contribute to negative interactions with micro and mac-roorganisms in case of environmental contaminations. Furthermore, several types of IL are excellent pesticides and herbicides – such applications correspond to a di-rect introduction of these compounds into the environ-ment. Considering the fact, that IL exhibit surface active properties and may therefore easily penetrate the soil matrix and transport via groundwater, it should be clear that assessment of their influence of the environment is a priority.The aim of this study was to investigate the biodegradation rate of selected ionic liquids (ammonium and phosphoni-um-based IL) by autochthonous soil microorganisms and determine the qualitative changes in bacterial soil popu-lations as a response to the presence of such compounds during long-term (300 days) soil microcosm studies. Bio-degradability of ionic liquids was assessed by employing HPLC-MS and the Disulphine Blue Active Substances (DBAS) method as well as respirometric measurements (determination of emitted CO2). Total bacterial DNA was extracted from the soil prior to the experiments and upon finishing the experiments. Subsequent molecular analyzes (16s rRNA) allowed for identification of residual dominant autochthonic species, which were most likely involved in the biodegradation of the selected ionic liq-uids. Interestingly, the obtained results suggest that the

100 Eurobiotech 2013

toxicity and biodegradability of the studied compounds is associated with their structure, and that biotransforma-tion and sorption into the soil matrix play a crucial role for the fate of IL in the terrestrial environment.AcknowledgementsThis work was supported by the grant number 2011/03/B/NZ9/00731 from The National Science Centre.

P7.34

Phytotoxic effect of rhamnolipids supplementation during biodegradation of diesel oil in soilAnna Parus1, Marta Woźniak1, Mateusz Sydow1, Alicja Szulc1, Katarzyna Mucha2, Joanna Wojtera-Kwiczor3, Anna Gielnik3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Faculty of Materials Science, Technology and Design, Kazimierz Pulaski University of Technology and Humanities in Radom, Radom, Poland; 3Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland

This study aimed at assessing the influence of rhamno-lipids on the phytotoxicity of diesel oil-contaminated soil samples. Standard tests evaluating the seed germination and growth inhibition of alfalfa, sorghum, mustard and cuckooflower were carried out at rhamnolipids concen-trations ranging from 0 to 1.200 mg/kg (wet soil). Diesel oil content ranged from 0 to 25 ml/kg of wet soil. Ob-tained results confirmed that the sole presence of rham-nolipids may be phytotoxic at various levels, which is es-pecially notable for sorghum (the germination index de-creased to 41%). The supplementation of diesel oil-con-taminated soil samples with rhamnolipids contributed to a significant increase of their phytotoxicity. The most notable toxic effect was observed after a rhamnolip-ids-supplemented diesel oil biodegradation, carried out with the use of a hydrocarbon degrading bacteria con-sortium. The supplemention of rhamnolipids (600 mg/kg of wet soil) resulted in a decrease of seed germination of all studied plant species and an inhibition of microbial activity, which was measured by the 2,3,5-triphenyltetra-zolium chloride (TTC) tests. These findings indicate that the presence of rhamnolipids may considerably increase the phytotoxicity of diesel oil. Therefore, their use at high concentrations during in situ bioremediation processes should be carefully planned in a terrestrial environment.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.35

Removal of atrazine in the presence of wetland plant speciesAnna Parus1, Daria Pęziak1, Aleksandra Piotrowska1, Alicja Szulc1, Łukasz Ławniczak1, Joanna Wojtera-Kwiczor2, Roman Marecik3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poznan, Poland; 3Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland

This study focused on assessing the phytoremediation potential of wetland plants toward atrazine in an aquat-ic environment. Changes in plant biomass and atrazine content were investigated for three plant species: sweet flag, broadleaf cattail, and narrow-leaf cattail. Atrazine removal and shifts in plant biomass were assessed. Two mathematical models were built to describe atrazine tox-icity toward the studied plant species and fate of atrazine during long-term phytoremediation. Sweet flag exhib-ited the highest tolerance toward atrazine as well as the most efficient atrazine removal rate. The average atrazine half-life was significantly reduced from about 400 days to 5  days. The highest studied initial concentration of atrazine (20 mg/l) was reduced by more than 99% after 40 days.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.36

Utilisation of betaine in vinasse stillage by a mixed culture of aerobic bacteria under non-controlled pH Agnieszka Ryznar-Luty, Edmund Cibis, Małgorzata Krzywonos

Wroclaw University of Economics, Department of Bioprocess Engineering

The aim of this work was to examine how temperature and the initial pH influence the efficiency of an aerobic biodegradation of betaine in vinasse stillage. Betaine ac-counted for as much as 37.6% of total organic carbon. In the study two series of batch biodegradation processes were carried out in a stirred tank reactor by a mixed cul-ture of Bacillus bacteria. The temperatures applied were 27, 36, 45, 54 and 63°C. The pH was not controlled, and the initial pH of the culture medium amounted to 6.5 in first series and 8.0 in the second series. Efficiency of biodegradation was expressed in terms of reduction in SCODsum, which is a sum of SCOD (soluble chemical ox-ygen demand, i.e. COD determined after suspended sol-ids separation) and theoretical COD of betaine. The high biodegradation efficiency obtained in the processes was attributable to the assimilation of betaine by the bacterial strains used in the study. Maximal reduction in SCOD-sum (85.41%), BOD5 (97.91%) and TOC (86.32%) was achieved during biodegradation at 36°C and pH0 = 8.0. Under the same temperature and pH regime, betaine was removed completely, while biodegradation proceeded at the fastest rate (1.1684 g O2/(l∙h) for SCODsum removal). During biodegradation the main organic pollutants were removed at the same time, but with diverse rate of remov-al. An exception to this rule was the assimilation of beta-ine, which intensified only after the medium was lacking in easily available carbon sources.AcknowledgementsThe study was financed by the Polish Ministry of Science and Higher Education under Project No. 2P06T 045 30.The study was financed by the Polish Ministry of Science and Higher Education under Project No. 2P06T 045 30.

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P7.37

Bacterial changeability monitoring of activated sludge bacterial communities in SBR bioreactors treating reject water with PCR-DGGEAleksandra Ziembińska, Grzegorz Cema, Anna Meresta, Agata Karło, Lesław Płonka

The Silesian University of Technology, Faculty of Environmental Engineering and Energy, Environmental Biotechnology Department, Akademicka 2, 44-100 Gliwice, Poland

The microbial community and physiochemical process-es were monitored in two SBR systems differ in opera-tional parameters. PCR-DGGE of total bacterial com-munity and Anammox bacteria together with sequenc-ing of the dominant Anammox genotypes were used as a tool for bacterial biocenoses analysis. Both SBRs were treating reject water with high ammonia concentration (> 600 mg/l) for over 370 days. After this period of time the ammonia concentration decreased to 250–300 mg/l. PCR-DGGE monitoring enabled to present the biodi-versity shifts linked with community rearrangement and bacterial biocenoses qualitative change. The analysis re-vealed that the drastic decrease of ammonia concentra-tion in the influent to adapted biocenoses caused slight qualitative change in community composition, mainly in low GC part of the bacteria genotypes but biodiver-sity index slightly responded to such a change. It was also stated that bacteria which DNA was amplified with Anammox-bacteria targeted primers and underwent se-quencing were not identified as Planctomycetes probably because this group of bacteria are still not well known and Genbank still does not possess enough 16S rRNA gene sequences belonging to these microorganisms. Difference in the SBRs working scheme can be the reason that the community structure differs but with no drastic influence on its performance. AcknowledgementsThis work was supported by Ministry of Science and Higher Education. Grant number N523751740.

P 7.38

Semiquantitative ICP-MS analysis as a tool in the study of new hyperaccumulatorsKarina Krzciuk

Geochemistry and the Environment Div., Institute of Chemistry, Jan Kochanowski University, 15G Swietokrzyska St., 25-406 Kielce, Poland

The interest in the study of hyperaccumulation increas-es from year to year. Natural plant species that are able to take up and accumulate high concentrations of trace elements are called “hyperaccumulators” [1]. Hyperaccu-mulators have found their application in environmental biotechnology (phytoremediation, phytomining) [2], as well as in nanotechnology [3]. According to the recent-ly published data, about 577 plant species show hyper-accumulative properties [4]. Most of them, about 78%, are hyperaccumulators of Ni. Other known species hy-peraccumulate Cu, Co, Se, Pb, Zn, Mn, As and Cd [4]. However, there is a need to widen the scope of data for plants accumulating other elements, including anthropo-genic pollutants (Cr, Pb, B) or elements of commercial value (Cu, Au, rare earth elements). Semiquantitative mode in inductively coupled plasma mass spectrometry (ICP-MS) is a good tool for the search of new hyperac-cumulators. Semiquantitative analysis is usually used for a rapid screening before quantitative multi-element anal-ysis of unknown samples and it can be used for preselec-tion of the samples with high-concentrations of elements. This approach may help both for identification of new hy-peraccumulators and for pinpointing a larger number of elements accumulated by plants. The reliability of semi-quantitative analysis by ICP-MS is high due to its good accuracy (typically 30–50%) and reproductibility.AcknowledgementsThis work was supported by project from the program “Knowledge and Economics – a development of scientific and business competence for the growth of regional economy competitiveness”.References[1] Baker A.J.M., Brooks R.R., Terrestrial higher plants which hyperaccumulate metallic elements – A review of their distribution, ecology and phytochemistry. Biorecovery 1987; 1: 81–126.[2] Brooks R.R., Plants that hyperaccumulate heavy metals. Their role in phytoremediation, microbiology, archaeology, mineral exploration and phytomining. CAB International: Wallingford, 1998.[3] Qu J., Luo C., Cong Q., Yuan X., Carbon nanotubes and Cu–Zn nanoparticles synthesis using hyperaccumulator plants. Environmental Chemistry Letters 2012; 10: 153–158.[4] Van der Ent A., Baker A.J.M., Reeves R.D., Pollard A.J., Schat H., Hyperaccumulators of metal and metalloid trace elements: Facts and fiction. Plant Soil 2013; 362(1-2): 319–334.[5] Chen H., Dabek-Zlotorzynska E., Rasumssen P.E., Hassan N., Lanouette M., Evaluation of semiquantitative analysis mode in ICP-MS. Talanta 2008; 74: 1547–1555.

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P7.39

Supporting the process of biosorptionPaulina Olesiak, Longina Stępniak

Politechnika Częstochowska, Wydział Inżynierii Środowiska i Biotechnologii, Instytut Inżynierii Środowiska

Microorganisms are used in environmental processes from antiquity, but until recently unconsciously. Origi-nally it was believed that the presence of undesirable mi-croorganisms in the process. Only current trends – pre-vailing in the second half of the twentieth century – as a matter of microorganisms include the desired changes in the processes of substance. They allow you to speed up or initiate many decomposition or synthesis. They are used widely in many industries, but research contin-ues them what time the extensive use of the following processes. The microorganism used on a large scale are mainly bacteria in the treatment and purification of waste water and sewage. The object of this study was to present the process of biosorption as one of the methods used currently in the process of water treatment. This process brings many benefits, but for the purpose of even greater intensification apply modifications such as ozonation or sonication.The subject of the research presented in the work was to evaluate the effectiveness of the use of biological de-posits for water from organic substances. As an enabler, said process used ultrasonic field – towards a solution subjected to sorption on the activated carbon bed. For this purpose, used ultrasonic disintegrator Sonics Vibra Cell (750 W, 20 kHz). The substrate studies were humic compounds. Biological filter (in imitation of a natural colonization by microorganisms in the Reservoir Water Treatment Stations), was prepared using biopreparation, or biota sludge. The filtration was carried out in columns, with a constant flow rate. Samples of effluent were collect-ed each day. To evaluate the effectiveness of the ongoing process of the following parameters were analyzed: UV254 absorbance, the amount of dissolved organic carbon DOC, oxidizability, as well as color, turbidity and pH. In the case of biofilters also evaluated the overall number of microorganisms and the dissolved oxygen level – in order to determine the ratio S – providing the advantages of the process occurring on the bed (sorption / biodegradation).Keywords: biosorption, ultrasound, humic substancesAcknowledgementsThe study was supported by Czestochowa University of Technology internal grant BS/MN-401-321/11. The autor Paulina Olesiak received a grant for the project DoctoRIS – Scholarship program for innovative Silesia co-financed by the European Union under the European Social Fund.

P7.40

Effect of tannic acid on Acidithiobacillus ferrooxidans biofilm formed on the concreteWeronika Dec, Beata Cwalina, Paweł Stachura, Joanna Michalska

Silesian University of Technology

Rapid biodeterioration of concrete in sewage collection systems has a major impact on health and environmen-tal safety, especially in countries with warm climate. Mi-croorganisms that produce sulfuric acid accelerate the destruction of concrete sewer pipes in a process termed “Microbially Induced Concrete Corrosion” (MICC). Amongst many microorganisms participating in the ma-terials’ deterioration, the acidophilic, sulphur-oxidizing bacteria of Acidithiobacillus genus (especially of A. fer-rooxidans species) are especially important. Tannic acid (TA) belongs to tannins (a type of polyphenol) which are considered to be bacteriostatic or bactericidal agents.The aim of this work was to assess the growth and activ-ity of A. ferrooxidans biofilm formed on concrete in the presence of TA.Experiments were carried out in the laboratory. Test sam-ples of materials were placed into 6-well microtiter plates containing liquid medium 9K (of Silverman & Lund-gren), then inoculated with A. ferrooxidans. A control samples were the sterile systems without bacteria. Stock solutions of TA (1, 5, 10, 50, 100 mg l–1) were prepared in liquid medium 9K. The biocide was introduced into the system at 48 h of incubation (when mature biofilm was obtained). The plates were incubated at 28°C at agitation speeds 120 rpm under appropriate pH conditions.Biofilm quantification has been carried out using follow-ing methods:– crystal violet staining, which is one of the more expe-

dite methods to quantify biofilm biomass and may be used directly, without disruption of the biofilm;

– protein concentration assay in the medium;– XTT assay; XTT is yellow salt that is converted to dark

blue formazan in the presence of metabolic activity (re-spiratory chain in cells) without disruption of the biofilm;

– extraction of extracellular polymeric substances (EPS) from biofilms;

– scanning electron microscopy (SEM) observations.The results of investigation showed, that TA inhibits A. ferrooxidans biofilm formation. The concentration of 10 mg l–1 inhibits biofilm activity by 27% but the highest activity of biofilm was observed in the media with 1 mg l–1 (compared with control). Significant effect of TA on concentration of the extracellular polysaccharides and proteins was observed whereas this substance did not in-fluence significantly the biofilm biomass.AcknowledgementsThe work is realized with financial support from Polish National Science Center, grant No. NN523614839, using equipment purchased in the project: “BIO-FARMA Silesia. Centre for Biotechnology, Bioengineering and Bioinformatics”, co-financed by ERDF OP IG, 2007–2013. W. Dec is a scholar in the Project SWIFT (Supporting Innovative Grants Technology Forum).

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P7.41

Textile dyes synthesis by alkalic laccase from soil-dwelling fungus Rhizoctonia praticolaKamila Wlizło, Jolanta Polak, Anna Jarosz-Wilkołazka

Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19 St., 20-033 Lublin, Poland

Laccase, (EC 1.10.3.2) is multi-copper-containing en-zyme which catalyses oxidation of a wide range of sub-strates including phenolic and aromatic compounds which may be transformed to potentially textile dyes. First of all, laccase is widespread in ligninolytic fungi, especially in white -rot strains of Basidiomycota which produce extracellular laccase during the growth on me-dia about low pH value, optimal for the activity of ligni-nolytic enzymes. Nevertheless there is an exception like a soil-dwelling and plant-pathogenic fungus Rhizoctonia praticola. This species is well known because of produc-ing a large amount of extracellular laccase which is active in alkaline conditions.The aim of presented work was the screening of differ-ent organic precursors to obtain the best substrates for transformation by alkalic laccase to coloured products. The process was conducted on solid growing medium with addition one or mixture of two different phenolic compounds as dyes precursors. During the transforma-tion the dyes appearance and the growth of the mycelium were measured. As it came out not every phenolic precur-sor may be an suitable substrate for laccase from R. prati-cola. Some of the precursors could not be transformed to dyes probably because of their structure. Additionally, the efficiency of the transformation and mycelium growth were different for each precursor or their mixture. What is more interesting the process conducted only in places where the ground was covered by the mycelium.This study allowed qualify which precursors may be used in further studies of the transformation by laccase from R. praticola and define potentially what kind of structure is decisive in this process.AcknowledgementsThis work was partially supported by the National Science Centre (NN 302 633040).

P7.42

Immobilization of cellobiose dehydrogenase from Cerrena unicolor to silica beads supportsJustyna Sulej, Monika Osińska-Jaroszuk, Jerzy Rogalski

Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19 St., 20-033 Lublin, Poland

Cellobiose dehydrogenase (cellobiose:acceptor 1-ox-idoreductase, EC 1.1.99.18) is the only currently known extracellular flavocytochrome produced by various spe-cies fungi under cellulolytic culture conditions [1]. CDH comprises two distinct domains, one containing FAD and one containing b-type heme as cofactor, connected by a flexible linker region consisting a hydrophilic poly-peptide [2]. The flavoprotein domain of CDH catalyzes the oxidation of cellobiose (Glc-β-1,4-Glc) and other β-1,4-linked disaccharides or oligosaccharides at the C-1 position to the corresponding lactones [3]. The exact bio-logical role of CDH has not been fully elucidated yet, but our current knowledge points to its participation in the degradation and modification of polymers such as cel-lulose, hemicellulose, and lignin by generating hydroxyl radicals via the Fenton reaction [4].Due to the potential applications of CDH in biotech-nology, for example, detection of soluble cellodextrins as biosensors, monitoring cellulose reactions, produc-tion of lactobionic acid or bioremediation, the effective production of CDH has become an important. Current advancements in biotechnology have promoted the us-age of immobilized enzymes for a wide range of appli-cations not only in the field of biotechnology, but also in pharmaceutical, environmental, food and biosensor industries.The purpose of the present study was to isolate, purify and immobilization the novel extracellular cellobiose dehydrogenase from the basidiomycete fungus Cerrena unicolor. CDH was covalent immobilized to the silica gel and porous glass beads activated by either silaniza-tion (APTES) with immobilization yield ranging from 4 to 98 %. The activity of unbound and immobilized to silica beads supports CDH was determined. The pH-ac-tivity and temperature-activity profiles were evaluated. Affinity changes, exhibited by kinetic parameters, pH and thermal stability and operational stability were also tested.References[1] Zamocky M., Ludwig R., Peterbauer C.K., Hallberg B.M., Divne C., Nicholls D., Haltrich D. (2006) Cellobiose dehydrogenase – A flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi, Current Protein and Peptide Science, 7 (3): 255–280.[2] Stoica L., Dimcheva N., Haltrich D., Ruzgas T., Gorton L. (2005) Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes, Biosensors and Bioelectronics, 20 (10): 2010–2018.[3] Desriani, Ferri S., Sode K. (2010) Functional expression of Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain in Escherichia coli, Biotechnology letters, 32: 855–9.

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[4] Harreither W., Sygmund C., Augustin M., Narciso M., Rabinovich M.L., Gorton L., Haltrich D., Ludwig R. (2011) Catalytic properties and classification of cellobiose dehydrogenases from Ascomycetes. Appl Environ Microbiol 77: 1804–1815AcknowledgementsThis research was supported by the National Science Centre in Poland (Grant No. 2011/01/N/NZ1/03458) and the research program BS/UMCS.

P7.43

The use of Miscanthus gigantues and Phalaris arundinacea in the process of phytoremediation of soilsKarolina Rosikoń, Małgorzata Kacprzak

Czestochowa University of Technology, Institute of Environmental Engineering, Brzeznicka 60a, 42-200 Czestochowa, Poland

The introduction of vegetation in degraded areas of heavy metal effect allows their remediation, or restoration of the utility and value of nature. Degraded land as they are excluded from food crops for consumption, ideal for the cultivation of energy crops such as Miscanthus giganthus (miscanthus) and Phalaris arundinacea (reed canary grass). These plants have the ability to phytoremediation, collect and accumulate contaminantion in their tissues, mainly heavy metals. Depending on the plant species as well as the absorbed of metal, they can be accumulated in large quantities in the aerial parts or roots. In addition, both miscanthus and canary through a well developed root system can prevent soil erosion, and therefore serve as both energy and reclamation.This paper present the influence of various factors, such as plant species, type of absorbed metal, fertilizer applied and the growing season, in the process of phytoremedia-tion of Cd, Ni and Zn.Research show that after first year of vegetation Cd (0.344 mg kg–1 d.m.) and Zn (29.47 mg kg–1 d.m.) were the most absorbed metals by the biomass of miscanthus grown on soil fertilized with municipal sludge (OW). However, in the case of the reed canary grass, the cor-relation can be attributed to the Ni (3.76 mg kg–1 d.m.) and Zn (122.6 mg kg–1 d.m.). In turn, after the second year of vegetation all of the tested metals are character-ized by a lower absorbtion as compared to the first year. In the case of reed canary grass Cd and Zn content in the biomass after the second year of vegetation was similar to the control, while the Ni concentration is was lower in relation to the control. In the case of miscanthus, it was observed that the concentration of Ni and Zn in the bio-mass after the second year of vegetation was similar to the control, while the Cd content varied in dependence on the applied fertilizer.On soils with high metal concentration of both reed canary grass and miscanthus are able to collect metals, which can result in quality biomass (unfavorable from the point of view of energy).AcknowledgementsThe study was supported by Czestochowa University of Technology internal grant BS/MN–401–317/11.The author Karolina Rosikoń received a grant for the project DoktoRIS – Scholarship program for innovative Silesia co-financed by the European Union under the European Social Fund.

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P7.44

Identification of lipid derivatives in HepG2 cellsJoanna Gdula-Argasinska1, Aneta Garbacik2, Malgorzata Tyszka-Czochara1, Michal Wozniakiewicz2

1Jagiellonian University Medical College Faculty of Pharmacy Department of Radioligands; 2Jagiellonian University Faculty of Chemistry Laboratory for Forensic Chemistry

Polyunsaturated fatty acids (PUFA) are highly susceptible to oxidative stress produced hydroperoxide species. Be-sides non-enzymatic lipid peroxidation, enzyme-induced peroxidation, cyclooxygenases and lipoxygenases is a process that leads to hydroperoxides specifically trans-formed into a series of more stable metabolites – eicosa-noids or docosanoids from C18 to C22 PUFA.The aim of our study was to assessed the effect of PUFA supplementation with added of PAHs on the HepG2 cells. Our study describes a rapid and simple technique for the analysis of PGF2a, PGF3a from cultured cells using UH-PLC/MS-TOF. This method permits detection of selected individual prostaglandins derived either from arachidon-ic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts.AcknowledgementsThis project was possible through the support given by National Science Center, Poland to the author Joanna Gdula-Argasinska DEC-2011/01/B/NZ7/00038.

P7.45

Removal heavy metals from wastewaters by yeast biomassSławomir Wierzba, Adam Latała

Opole University, Chair of Biotechnology and Molecular Biology

Conventional methods for removing metals from aque-ous solutions include chemical precipitation, chemical oxidation or reduction, ion exchange, filtration, electro-chemical treatment, reverse osmosis, membrane tech-nologies, and evaporation recovery. These processes may be ineffective or extremely expensive;especially when the metals in solution are in the range of 1–100 mg/l, the production of toxic chemical sludge and its disposal/treatment becomes a costly affair and is not ecofriendly. Therefore, removal of toxic heavy metals to an environ-mentally safe level in a costeffective and environment friendly manner was of great importance. The use of bio-sorbents with microbial origin especially bacteria, algae, fungi and yeasts is a considerable alternative to the exist-ing methods because of their good performance, low cost and large availability. This work presents some results on the use of Saccharomyces cerevisiae cells biomass for removal of lead, zinc, nickel, cadmium and copper ions from contaminated industrial wastewater. Yeast biomass was immobilized in the process of flocculation by using calcium chloride and hydrogen peroxide. Process con-ditions: pH 5.0, temperature 20°C, the concentration of biomass, 1.0 g/l, contact time 30 minutes of flocculation, aeration and continuous operation. The initial content of metal ions ranged from 2.2 mg/l (cadmium) to 83.1 mg/l (lead). Analysis of microbiological and physicochemical industrial wastewaters showed high efficiency the meth-ods immobilization of insoluble aggregates of cells with calcium chloride and hydrogen peroxide in removing heavy metals. Reduction of concentration of Pb(II) was 98.0%, Cd (II) – 96.0%, Cu (II) – 95.9%, Zn (II) – 95.2% and Ni (II) – 92.7%. The results indicate the possibility of using the aggregation of cells in flocs during flocculation in wastewater treatment from heavy metal ions.

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P7.46

Bacillus spp. and their metabolites present ecofriendly alternative to synthetic chemicals for plant growth enhancement in many different applications. Extensive research has demonstrated that these microorganisms and their metabolites could have an important role in agriculture and horticulture in improving crop productivityKatarzyna Grata, Małgorzata Nabrdalik

Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland

Bacillus spp. and their metabolites present ecofriendly alternative to synthetic chemicals for plant growth en-hancement in many different applications. Extensive re-search has demonstrated that these microorganisms and their metabolites could have an important role in agri-culture and horticulture in improving crop productivity. The aim of the research was to assess a potential biologi-cal activity of cell-free supernatants (CFS) obtained from 4, 6, 8, 10 and 24-hour culture of Bacillus subtilis against 4 pathogenic strains of Rhizoctonia solani marked as R1, R2, R3 and R4. The bacterium was isolated from soil fer-tilized with urea phosphate while R. solani strains were isolated from field cultivation of sugar beet. The antago-nistic properties of bacterial egzometabolites were deter-mined by the dual-culture technique on Czapek-Dox me-dium, where the carbon source was sucrose. Plates were incubated at 26°C for 4–6 days and the fungal growth was measured every day and compared to control. The influ-ence of metabolites produced by B. subtilis on the growth of R. solani was evaluate on the basis the growth rate in-dex .On the basis of obtained results, it has been proved that fungistatic activity of B. subtilis is varied and depends ob the age of the bacterial culture and susceptibility of the fungus. Taking into consideration all the analysed param-eters, R. solani R1 and R4 were the most sensitive but R3 lest sensitive to the metabolites produced by B. subtilis. The highest decrease, amounting 84%, in the value of the growth rate index of R. solani R4 was obtained for 10-hour culture of B. subtilis. The growth rate index of R. solani R1 and R3 was also strongly inhibited (amount-ed 82%), when were applied 6-hour and 4-hour culture of B. subtilis, respectively. Moreover, on the basis of ob-tained results, it shows that the most favourable is to ap-ply young 8-10-hour bacterial cultures to inhibit the lin-ear growth of R. solani. B. subtilis may find a wide range of application, in the process of plant protection against diseases caused by R. solani.

P7.47

Traditional methods of plant protection employing chemical substances may be replaced with a biological alternative based on non-pathogenic microorganisms. Investigations into properties of such microorganisms in order to find the most suitable bacterial strains brought the attention to the activity of Pseudomonas fluorescensMałgorzata Nabrdalik, Katarzyna Grata

Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland

Traditional methods of plant protection employing chemical substances may be replaced with a biological alternative based on non-pathogenic microorganisms. Investigations into properties of such microorganisms in order to find the most suitable bacterial strains brought the attention to the activity of Pseudomonas fluorescens.The aim of conducted research was to determine the influence of metabolites of Pseudomonas fluorescens on the growth of 4 pathogenic strains of Rhizoctonia solani marked as R1, R2, R3 and R4 infesting sugar beet. The antagonistic properties of metabolites were assessed after 4, 6, 8, 10 and 24 hours of culturing of Pseudomonas flu-orescens with a culture-plate method on PDA medium. The bacterial strains were cultured at 25°C for 4–7 days. Fungistatic activity of Pseudomonas fluorescens was de-termined on the rate of mycelial growth inhibition.Obtained results have proved that the strains of Rhizoc-tonia spp. under study were both sensitive and resistant to Pseudomonas fluorescens metabolites. The highest inhibition of the linear growth of fungi was noted for Rhizoctonia solani R1 and R4. In both cases the highest inhibition of the growth rate, amounting almost 60%, was obtained after 4 hours of culturing and the lowest, amounting around 30%, was noted after 24 hours of cul-turing. Whereas the following strains of Rhizoctonia so-lani R2 and R3 were resistant to metabolites produced by Pseudomonas fluorescens regardless of the culturing time length. Conducted research confirmed fungistatic properties of Pseudomonas fluorescens strains against Rhizoctonia sola-ni strains. However, due to the fact that differences in the mycelial growth inhibition have been recorded among strains of Rhizoctonia solani it allows to assume that the sensitivity to metabolites is characteristic for individual strains. The tests also showed that growth inhibition of the mycelium depends not only on the type of metabo-lites produced by a specific bacterial strain but also on the length of culturing.

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P7.48

Isolation of biosurfactant producing strains from environmental samplesMarta Woźniak1, Kamila Myszka2, Mateusz Sydow1, Alicja Szulc1, Łukasz Ławniczak1, Aleksandra Piotrowska1, Daria Pęziak1, Anna Parus1, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland

In this study we carried out research on aerobic bacteria selected from cow, chicken and pig feces as well as from manure. According to the literature reports, the main iso-lation sources of microorganisms capable of biodegrad-ing petroleum hydrocarbons are oil-contaminated soil, water tanks or sediments. Both livestock feces and ma-nure are a rich source of gram-negative bacteria, which have a high potential to biosynthesize biosurfactants. In the last few decades, the ability to employ biosurfactants in bioremediation processes of petroleum contamination has attracted a lot of scientific attention. Therefore, from this point of view, the research has shown a pioneering approach to the subject of isolation of microorganisms capable of growth on hydrocarbons from uncommon re-search materials.Using 4 screening techniques, we selected a bacterial con-sortium consisting of three strains: Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli. The current literature reports concerning the bioremediation and the role of biosurfactants in these processes has focused pri-marily on the use of monocultures, occasionally mixed cultures. The present study underlines the impact of bac-terial consortia, which increase the efficiency of biodegra-dation in comparison with monocultures. Pseudomonas aeruginosa isolated from the tested consortium had the ability to secrete compounds that reduced surface tension. Both the consortium, as well as the isolated Pseudomonas aeruginosa strain, were capable of growth on hydrocarbons as hydrophobic carbon sources. The intensity of secretion of surface active compounds by Pseudomonas aeruginosa species can be correlated with the respective sequence of hydrocarbons metabolization. The influence of strains in bacterial consortium caused stimulation of Pseudomonas aeruginosa strain to an increased biosurfactants secretion during cultivation on less available carbon sources. In ad-dition, some diesel components inhibited secretion of sur-face-active agents by the consortium and stimulated their synthesis by the Pseudomonas aeruginosa strain. During cultivation in the bioreactor, the consortium delayed the release of surface active compounds by the Pseudomonas aeruginosa strain. Most likely, it was a result of preliminary competition among the microorganisms for nutrients in the new environment. Ultimately, the consortium stimu-lated Pseudomonas aeruginosa in terms of more efficient production of surface-active agents. AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.49

Substrate preferences in an environmental microbial consortium degrading diesel oilMateusz Sydow1, Daria Pęziak1, Aleksandra Piotrowska1, Marta Woźniak1, Łukasz Ławniczak1, Anna Parus1, Roman Marecik2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland

The microbial populations inhabiting soil or aquatic en-vironments are composed of diverse, synergistic or antag-onistic communities. The presence of multiple bacterial species in an environmental community allows for an efficient removal of a wide range of compounds. Large-scale pollution due to man-made chemical substances is the current global concern. From the viewpoint of man and his impact on the environment, the most import-ant form of pollution are oil spills. Diesel oil is still the world’s first volume-produced fuel. The composition of this mixture comes close to 3,000 different hydrocarbons. Among them are aliphatic, cycloaliphatic and aromatic compounds. The straight-chain alkanes fraction is the easiest to decompose by bacteria, whereas cyclic and aro-matic compounds are degraded over a relatively long pe-riod of time. Moreover, if the pollution consists of many chemical compounds, as in the case of diesel oil, gradual changes in mixture composition are observed.Relatively little is known regarding the determinants of microbial population dynamics in soil contaminated with complex hydrocarbon mixtures such as diesel oil or petroleum. The ability to metabolize oil is displayed by many different microorganisms and some of them are more versatile than others. Many bacterial species prefer specific carbon sources over complex mixtures of com-pounds. Other bacteria are capable of using many carbon sources.The aim of this study was to evaluate quantitative and qualitative changes within the composition of a micro-bial consortium resulting from selective cultivation on the chosen diesel oil fractions and to assess the impact of such changes on the biodegradation efficiency of com-mercial diesel oil. During the research, specific substrate preferences as well as quantitative and qualitative chang-es within the microbial consortium were determined by using relative quantitative real-time PCR method. More-over, all mineralization efficiencies of diversified consor-tia were compared to utilization capacity of the initial bacterial community with the use of mathematical mod-eling methods. The obtained results demonstrate that cul-tivation on specific carbon sources did not significantly affect diesel oil biodegradation efficiency in soil.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

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P7.50

Biodegradation of diesel/biodiesel blends in urban soilŁukasz Chrzanowski1, Piotr Lisiecki1, Grzegorz Framski2, Anna Syguda1, Roman Marecik3, Kamila Myszka3, Jacek Staniewski1, Łukasz Ławniczak1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 3Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, Poznan, Poland

Biodiesel has gained a lot of scientific attention over the last few years. It is commonly believed that the addition of biodiesel to diesel fuel may potentially enhance the biodegradation of specific hydrocarbon fractions present in diesel. Except for assumptions that fatty acid methyl esters are very similar to alkanes and this similarity would accelerate degradation of the aliphatic fraction of diesel, no hard proof has been published so far. Thus, the aim of our study was to evaluate the long-term biodegradation rate of both aromatic and aliphatic hydrocarbon frac-tions in urban soil spiked with diesel/biodiesel blends. Additionally, we tried to evaluate the effectiveness of bioaugmentation with a previously isolated (non-autoch-thonous) bacterial consortium of high affinity towards hydrocarbons. GCxGC-TOF-MS studies of residual hy-drocarbons as well as respirometric studies were exten-sively applied during this project. The obtained results suggest that all diesel/biodiesel blends were degraded in the extent of > 95% within 450 days. Moreover, no effect of the externally added bacterial consortium was noticed. The most prominent observation is that biodiesel did not affect the biodegradation rate of aromatic and aliphatic fractions.AcknowledgementsThis work was supported by the grant number 32-400/2013 DS-MK.

P7.51

Identification and phylogenetic analysis of Rhizopus spp. based on 28S rRNA geneAdam Kuzdraliński, Sylwia Kowalczyk, Elwira Komoń, Agnieszka Glibowska, Jakub Wyrostek, Zdzisław Targoński

University of Life Sciences in Lublin, Poland

Background. Many species of genus Rhizopus are very important due to their biotechnological relevance. These fungi are becoming increasingly important for biotech industry due to their ability of production of a wide spectrum of metabolites such as ethanol and lactic and fumaric acids. The correct identification and phylogeny of fungi is very important in plant pathology, biotechnol-ogy, clinical settings and environmental studies.Objective. We aimed to establish the molecular phyloge-ny of the Rhizopus oryzae, R. stolonifer and R. microspo-rus belonging to the genus Rhizopus, based on the se-quencing of the large subunit gene (28S) and to compare the results with other classification studies of the genus. We have also established a PCR-based method for screen-ing this fragment of the large subunit gene.Methods. Isolates were obtained from the Leibniz Insti-tute DSMZ-German Collection of Microorganisms and Cell Cultures, Agricultural Research Service Culture Col-lection, Biological Resource Center Culture Collection and from southeastern Poland from different substrates such as cereals, bread, soil, water and apples. We devel-oped a direct DNA extraction method, which offers high efficiency of DNA isolation from fungi. We designed PCR primers by comparing Rhizopus species sequences pub-lished in the NCBI GenBank database. They were intend-ed to be specific for genus Rhizopus and to flanking the most polymorphic region of 28s rRNA gene. The amplifi-cation products were extracted and purified from agarose gel. The sequencing of both DNA strands was performed and DNA sequences were edited and aligned. All fungal DNA sequences were initially aligned using the ClustalW. The molecular diversity and its variance were estimated. Consensus sequences were constructed for each species. Phylogenetic analyses of the aligned sequences were per-formed with PAUP 4.0 b10.Results. A partial sequence of 28s rRNA gene has been amplified by PCR using our primers in all isolates belong-ing to the genus Rhizopus. The amplified region of the 28S rRNA gene included 5 haplotypes. Analysed sequenc-es of R. oryzae represented 2 haplotypes, 2 haplotypes of R. stolonifer, and 1 haplotype in case of R. microsporus. R. stolonifer sensu lato has proven to be most intraspe-cies variable fungus. Species of R. oryzae, R. delemar and Amylomyces rouxii showed high similarity, as expected. R. oryzae had the highest number of parsimony-informa-tive and variable sites but only if we divided R. stolonifer sensu lato into separate clades accordingly to previous intraspecies alignment. Comparative alignment of this clade might also suggest that these isolates belonged to

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one of the two varieties of R. stolonifer, i.e. lyococcus or reflexus. Based on the alignment data of partial consen-sus sequences of 28s rRNA gene, the phylogeny analysis was carried out separately for all species and for both clades of R. stolonifer. Occurrence of separate clades in R. stolonifer group revealed that genetic polymorphism of R. stolonifer sensu lato is caused by the presence of genetically distant varieties. Genetic variation of both R. stolonifer clades is lower in comparison to R. oryzae and R. microsporus species. All clades except for R. sto-lonifer (with R. stolonifer var. lyococcus and reflexus) ap-peared monophyletic.Conclusions. We postulate a division of R. stolonifer var. stolonifer and R. stolonifer var. lyococcus and reflexus into separated species. Phylogeny analysis of 28s rRNA gene of R. oryzae, R. delemar and A. rouxii showed lim-ited potential of discrimination. Domesticated species of R. oryzae and R. microsporus formed a polyphyletic clus-ters.AcknowledgementsThis study was prepared within the framework of the project PO IG 01.01.02-00-074/09, co-funded by The European Union from The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007–2013.

P7.52

Metal cross-talk in Indian mustard results in induced uptake of Cd and Pb when supplemented with ZnAgnieszka Kutrowska1, Aneta Piechalak1, Arleta Małecka1, Anetta Hańć2, Danuta Barałkiewicz2, Barbara Tomaszewska1

1Department of Biochemistry, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland; 2Department of Trace Element Analysis by Spectroscopy Method, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 90, 61-614 Poznan, Poland

Trace metal toxicity towards plants varies with each metal considered. Comparison of two essential (zinc and copper) and two non-essential elements (lead and cadmium) results in following generalized sequence of metal toxicity Cd > Pb > Cu > Zn (Singh 2008). Detri-mental influence of metals depends on the plant spe-cies, nature of an element and correlates with metal bioavailability in soil solution (affected by pH, organic matter and cation exchange capacity of the soil). For instance, phytoextraction of lead is difficult due to its low bioavailability and solubility (Jadia and Fulekar 2009). However, trace metals in soil solution are never present as separate ions. The mixture of different el-ements in varying concentrations stimulate cross-talk both before and after uptake by plant. Resultant effect can comprise of decrease in metal toxicity and concen-tration in planta, e.g. inhibited uptake after competi-tion for metal transporters or, conversely, increased import due to stimulation of transporters (Rascio and Navari-Izzo 2011). Therefore, metal cross-talk could be used for improvement of phytoextraction, if its ef-fect led to increased metal transport to aboveground plant tissues.In order to assess interactions between four trace met-als: Zn, Cu, Cd, Pb, we have measured metal uptake and distribution in seedlings of Indian mustard (Brassica juncea), a known hyperaccumulator. Plants were grown hydroponically and subjected to trace elements in fol-lowing concentrations: 50 μM of Zn2+, Cu2+, Cd2+ or Pb2+ of each, or mixtures of two metal ions (variants ZnCd, ZnCu, ZnPb, CuCd, CuPb, CdPb) with 25 μM of each metal. With use of two methods, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Laser Abla-tion Mass Spectrometry (LA-MS), we have determined i.a. that in variants ZnCd and ZnPb concentration of Cd and Pb were significantly higher than in seedlings treated with single metals, respectively. Our results suggest that presence of zinc ions could improve efficiency of phytore-mediation. Additionally, because Zn is an essential metal, often exogenously supplied to increase crop productivi-ty, its use in phytoextraction of Cd and Pb in controlled quantities should not pose additional threat to the envi-ronment.AcknowledgementsThis research was supported by grant MNiSW N N305 381138.

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References[1] Jadia C.D., Fulekar M.H., Phytoremediation of heavy metals: Recent techniques. African Journal of Biotechnology, 2009, 8(6): 921–928.[2] Rascio N., Navari-Izzo F., Heavy metal hyperaccumulating plants: How and why do they do it? And what makes them so interesting? Plant Science, 2011, 180: 169–181.[3] Singh V.P., Metal Toxicity and Tolerance in Plants and Animals. Sarup & Son, 2008.

P7.53

High density polyethylene as a carbon source for Achromobacter xylosoxidans Anna Kowalczyk1, Marek Chyc2, Przemysław Ryszka3, Kazimierz Strzałka1, Dariusz Latowski1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland; 2Silesian Environmental Doctoral Study, Plac Gwarkow 1, 40-166 Katowice, Poland; 3Institute of Environmental Sciences, Faculty of Biology and Earth Sciences, Gronostajowa 7, 30-387 Crakow, Poland

Disposal of waste is one of the most important current issues of environmental protection. Particularly burden-some waste are polymers, including high density poly-ethylene (HDPE), strongly resistant to degradation. Used HDPE is disposed to landfill, often burned, which leads to contamination of the air with toxic compound, such as polycyclic unsaturated hydrocarbons. Less frequently the HDPE is recycled, which is still costly method of uti-lization.The present study deals with degradation of the chemical structure of HDPE with the use of microorganisms, as it is resistant to degradation in environment.The biological material was isolated from the soil, and the substance to be degraded was HDPE in the form of foil. Disinfected with ethanol foil samples were subjected to the action of microorganisms suspended in a liquid Davis Minimal Broth medium for 6 weeks. The chemical composition of the medium was modified. There were 2 assays carried simultaneously. First of them contained: Davis Minimal Broth medium with 30% of recommend-ed content of glucose to enhance the growth of micro-organisms, foil samples and previously isolated micro-organisms. The second one contained: Davis Minimal Broth medium without glucose, foil samples and micro-organisms, so that the only carbon source for microor-ganisms was polyethylene. Part of foil samples in both assays were irradiated with UV light, to accelerate HDPE degradation. Control assays included foil (also samples irradiated) suspended in proper medium with no mi-croorganisms. The populations of microorganisms were controlled by measurement of OD650 every second day throughout the experiment. After 6 weeks the foil sam-ples were analyzed with Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) method and the results revealed degradation of chemical structure of HDPE polyethylene in the microorganisms supplemented media. FTIR research showed formation of carbonyl groups in structure of HDPE, which prob-ably induces formation of another groups, such as hy-droxyl or ether groups and also skeletal vibrations. The study also demonstrated, that in assays with glucose addition microorganisms growth is improved and the degradation of foil samples is more effective. There was showed no correlation between irradiating with UV and the extent and HDPE degradation.

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The microorganisms were identified by analysis of the 16S ribosome subunit coding sequences. They were di-agnosed as Achromobacter xylosoxidans. This is the first communication showing that these bacteria can degrade HDPE, although it was documented that some chemical substances like benzoate, bisphenol A, ethylbenzene or p-nitrophenol could be metabolized by these microor-ganisms.

P7.54

The influence of vermicomposting on heavy metals contained in sewage sludgeHanine Suleiman1, Agnieszka Rorat2, Barbara Płytycz3, Małgorzata Kacprzak2, Franck Vandenbulcke1

1Univ Lille Nord de France, LGCgE, EA 4515, Univ Lille 1, EcoNum-Ecotox, F-59655 Villeneuve d’Ascq, France; 2Institute of Environmental Engineering, Czestochowa University of Technology, Czestochowa, Poland; 3Institute of Zoology, Jagiellonian University, Crakow, Poland

Vermicomposting is a biotechnological method used to convert organic wastes to fertilizer using earthworms. Sewage sludge produced in wastewater treatment plants is a rich waste in organic matter. Sewage sludge may be valued in agriculture as fertilizer but its heavy metal con-tent presents a constraint.The aim of this study was to evaluate the changes in sew-age sludge (its composition and mainly its heavy metals content) after vermicomposting. Two earthworm’s spe-cies were used (Eisenia fetida and Eisenia andre) sepa-rately and in mixture. Three sewage sludges differ mainly in their heavy metals’ content were used.  Each sewage sludge was mixed with added material (commercial com-post) by a ratio (1:3). The process was observed after three time periods (3, 6 and 9 weeks) by analyzing of heavy metals as a main stress factor and of its total carbon, to-tal nitrogen and total phosphorus content to evaluate its value as fertilizer.

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P7.55

The effect of phosphate starvation and mycorrhizae on the gene expression of phosphate transporters in greenhouse tomatoIwona Kowalska1, Marzena Wińska-Krysiak2, Anna Konieczny1

1Department of Soil Cultivation and Fertilization of Horticultural Plants, Faculty of Horticulture, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland; 2Laboratory of Basic Research in Horticulture, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland

The symbiosis of arbuscular mycorrhizal fungi (AMF) allows for the water and mineral supply of a plant to be enhanced, as well as creates increased toleration for heavy metals, salt, water and thermal stress. In soilless cultiva-tion, including the hydroponics, an increase in the effec-tiveness of nutrient and water uptake by the roots system developed in the limited area is especially important.Current studies suggest that in this kind of cultivation there is a diversified level of the colonization of roots by AMF as a result of both the presence of easily accessible forms of P in the culture, and of weak aeration of the root environment. The hypothesis of the experiment assumes that through the colonization of AMF on the tomato roots, it is possible to decrease the concentration of P in the nutrient solution while maintaining a similar feeding status in accordance with the recommended levels.An increased P uptake by the plant as a result of mycor-rhizis will enhance the gene expression of P transporters in a plant. Above hypothesis was verified in a tomato cultivation on two different kinds of medium, since the development of AMF may depend on the physical and biological characteristics of environments, such as air access.Tomato plants cv. ‘Admiro F1’ were grown hydroponical-ly in cultivation rows located in the foil tunnel, in 2012 year. The experimental factors were the concentration of P in the nutrient solution (15 or 50 mg dm–3), type of substrate (rockwool or coconut fiber) and root inocula-tion by AMF. AMF colonization and gene expression of P transporters (LePT1-LePT5) were determined in two periods (I – fruit setting on the 6rd cluster, II – the end of cultivation). Plant material was lyophilized and RNA was isolated by the method of Gasic et al. (2004). RT-PCR was performed with primers designed for amplifications of genes LePT1-LePT5 and 18S rRNA in tomato (Balestri-ni et al. 2007). Synthesis of cDNA and RT-PCR reactions were carried out using a Reverse Transcription System Kit (Promega, USA).Inspection of microscopic preparations of the roots re-vealed the presence of mycorrhiza in the roots of inocu-lated plants, as well as the influence of the substrate type and level of P in the medium on the presence of AMF. Expression of LePT depended mainly on the concentra-tion of P in the medium, the presence or absence of AMF,

stage of growth of tomato plant as well as the type of sub-strate.The level of transcripts was strongest for LePT4 and the weakest for LePT1. Gene expression LePT2 and LePT4 had a similar profile. These genes were strongly expressed in plants growing with AMF, regardless of other factors studied. Gene expression LePT3 in plants growing on coconut fiber and rockwool after application of 15 mg P dm–3 in the medium and AMF was higher than in the plants growing on the same medium, but without the AMF. A similar expression profile was obtained for the LePT5 gene.AcknowledgementsThe research studies were financed by the National Science Centre within the Project No. N N310 725040.References[1] Balestrini et al. (2007) Mol Plant Microbe Interaction 9: 1055–1062.[2] Gasic et al. (2004) Plant Mol. Biol. Report 22: 437a–437g.

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P7.56

Effect of composting proces on the changes of some organic compoundsMarcin Milczarek, Ewa Neczaj, Dorota Kusal

Czestochowa University of Technology, Institute of Environmental Engineering, Brzeznicka 60a, 42-200 Czestochowa, Poland

Sewage sludge is a product of wastewater treatment pro-cesses; nowadays, about 8 million tones are produced each year in the EU member states. A preferred method of sewage sludges utilization is its application in soil fer-tilization and reclamation. The above has an unfavorable impact on the environment, it opens the gates to environ-ment pollution by organic and inorganic compounds and pathogens. Sewage sludge pollution with polycyclic aro-matic hydrocarbons is common. Their content in sewage sludge can range across very wide borders. The standards valid in the European Union forbid the usage of sewage sludge with the sum content of 11 PAHs exceeding 6 mg kg–1. This article presents results of many authors, who composted organic wastes such as sewage ludge, green wastes, organic fraction of municipal solid wastes. Studies have shown (Semple et al., 2001; Antizar-Ladislao et al., 2004) that composting of soils polluted with PAH results in a significant reduction of PAH content. Some recent studies (Lazzari et al., 2000; Amir et al., 2005; Oleszczuk, 2006) showed also that sewage sludge composting can significantly reduce PAH content in this material. The above issue should be considered not only in the case of sewage sludges in which valid norms are exceeded but also in the case of sludges and organic fraction of munic-ipal solid waste in which the content level of these com-pounds is increased. Such an approach will allow for the limiting of PAH accumulation in fertilised soil and, at the same time, influence the improvement of sewage sludge fertilising properties. Microbial degradation represents the major route suitable for the ecological recovery of PAH-contaminated sites, with better results obtained for the degradation of low molecular weight PAHs (2–4 rings). The refractory nature of some PAHs is related to their limited water solubility, to their diverse and com-plex structure and to the requirement of providing ox-ygen to start degradation. Environmental conditions which can affect the biodegradation rate are: high tem-perature which increases the rate of enzymatic reactions and water solubility as well, good availability of nutrients, high moisture level, good oxygenation, presence of alter-native substrates required for fulfilling co-oxidation reac-tions, increased microbial variety and pH near neutrality. It seems that composting process is very good to remove some organic compounds from wastes or change them to less toxic form.AcknowledgementsThis article was financed by the project’s own Minister of Science and Higher Education BS/MN-401-313/11.

P7.57

Isolation and characterization of plant growth-promoting rhizobacteria (PGPR) from isolated from Arabidopsis thaliana (L.) Heynh. and Morus alba L. grown on heavy metals rich acid soiAnna Grobelak, Anna Napora, Anna Grosser, Małgorzata Kacprzak

Institute of Environmental Engineering, Czestochowa University of Technology, Brzeznicka 60a, Czestochowa 42-200, Poland

Plant growth-promoting rhizobacteria (PGPR) are nat-urally occurring soil bacteria and benefit plants by pro-viding growth promotion. This study was designed to isolate and characterize PGPR bacteria from Arabidopsis thaliana (L.) Heynh. and Morus alba L. (white mulber-ry). Plants were grown under extreme soil contamina-tion with heavy metals (Zn 980 mg/kg, Pb 1400 mg/kg, Cd 16 mg/kg) near zinc smelter area in Silesia region. A large number of bacteria were isolated from roots with three time levels of surface sterilization (0, 2, 10 min.) using ethanol. For enrichment and isolation of root-as-sociated bacteria the following media were used: Congo Red agar and nitrogen-free base (NFb) media to isolate a free-living diazotrophic bacteria, Luria agar (LA) to iso-late nutritionally demanding bacteria, and yeast extract mannitol agar (YEMA) to isolate Rhizobiaceae bacteria. Isolates were grown until exponential growth phase to evaluate the atmospheric nitrogen fixation, phosphate solubilization, siderophores, indole-3-acetic acid produc-tion, as well as antifungal, protease, and 1-aminocyclo-propane-1-carboxylate (ACC) deaminase activity. A total number of 26 for A. thaliana and 25 for mulberry isolates were obtained from the enrichment cultures and tested. All bacteria were able to grow and to produce IAA, and the highest concentrations were found for bacteria isolat-ed from mulberry plants. Only few of them were able to produce siderophores, and solubilize phosphate. ACC de-aminase activity was positive solely for three strains. The growth of Alternaria alternta, Fusarium oxysosporum and F. culmorum was inhibited over 50% for eight bac-teria strains. To determine and confirm the plant growth promoting potential, some bacteria strains were chosen for plant inoculation in the growth chamber experiment.AcknowledgementsThe project was supported by National Science Centre grant UMO-2011/ 03/N/ NZ9/ 02034.

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P7.58

Biogenic catalysis in sulphide minerals weathering processes and acid mine drainage genesisMaria Kusnierova1, Maria Prascakova1, Anna K. Nowak2, Zbigniew Wzorek2

1Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, SK-04353 Kosice, Slovak Republic; 2Institute of Chemistry and Inorganic Technology, Crakow University of Technology, Warszawska 24, 31-155 Crakow, Poland

From a great group of environmental processes partic-ipating on the natural material circle is possible to use mainly the processes of bioleaching and biogenesis in raw materials processing. The bio-oxidation reactions are the main basis for bioleaching procedures often parallel participating in the leaching processes. During leaching processes of the polycomponent sulphide substrates also the factor of processes selection plays an important role being in a direct relation to the electric properties and galvanic effect occurring between the individual compo-nents of the leaching substrate.This work gives summary of results from research fo-cused on possibilities of using biotechnological proce-dures for Slovak sulphides ores treatment. The object of the research is an extraction of valuable metals, unde-sirable admixtures and degradation of crystal lattice of sulphides for subsequent chemical leaching processing of precious metals.Further, the results of experiments on existence of bio-genic processes in situ on waste dumps from exploitation containing residual sulphides are presented. Outcome of this processes is acid mine drainage waters generation. These waters are strongly mineralized and of low pH that’s why they are very aggressive. The heavy and toxic metals contents as well as Cu, Zn, Fe, As, Cd etc. in out flowed waters from old mines loadings are high over the lawful limits.AcknowledgementsThis work was supported by the Operational Programme Research and Development through the project: Centre of Excellence for Integrated Research of the Earth’s Geosphere (ITMS: 26220120064), which is co-financed through the European Regional Development Fund, SGA project No. VEGA-2/0086/10 and within the SRDA project No. APVV-0252-10.

P7.59

Isolation of saponins from plants and their influence on hydrocarbon bioremediationZuzanna Szczepaniak1, Justyna Staninska2, Andrzej Olszanowski3

1Institute of Food Technology of Plant Origin, Poznan University of Life Sciences, Poznan, Poland; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland; 3Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Poland

A very dynamic development of the industry in recent years is the major cause of water and soil pollution. The most common sources of contamination are hydrocar-bons, especially oil derivatives. Increasing environmental public awareness has led to development of many reme-diation methods – technologies aimed at removing con-tamination from soil and groundwater. Apart from tradi-tional, physicochemical techniques, there is a big interest in biological methods that lead to degradation of hazard-ous compounds carrying out by plants (fitoremediation), or microorganisms (bioremediation). Unfortunately, low solubility of hydrophobic compounds in water results in very limited bioavailability of these substances for the mi-croorganisms, and thus significantly reduces the biodeg-radation process. In addition, the high molecular mass of toxic compounds, such as polycyclicaromatic hydrocar-bons (PAH) leads to even lower solubility. The aim of this study was to isolate saponins – natural surfactants occur in plants - from soup nut shells and ivy leaves, as well as the characteristics of the obtained extracts and their ef-fect on diesel oil biodegradation efficiency. Moreover the study was aimed at determination of bacterial cell surface properties, such as zeta potential and cell hydrophobici-ty. It allowed to demonstrate that saponins isolated from plants are not a toxic factor. On the contrary, they are an easily assimilable carbon source. It has been proven, that addition of saponins in most cases results in a higher efficiency of diesel oil biodegradationa. Among all test-ed concentrations of saponins (120, 240, 360, 400 mg/l) in most cases, the lowest concentration of saponins (120 mg/l) did not significantly affect the efficiency of oil biodegradation, whereas the concentration of 240 mg/l turned out optimal. This is probably not related to the change hydrophobicity cells, since no strict relationship between the degree of bacterial adhesion to hydrocar-bon, and the efficiency of hydrocarbon biodegradation has been proven. These results indicate the possible ap-plication of saponins as natural surfactants isolated from plants in biodegradation process.

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P7.60

Biodegradation of soot components by soil bacteria Barbara Kalicka1, Marek Chyc2, Monika Bojko1, Kazimierz Strzałka1, Dariusz Latowski1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland; 2Silesian Environmental Doctoral Study, Plac Gwarkow 1, 40-166 Katowice, Poland

Reducing the level of soot released into the atmosphere should be one of the most important challenges aimed at reducing emissions of air pollutants. It has been sug-gested that the soot particles in the air contribute to the enhanced global warming effect to a degree comparable to the greenhouse gases such as methane, halocarbons, volataile organic compounds (VOCs) and tropospheric ozone. In this study were compared biodegradation of two types of soot: unmodified (obtained only from burn-ing hard coal in a central heating boiler) and soot SAS (obtained from burning coal with an adequate amount of new marketed fuel additive labelled SAS (an acronym for Safety And Strong). SAS is an innovative and effective fuel additive, obtained mainly from a waste material, based on iron oxide and inorganic salts. This invention reduces the emmision of particulate matter, CO, CH4 and unburned VOCs exhaled with the flue gases into the atmosphere. SAS maintains the surface of the fireside heat exchanger free of soot thereby increasing the thermal efficiency of the heating system.6.00% w/v solutions of soot in basic salt medium (BSM) where soot was the only carbon source were autoclaved and inoculated with one of two types of bacteria: Pseu-domonas aeruginosa or Bacillus subtilis. Results obtained after 4 weeks of incubation of soot samples (30°C, RPM 150) exposed to bacteria were compared to the samples stored under the same conditions but without the par-ticipation of microorganisms by static headspace gas chromatography with mass spectrometry detection (HS-GC-MS) of the soot residue. Biodegradation was concluded by analysing sum of 4 aromatic compounds (phenol, naphthalene, phenanthrene, anthracene). The highest level of biodegradation (about 80%) was obtained for soot SAS samples with Pseudomonas cultures. From the results it can be concluded that soot obtained from carbon modification with SAS fuel additive will be more quickly removed from the environment. Unmodified soot, SAS and four other types of soot were also tested for microbial content. The microorganisms were present in all analysed kinds of soot. This suggests that soot can be treated as source of microorganisms potentially useful in biodegradation of soot components. All analysed types of soot were non-toxic for Pseudomonas and Bacillus strains applied in our studies, even with high contents of soot in medium.

P7.61

Biodiesel-derived crude glycerol as a substrate for fumaric acid production by fungus of the genus RhizopusElwira Komoń, Jakub Wyrostek, Adam Kuzdraliński, Zdzisław Targoński

University of Life Sciences in Lublin

Crude glycerol is the waste byproduct of biodiesel pro-duction process and its conversion mainly to organic acids brings significant economic benefits. Fungus of the genus Rhizopus are well known producers of key me-tabolites such as lactic acid, fumaric acid and ethanol as co-products. Most of researches was focused on simple carbon sources like glucose or fructose to study meta-bolic profiles of this fungus. Previous cultures that were conducted with supplementation of pure glycerol showed minimal or no consumption of this carbon source by in-vestigated fungi, which affected on low fumaric acid pro-duction. Crude glycerol contains lots of inorganic salts and MONG (matter organic non glycerol) which can be used for enhancing biomass growth and modify metabol-ic profiles. In this study ability to utilization of crude glycerol by six strains of Rhizopus oryzae was studied. Preselected cul-tures showed that optimal amount of crude glycerol in fermentation medium is 30 g/L. Each of selected fungus were grown in shaking flask culture in triplicate for 168 h. To avoid excessive acidification of cultures sodium car-bonate as a neutralization factor was added to reach 2% as the final concentration. After HPLC analysis of broth samples the glycerol consumption rate was observed to reach 0,16 g/L/h. Best result of final amount of fumaric acid was observed in R oryzae ATCC 20344 culture, that concentration reached 15 g/L. Some of studied fungus showed weak biomass growth in crude glycerol supple-mented medium. HLPC analysis of those samples proved that neither significant utilization of propanetriol nor fu-maric acid production was occurred.AcknowledgementsThis study was prepared within the framework of the project PO IG 01.01.02-00-074/09, co-funded by The European Union from The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007–2013.

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P7.62

Effectiveness of eco-friendly, natural products in in vitro rooting of Prunus domestica L. microshootsAlina Wiszniewska, Barbara Nowak, Anna Kołton

Department of Botany and Plant Physiology, Faculty of Horticulture, University of Agriculture in Crakow

Development of novel, effective chemical preparations – biostimulators, that could enhance plant production, plays an important role in sustainable agriculture. Recent studies confirm that natural biopreparations may induce plant defense reactions and stimulate development of plant organs, with limited influence on the environment (Pacholczak et al. 2012). Biotechnological approach al-lows to test the effectiveness of biostimulators that are composed of environmentally friendly, natural prod-ucts, such as algae extracts, plant extracts and cell exu-dates. Therefore the aim of our study was to evaluate the root-promoting activity of two natural products, i.e. pine-apple pulp and conditioned medium containing green algae exudates, during in vitro rooting of plum (Prunus domestica L.) microshoots. The long term purpose of the research is to limit the level of synthetic growth regula-tors exploited during rooting phase in micropropagation of this fruit tree.In vitro shoots of two Polish plum varieties, ‘Węgierka Zwykła’ and ‘Węgierka Dąbrowicka’, were used as plant material. Root induction was conducted for 7 days on fourculture media: 1) control medium WPM (Lloyd and McCown 1981) containing 5 mg/l IAA and 2 mg/l IBA (according to Nowak and Miczyński 1996); 2) WPM medium containing 10 ml/l of dialyzed pineapple pulp (low-molecular weight fraction Dialyzate and condi-tioned medium were produced and kindly provided by Professor Z. Tukaj and Dr K. Grabski, from the Depart-ment of Plant Physiology, University of Gdańsk, Poland.After one-week long induction, shoots were transferred to hormone-free MS medium (Murashige and Skoog 1962). Root formation was observed for 4–6 weeks and effectiveness of rooting was evaluated on the basis of bio-metric measurements.The study also aimed at determination of optimal time needed for rooting induction on tested medium variants. Peroxidase activity and phenolic profile were determined using 5-mm long shoot bases. Peroxidase activity was an-alyzed during 7 days, starting from 1. day (24 h) after cul-ture establishment. Phenolic profile was determined after 7 days of induction phase.Results revealed that rooting ability in plum depends strongly on the genotype and applied organic medi-um supplements. In ‘Węgierka Dąbrowicka’ the highest rooting percentage was obtained on medium containing dialyzate of pineapple pulp. Microshoots of ‘Węgierka Zwykła’ rooted decidedly less effectively,on every tested medium. Moreover, the results of physiological analyses

suggested that rooting protocol for ‘Węgierka Zwykła’ could be modified in order to achieve higher percentage of rooted shoots. Details concerning obtained results will be presented and discussed during poster session.AcknowledgementsThe study was financed by Polish Ministry of Science and Higher Education.References[1] Lloyd G., McCown B. (1981) Commercially-feasible micropropaga-tion of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Comb Proc Intl Plant Prop Soc 30, 421–437.[2] Murashige T., Skoog F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15, 473–497.[3] Nowak B., Miczyński K. (1996) Regeneration capacity of Prunus domestica L. cv. Węgierka Zwykła from leaf explants of in vitro shoots using TDZ. Folia Hort 8/2, 41–49.[4] Pacholczak A., Szydło W., Jacygrad E., Federowicz M. (2012) Effect of auxins and the biostimulator Algaminoplant on rhizogenesis in stem cuttings of two dogwood cultivars (Cornus alba ‘Aurea’ and ‘Ellegantissima’). Acta Sci Pol, Hortorum Cultus 11(2), 93–103.

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P7.63

Monoaromatic hydrocarbon degradation by selected bacterial strainPrzemysław Petryszak, Paweł Kaszycki

Departament of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

Dynamic industrial expansion leads to overproduction of various toxic and environmental nuisance compounds. The anthropogenic contamination with aliphatic and ar-omatic hydrocarbons is caused by a variety of petroleum processing technologies and results mostly from improp-er use, transport and storage of diesel fuel and oil-derived substances. This leads to the release of huge amounts of toxic xenobiotics such as low- and high–molecular n–al-kanes as well as aromatic hydrocarbons, especially those belonging to the BTEX group (benzene, toluene, ethyl-benzene, xylene), naphthalene and many other chemical derivatives. Since the above pollutants tend to negatively affect the natural – soil and water – environments, the development of effective and relatively cheap decontami-nation methods is necessary.In search for optimized bioremediation strategies, bac-terial decomposition of aromatic hydrocarbons utilized as carbon and energy sources was studied. The possibil-ity and conditions for effective biodegradation of BTEX substances (applied as single xenobiotics or in a mixture) were evaluated. Various strains of the bacterial culture collection of Biochemistry Department, earlier isolat-ed from hydrocarbon-contaminated soil and belonging mainly to the genus Pseudomonas, were tested. The se-lected strains showed high level of tolerance for xenobi-otic action, which was expressed either as a growth inhi-bition-rate index, or as an effectiveness of 2,3,5-triphen-yltetrazolium chloride reduction. Bacterial biodegrada-tion efficiency was proved by gas chromatography anal-yses of monoaromatic hydrocarbon content in culture media. Biodegradation kinetics of the most recalcitrant xenobiotics were presented. The possibility of adaptation of the selected strains to toxic environmental conditions, i.e. to sub-lethal concentrations of monoaromatics, was shown employing qualitative and quantitative analyses of fatty acid methyl esters. SDS-PAGE electrophoretic pro-files obtained upon biodegradation processes were com-pared to reveal protein changes as induced by the pres-ence of xenobiotics.AcknowledgementsThe work was financially supported by the University of Agriculture Research Grant for Young Scientists no. BM/2012/4519 and Grant for Scientific Research no. 3500, approved by the Polish Ministry of Science and Higher Education.

P7.64

The improvement of fumaric acid production trough coutilization of glycerol and pectin by Rhizopus oryzae ATCC 20344Jakub Wyrostek, Elwira Komoń, Adam Kuzdraliński, Zdzisław Targoński

University of Life Sciences in Lublin

Fumaric acid is commercially important element of the components of food, medicines and industrial materials, it is used for food acidification and the production of syn-thetic resins. Rhizopus oryzae is a zygomycete filamen-tous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus able to overproduce fumaric acid, lactic acid and ethanol.The effects of substrats and their mixtures on the fumaric acid production by Rhizopus oryzae ATCC 20344 were studied, with the aim of improving the efficiency of fu-maric acid production. The amount of fumaric acid pro-duced on mixtures of the carbon sources glycerol and pectin was different from the produced on each of those substrates separately. The fumaric acid amount was ob-served to reach 1,96 and 1,2 g/L for glycerol and pectin supplemented medium, respectively. After cofermenta-tion in different variants of glycerol-pectin medium with pectin after enzymatic hydrolysis and with non-hydro-lyzed pectin the amount was 4,2 and 19,6 g/L. The present study aimed to put forward a theory for glycerol/pectin cofermentation.AcknowledgementsThis study was prepared within the framework of the project PO IG 01.01.02-00-074/09, cofunded by The European Union from The European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007–2013.

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P7.65

Biostimulation of xenobiotic-degrading bacterial consortia with oxygen-releasing compoPaweł Kaszycki, Paula Banaś, Przemysław Petryszak

Departament of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Craków

Bioremediation is a powerful method to deal with envi-ronmental pollution. Particularly toxic and recalcitrant xenobiotics can be biodegraded employing microbial communities that reveal specialized metabolic path-ways. Biochemical processes leading to the reduction of total load of chemical pollutants such as oily products are very complex. In general, the maximum contami-nant removal efficiency is obtained at aerobic condi-tions where oxygen serves as the final electron acceptor in biooxidation reactions. Since water and soil are main targets for hydrocarbon discharge by industrial activi-ties, oxygen level often becomes a limiting factor ham-pering biodegradation activity of autochthonous and/or allochthonous microorganisms. To overcome the bot-tle-neck effect of oxygen consumption some methods of soil bioremediation suggest microflora biostimulation with oxygen-releasing compounds. Wastewaters can be treated using consortia aerated with air-compressors, however this approach may not applicable for in-situ cleanup projects.In this study a microbial consortium ZB-01 was tested for its susceptibility for aerobic metabolism stimula-tion with two variant oxygen-supplementing commer-cial products, differing in the mechanism and intensi-ty of oxygen release. The ZB-01 is a highly specialized biocenosis constructed at Biochemistry Department of University of Agriculture in Crakow. It consists of a number of strains able to biodegrade aliphatic and ar-omatic hydrocarbons, both in water and soil environ-ments. The two sources of oxygen were: an EHC-OTM preparation (Adventus Americas Inc.), applied predom-inantly for reclamation of soil environment, and oxygen tablets, OxyTABS (JBL GmbH & Co. KG), used mainly to maintain proper aeration for live in small ponds and fish tanks. For both substances, optimized concentra-tions were established as determined by bacterial sur-vivability experiments. The preparations showed a stim-ulating influence on microorganisms’ metabolism mea-sured with triphenyltetrazolium chloride (TTC) used to reveal dehydrogenase activity. Also, bacterial frequency determinations proved proliferation of microbial cells under oxygen supplementation conditions. Oxygen lev-el was monitored as a DO (dissolved oxygen) parameter. Based on the comparative tests the best effect was found for EHC-OTM applied at 0.1%.It is concluded that the use of oxygen-releasing com-pounds to provide aerobic conditions in aqueous media appear favorable in terms of both, short-time bacterial

metabolism stimulation prior to application at contam-inated sites, and optimization of long-term consortia storage.AcknowledgementsThe work was financially supported by the grant for scientific research No. 3500, approved by the Polish Ministry of Science and Higher Education.

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P7.66

Analysis of physico-chemical properties of porous carriers for bacteria immobilizationDagmara Leśniak, Paulina Worsztynowicz

Poznan University of Life Science, Department of Food Biotechnology and Microbiology

Introduction. Using crude glycerol from a biodiesel pro-duction process for producing 1,3-PD is a good solution from the economical as well as ecological point of view. Biotechnological production of 1,3-PD from waste bio-mass is a promising and attractive alternative to the tra-ditional chemical synthesis. The productivity of 1,3-PD can be improved through the application of fermentation with cell immobilization on porous carriers in packed bed reactor (PBR).AIM. The aim of this study was to investigate the influ-ence of physico-chemical properties of porous carriers used in PBR to immobilize Clostridium butyricum cells.Materials and methods. An analysis of the surface prop-erties was made using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). SEM is a type of electron microscope that produces im-ages of a sample by scanning it with a focused beam of electrons used for analysis of sample’s surface topography and composition. EDS is an analytical technique used for the elemental analysis or chemical characterization of a  sample. All samples were cleaned of any organic resi-dues (300oC for 3h), cut to appropriate size, and mounted on a specimen holder for viewing in the SEM. Results. Using the signal of secondary electrons (acceler-ating voltages of 5–15 kV), image of: pumice, expanded clay (LCA), Sera Siporax Mini, Eheim Substrat Pro and coral gravel were made. Images allowed for assessment of topography and porosity of each carrier, which are re-lated to the adsorption of bacterial cells to the surface. EDS analysis have shown that most common element was silicon (Si).Conclusions. The area of porous carriers available for the adhesion of microorganisms, affect the amount of immo-bilized biomass, which in turn affects the efficiency of the bioconversion process. Impact on the bacterial adhesion efficiency has a content of such elements as magnesium and calcium, and any scratches and surface irregularities.AcknowledgementsThis work was funded within the framework of project No. 01.01.02-00-074/09 co-funded by The EU from the European Regional Development Fund within the framework of the Innovative Economy Operational Programme 2007–2013.

P7.67

The cultivation of endophytic methanotrophs isolated from different species Sphagnum as perspective for environmental bioengineeringSesja: Environmental BiotechnologyZofia Stępniewska, Agnieszka Kuźniar

The John Paul II Catholic University of Lublin, Department of Biochemistry and Environmental Chemistry, Konstantynow 1I St., 20-708 Lublin, Poland

Enriched cultures of microorganisms are essential step in the production of inoculum of these organisms for bio-technology and bioengineering. A potential application of methanotrophs is widely studied as microorganisms for the removal of methane produced from landfills, coal mines as well as biodegradation of toxic compounds. Therefore, searching for new sources of methanotrophs can contribute to increase the possibilities of biotechnol-ogy and bioengineering.Enrichment cultures of endophytic methanotrophs were initiated in NMS medium as most widely used medium for cultivation of methanotrophic bacteria from various environments is that proposed in 1970 by Whittenbury. Incubation was carried out at 10, 20, 30 and 37°C with vigorous shaking on a shaker (180 rpm). The source of carbon and energy for endophytes were methane at the concentrations range between 1–20%. The medium NMS were inoculated homogenized Sphagnum sp. materials that originated from Moszne peatbog (the Poleski Na-tional Park, Eastern Poland). It have been demonstrated that in natural conditions exist methanotrophic bacteria in cooperation with other microorganisms and their in pure cultures are unstable for extended periods of time (Hoefman et al., 2010). Therefore, in these studies we in-vestigated consortium of whole microorganisms commu-nities. During the incubation, growth of the cultures and dynamics of gases were controlled, respectively by the op-tical density measurements at the wave length of 600 nm and concentration of gases in headspaces with the use of gas chromatograph (SIMADZU, GC 2010).It appeared that the consortium of endophytic bacteria grew only at 20 and 30oC temperature which is charac-teristic for mesophilic methanotrophs. We determined that the length of phase adaptation of endophytes was ranged from 5 to 12 days. During these time the bacte-ria adapted to the new conditions in the environment. Then, there was rapid growth of endophytic population (phase logarithmic) that it was reflected by an increase of optical density in the range from 0.3 to 2.0, depend-ing on methane concentration (1–20%). After phase logarithmic, it have been observed the lack of carbon/energy sources and/or increase of waste products con-centration to the level of harm for methanotrophic con-sortia. These time was called as a stationary phase of enrichment cultures.

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During the culture endophytes, the measurements of gases concentration showed a steady loss of methane and oxygen, as well as the accumulation of carbon dioxide as a CH4 oxidation product.The use of FISH has made possible characterization of endophytic consortia. I turned out that the population of endophytic consist with type I and II methanotrophs as well as associated non methanotrophic bacteria.Furthermore, we determined the potential activity of ex-amined bacteria for methane oxidation that it was ranged up to 4,7 µMCH4 per ml population of endophytes and day.The interest in methanotrophs has largely been due to their biotechnological potential for the production, in particularly single cell protein, methanol, osmopro-tectants (for example ectoine) vitamins and other bio-technological products.AcknowledgementsThis work was supported by the National Science Centre grant in Poland (No. 2011/01/N/NZ9/06811).

P7.68

The physiological response of perennial grasses grown on heavy-metal polluted soilSesja:Krystyna Rybka1, Grzegorz Żurek1, Marta Pogrzeba2, Jacek Krzyżak2, Kamil Prokopiuk1

1Plant Breeding and Acclimatization Institute IHAR-PIB, Radzikow, 05-870 Blonie, Poland; 2Phytoremediation Team, Institute for Ecology of Industrial Areas, 40-844 Katowice, Poland

Phytoremediation requires plants that maintain good fitness on contaminated soils in parallel with the highest possible concentration of pollutants in aerial parts. For evaluation of species and varieties as potential phytore-mediators in breeding programs, low-cost, easy to carry and high throughput techniques are required. Chloro-phyll a (Chl a) fluorescence measurements fulfill those conditions and in parallel with detection of heavy metal (HM) ions concentration represent a good screening tool for evaluation of perennial grasses suitability to grow on lands of long history of industrial emission.Our experiment was located on contaminated agricultur-al soil of Silesia, southern Poland. Plots were established in the vicinity of a closed-down cadmium/lead/zinc/ ore mine and processing plant, which operated for more than 100 years and had significant impact on local soils. Refer-ence site (uncontaminated soil) was located in a distance of 25 km North from above, on agricultural land with similar forage crop history. HM ions amount in polluted soil exceeded by almost 2 orders of magnitude the con-centration of Cd and Zn ions and about 50 times of Pb ions when compared to reference place.To detect the effect of HM soil contamination on selected perennial grass cultivars: plant height, generative shoots abundance, biomass yield as well as total Pb, Cd and Zn ions concentration were measured in parallel with Chl a fluorescence transient. The HM ions negatively influ-enced fluorescence in all listed points: F0 (minimal), FM (maximal), FV (variable), F1–F5 (at times [ms]: 0.05, 0.15, 0.30, 2.0, 30) as well as total complementary area (Area) of Chl a fluorescence induction curve and forward elec-tron transport measured as (1-VJ)/(VJ)). Tall wheat grass cv. Bamar and tall oat grass cv. Wiwena were character-ized by unreduced yield, whereas rescuegrass cv. Broma. and smooth bromegrass cv. Brudzyńska significantly re-duced the biomass yield as well as F0, FV and FM. The high-est concentration of Cd and Zn ions in the biomass of cv. Rahela was not associated with yield reduction.All parameters detected during fluorescence measure-ment could be used for plant physiological state descrip-tion, but sometimes it is difficult to highlight those best suiting for data interpretation. One of the method of pa-rameter number reduction is PCA. Grouping of cultivars calculated on bases of 2 principal components, which ex-plained 85% of differentiation, gave logical results. Culti-vars were grouped in sub-classes as “polluted” and “refer-

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ence’”. Cultivars Bamar and Wiwena were characterized by positive correlation with component 2. Both principal components: 1st and 2nd, have all sub-components posi-tively correlated, which means that if increasing values were recorded, the position of scatter would move to-wards higher values on axes. The other statistical method, presented by Goltsev et al. (2012) is neutral network.To compare the dynamics of electron transfer chain in chloroplasts Strasser et al (2004) proposed double nor-malized curves enabling specification of the best stress tolerating cultivar (Gururani et al. 2012; Bussotti et al. 2011; Oukaroum et al. 2007). The usage of such an ap-proach pointed out cv. Rahela as outstanding cultivar under the HM ion stress. Contrary to expectation, cv. Ra-hela which sequestrated the highest concentration of HM ions in the aerial part, did not show PSII antennas group-ing under the stress, as other cultivars did. All cultivars except cv. Rahela accelerated metabolism, which is a typi-cal physiological response characteristic for plants slight-ly resistant or non-resistant to the stress. Resistant plants typically launch additional defense mechanisms and re-build its metabolism (Foyer et al. 2012, Zagdańska 1995). Such cultivars, which survive the stress by accelerating photosynthesis, as well as the whole metabolism, could be less resistant in front of additional environmental stresses such as i.e. drought or cold and could react more rapidly on them. Cultivar Rahela in fact deals the best with the stress. Its antennas were not damaged, might be due to redox guard in the form of ascorbate and proline which in other cultivars seemed not to be induced strongly enough to be reflected on duble normalized graphs.AcknowledgementsThe work was financed by the Ministry of Agriculture and Rural Development, as PW3-3-00-0-03. KR and GŻ are considered to be the first author.ReferencesGoltsev et al. (2012) Biochim Bioph Acta 1817: 1490–1498.Strasser et al. (2004) Kluwer.Gururani et al. (2012) Plant Physiol Bioch 58: 182–194.Bussotti et al. (2011) Envir Exp Bot.Oukarroum et al. (2007) Envir Exp Bot 60: 438–446.Foyer et al. (2012) JXB 63: 1637–166.Zagdańska (1995) Physiol Plant 95: 428–436.

P7.69

Utilisation of betaine in vinasse stillage by a mixed culture of aerobic bacteria under non-controlled pHAgnieszka Ryznar-Luty, Edmund Cibis, Małgorzata Krzywonos

Wroclaw University of Economics, Department of Bioprocess Engineering

The aim of this work was to examine how temperature and the initial pH influence the efficiency of an aerobic biodegradation of betaine in vinasse stillage. Betaine ac-counted for as much as 37.6% of total organic carbon. In the study two series of batch biodegradation process-es were carried out in a stirred tank reactor by a mixed culture of Bacillus bacteria. The temperatures applied were 27, 36, 45, 54 and 63°C. The pH was not controlled, and the initial pH of the culture medium amounted to 6.5 in first series and 8.0 in the second series. Efficiency of biodegradation was expressed in terms of reduction in SCODsum, which is a sum of SCOD (soluble chemical oxygen demand, i.e. COD determined after suspended solids separation) and theoretical COD of betaine. The high biodegradation efficiency obtained in the process-es was attributable to the assimilation of betaine by the bacterial strains used in the study. Maximal reduction in SCODsum (85.41%), BOD5 (97.91%) and TOC (86.32%) was achieved during biodegradation at 36°C and pH0 = 8.0. Under the same temperature and pH regime, betaine was removed completely, while biodegrada-tion proceeded at the fastest rate (1.1684 g O2/(l∙h) for SCODsum removal). During biodegradation the main or-ganic pollutants were removed at the same time, but with diverse rate of removal. An exception to this rule was the assimilation of betaine, which intensified only after the medium was lacking in easily available carbon sources.AcknowledgementsThe study was financed by the Polish Ministry of Science and Higher Education under Project No. 2P06T 045 30.

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P7.70

Use of biotechnological methods in reforestation of degraded landRafał Ważny1, Katarzyna Turnau1, 2

1Jagiellonian University, Malopolska Centre of Biotechnology, Gronostajowa 7, 30-387 Crakow; 2Jagiellonian University, Institute of Environmental Sciences, Gronostajowa 7, 30-387 Crakow

Presence of beneficial organisms in degraded soil is usu-ally limited, especially in soil polluted with heavy metals or in soil previously used for agricultural purposes. The main obstacle is usually the lack of well adapted mycor-rhizal fungi. Bioremediation and reforestration can be supported by inoculum production of mycorrhizal fungi. The introduction of the particular strain should be pre-ceded, however, by proper knowledge of fungal species in a given habitat, known receptivity of the soil understood as the ability of the fungus to survive under given condi-tion and to establish long lasting existence. Further, the succession of fungal taxa should be estimated and con-trolled. In such studies it is not possible to rely only on conventional studies but the use of molecular tools is nec-essary. The research carried out on Abies alba seedlings regenerating in forest sites established on agricultural land are presented. We aimed to determine whether the fungal communities of seedlings from degraded land dif-fer from fungal communities of seedlings growing in nat-ural forest and whether there was the need for some treat-ments like inoculation. Mean mycorrhizal colonization of seedlings was over 90%. A total of 49 ectomycorrhizal taxa were identified, 36 of which were identified in natu-ral forest and 23 in forest on degraded area. According to the analysis of similarity, the ECM fungal communities were different between these forests. Only 20% symbionts common to both sites suggests that succession of ECM fungus specific to fir regenerating on post-arable soil is still in progress. The use of mycorrhizal inocula offers the possibility to improve the reforestation process.

P7.71

Pilot study on feasibility of application of gas chromatography for the assessment of acrylamide concentration in sewage sludgeElżbieta Włodarczyk1, Marta Próba1, Łukasz Wojtal2

1Częstochowa University of Technology Faculty of Engineering and Biotechnology Institute of Environmental Engineering Brzeźnicka 60A St., 42-200 Częstochowa, Poland; 2Center for Research and Environmental Control, Owocowa 8 St., 40-158 Katowice, Poland

Background. Acrylamide (2-propenamide CH2=CH–CO–NH2) is a low molecular weight organic compound containing in its structure conjugated double bonds and a part of the amide. Due to the presence of characteristic functional groups, acrylamide is polar compound. It is well soluble in water and polar solvents such as metha-nol or ethanol. It is produced on an industrial scale for 60 years by catalytic hydrolysis of acrylonitrile. Currently, it is used, for the synthesis of modified polyacrylamides, which are used in industry, in the production of plastics, dyes, adhesives, cosmetics, mortar, as well as a coagulant for treatment of water, wastewater or sewage sludge con-ditioning.Objectives. Aim of this study was to determine the possi-bility of using gas chromatography to measurement of the acrylamide concentration in sewage sludge.Methods. Determination of acrylamide by gas chroma-tography is based on the standard: EPA Method 8032A “Acrylamid by gas chromatography”. To mark acrylamide in the sludge elementary test should be performed with ratio of liquid to solid part equal 10l/kg [mg/kg dry weight].The method permits for the determination of acrylamide (2-propenamide) in water in the range of 0.040 mg/l to 2.0 mg/l.It consists of: bromination reaction of the compound in the presence of dibromopropendial derivative, triple ex-traction with ethyl acetate, concentration of eluate sample up to the 1 ml volume, and analysis by gas chromatogra-phy using an electron capture detector (ECD).According to the manual of chromatograph software cal-ibration curve was made (calibration using the internal consistency of the NIST standard (Lot No. 51011).Calibration is considered valid if the correlation coeffi-cient is r Results. To ensure the quality of test results for each test series were performed: a blank sample (checking the purity of the reagents, water) and a control sam-ple (50 mg/l-checking the precision and stability of the measurement system) and a desired sample of sewage sludge water extract. Samples were performed twice (the difference between the results is not greater than 10%), and the average value of the two samples was 1.62 mg/ l.

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Conclusions. The presence of acrylamide in sewage sludge has been confirmed. Therefore, the use of poly-acrylamide materials, containing acrylamide, is becom-ing wider, more and more of the compound enters the natural environment (through sewage and sediments into groundwater and surface water). Acrylamide, as the toxic substance is not indifferent to human health. The need of further research in this area was highlighted.Keywords: acrylamide, polyelectrolyte, sewage sludge, gas chromatog-raphyAcknowledgementsBS/MN 401-307/12.

Plant genetic engineering

Lectures

L8.1

The ins and outs of multigene engineering of complex biosynthetic pathways inplantsChangfu Zhu1, Teresa Capell1, Paul Christou1, 2

1Departament de Produccio’ Vegetal i Cie’ncia Forestal, Universitat de Lleida-AGROTECNIO Center, Lleida, Spain, 2Institucio’ Catalana de Recerca i Estudis Avanc ats, Barcelona, Spain

The simultaneous transfer of multiple genes into plants enables researchers to study and modulate entire meta-bolic pathways, express multimeric proteins or protein complexes, and study complex genetic control circuits and regulatory hierarchies. Technical hurdles limiting the number of genes transferred to plants have introduced a  significant bottleneck to progress in plant biotech-nology for the past three decades. Early transformation methods for plants were developed with the implicit in-tention to introduce one or two genes (usually a prima-ry transgene and a selectable or screenable marker) and have been optimized on that basis. Although multigene transfer (MGT) can be achieved using such methods, they operate under the law of diminishing returns. The more transgenes are included, the less likely all of them will be integrated and expressed. Larger and larger populations of plants must be screened to identify rare individual lines with the sought-after genotype.More recently, researchers have attempted to address these limitations by developing new transformation methods that recognize the desire to introduce multiple transgenes into plants and express them in a coordinated manner. Essentially all these methods aim to achieve the creation of a SMART locus, i.e. one containing Stable Multiple Ar-rays of Transgenes. We will discuss the most recent strate-gies for MGT in plants using case studies to illustrate how these approaches have been applied in the dissection and modulation of complex metabolic pathways.

L8.2

Biotechnology of FlaxAnna Kulma, Jan Szopa

Wroclaw University, Linum Foundation

The development of molecular biology, especially the se-quencing of nucleic acid, resulted in the stream of data on gene structure. Study of gene structure forced the de-velopment of research on identification of their function. For evaluation of gene function, the most effective and actually the only method is the generation of an organism with overexpression or/and repression or/and comple-mentation of repression/overexpression of the gene with the subsequent analysis of modified organism. GM plants, generated for over three decades, provided the valuable information of gene function, and simultaneously indi-cated the advantages of transgenic plants.According to the European Commission, the challenge for biotechnology, as a scientific discipline, is searching for tools for agriculture and industry development and providing such a plant productivity, that will satisfy grow-ing food demand.To rise this challenge, the generation and application of GM plants is justified. At the turn of 2010/2011 the Euro-pean Commission released a study summing up the ten-year (2001–2010) research on GM plants (A decade of EU-funded GMO research). The aim of the study was to estimate the influence of transgenic plants on the broad-ly- taken environment, study of the risks connected with their release into the environment, determination of the putative horizontal gene transfer between GMO and non-GMO, study of the safety of food produced from trans-genic plants, etc. 50 projects realised for over 200 million of Euro by over 400 research groups were summarised. The main conclusion summarizing these research is, that the cultivation, processing and using of GM plants is not more risky than those of conventional cultivation. Even though, another study released by the European Com-mission (Europeans and Biotechnology in 2010) says, that over 60% (61–90% depending of the country) of the society does not accept GMO.From two decades we (Linum Foundation, www.leczeniel-nem.pl) have been generating GM potatoes and nowadays GM flax. At first, the aim was to identify the genes function and their significance for plant productivity and metabo-lism. The most attention was given to those genes, that are putatively key genes for plant infection resistance, are crucial for regulation of the synthesis of the compounds of biomedical functions (phenolic acids, flavonoids, ter-penes) or are applicable in industry (fatty acids) and in the

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environment protection (biodegradable polymers). We generated plants of elevated resistance to pathogen infec-tion, of increased accumulation of phenylpropanoids and terpenoids, of altered synthesis and accumulation of bio-polymers (pectins, lignins, cellulose, hemicellulose) and biodegradable polymers (polyhydroxybutyrate) and flax of altered fatty acids profile. We estimated the influence of GM flax cultivation on soil ecosystem and we revealed the intensified and beneficial interaction of mycorrhized organisms on flax root system. We showed the positive influence of GM flax seeds consumption on the broad range of parameters describing health condition of lab-oratory animals (mice, rats). We generated a few prepa-rations applicable in the prevention and treatment of people, including certified flax dressing LENPLAST for chronic wounds treatment, certified preparations of an-tiinflammatory and antibacterial function (LINFIX) and of skin regenerating function (OILFIX), diet supplement LINACTIVE beneficial in antiinflammatory and antican-cer prevention, admitted to the trade by GIS and we offer flax oil of the characteristics of ideal oil and other prepa-rations of antibacterial function (see www.pkblinum.pl).The favourable characteristics of GM flax and the prepa-ration generated from GM flax cannot be produced on the large- scale due to the difficulties in obtaining agree-ment on GM plants release into the environment for production. Although unjustified but still strong social reluctance to GMO and the restrictive regulations forced the elaboration of new technology using the knowledge resulting from GM plants analysis and using it to the generation of favourable altered plants omitting the in-troduction to their genome the heterologous genes. Two- year experiments led to the elaboration of alterGMO technology resulting in the profitable changes in plants without their modification by the vector transgenesis. Briefly, the first stage of the method comprises the induc-tion of the changes in the endogenous gene by its meth-ylation/demethylation or the changes in accumulation of gene derived products (mRNA). In the second stage, the plants are selected by molecular markers derived from the analysis of GM plants. In the third stage, the es-tablishment of introduced changes by the exposition of plants to the factors increasing DNA methylation level (mannitol, salicylic acid, jasmonates) or mobilizing the activity of the whole genome (infection by non-virulent microorganisms) takes place. The comparative analysis of flax, in which the lycopene cyclase gene was induced by a vector method (GMO) and non-vector method (alterGMO), revealed the two time higher effectiveness of the latter. Beside from the evidently higher effectiveness, the most important is, that the alterGMO method allow to generate the favorably altered plants, whose cultivation make the plant producer independent from the compli-cated and unstable procedure of obtaining agreement on their release into environment.

L8.3

RNAi in cereal functional genomicsWaclaw Orczyk1, Anna Nadolska-Orczyk2, Sebastian Gasparis2, Wojciech Zalewski2, Yuliya Yanushevska1

1Dept. of Genetic Engineering, Acclimatization and Plant Breeding Institute – Nat’l Research Institute, Radzikow, 05-870 Blonie, Poland; 2Dept. of Functional Genomics, Acclimatization and Plant Breeding Institute – Nat’l Research Institute, Radzikow, 05-870 Blonie, Poland

One of the most important biological achievements of last two decades of XXc. was discovery of novel class of RNA molecules and discovery of the process – RNA in-terference (RNAi) – where the molecules act as regula-tors. The impact of the discovery on basic and applied biology (recognized by Nobel Prize awarded in 2006) is enormous and is still growing.Experimental gene silencing and silencing-based func-tional gene analysis is one of the possibilities opened by the knowledge. Depending on the experimental de-sign the silencing can be specific to a particular gene or a group of genes. It is particularly important in allopoly-ploids (such as wheat or triticale) where a group of ho-mologous genes exist and their functional analysis might require this level of specificity.We have adapted and we have been currently using this strategy to study function of the genes selected based on their putative involvement in conferring important agricultural traits in cereals [1, 2, 3]. We tested this ap-proach by silencing expression of genes responsible for grain hardness: Pina / Pinb and Sina / Sinb in wheat and triticale respectively. Both Pina / Pinb genes are located in hardness locus in D genome of allohexaploid wheat (AABBDD). Wheat plants transformed with silencing cassettes showed 80–91% reduction of Pina / Pinb tran-script level compared with control, what was followed by significant reduction in PINA and PINB proteins in seed endosperm. Observed in the plants increase of kernel hardness, up to the level found in hard grain T. durum [4], was consistent with the earlier knowledge of the gene function based on mutant analysis. Sina / Sinb genes are believed to be Pin orthologs located in R genome in allo-hexaploid triticale (AABBRR). Transgenic triticale (with Pina silencing constructs) led to silencing of Sina / Sinb ortologs and to lowering SINA /SINB proteins. The unex-pected and surprising in the light of the previous results was grain hardness. It remained unchanged in transgenic plants. The results clearly confirmed the expected role of Pina / Pinb genes in endosperm texture of wheat kernels and contradicted the anticipated role of the genes and the proteins in triticale seeds (unpublished).HvCKX and TaCKX, existing in various species as a gene family, represent another group of tested genes. They encode cytokinin oxidase – an enzyme responsible for catabolism of cytokinin. Expression pattern of the genes (as well as the activity of the enzyme) is organ and devel-opmental dependent but detailed functions remain un-

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known. We found that HvCKX1 and HvCKX2 were spe-cifically induced in developing barley spikes. Silencing of the genes in barley strongly correlated with increased productivity shown as higher number and higher mass of kernels. The trait was inherited in the next generations. The results confirm the hypothesis that organ specific expression of the CKX genes influences traits related to plant productivity [3].Comparison of two different transformation methods (Agrobacterium-based and particle bombardment) re-vealed that the experimental design might have a big impact in final results. Introduction of the same silenc-ing cassette via Agrobacterium and by particle bombard-ment gave clearly different results. In our case depression of the CKX transcript level was observed only in Agro transformed plants. Particle delivery of the silencing construct led to inconsistent and self-contradictory re-sults [5].Current project, and unpublished results, is focused on identification and functional analysis of putative barley ortholog of brassinosteroid regulators OsGSK1 in rice and BIN2 / AtSK2 in Arabidopsis. Depressing transcript level of the tested gene to 0.09 – 0.22 of the transcript in control plants revealed the clear correlation with elevated salt tolerance of the seedlings. The structure of the gene and biological function of the gene will presented and discussed.AcknowledgementsThe research was financed by grants: 620/N-COST/09/2010/0 (AN-O), UMO-2011/03/B/NZ9/01383 (AN-O), N302 013 31/1517 (AN-O), 718/N-COST/2010/0 (WO), UMO-2011/01/B/NZ9/02387 (WO).References[1] Gasparis S., Bregier C., Orczyk W., Nadolska-Orczyk A. (2008) Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants. Plant Cell Rep, 27: 1721–1729.[2] Binka A., Orczyk W., Nadolska-Orczyk A. (2012) The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector systems and selection cassettes. J Appl Genet, 53: 1–8.[3] Zalewski W., Galuszka P., Gasparis S., Orczyk W., Nadolska-Orczyk A. (2010) Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity. J Exp Botany, 61: 1839–1851.[4] Gasparis S., Orczyk W., Zalewski W., Nadolska-Orczyk A. (2011) The RNA-mediated silencing of one of the Pin genes in allohexaploid wheat simultaneously decreases the expression of the other, and increases grain hardness. J Exp Botany, 62: 4025–4036.[5] Zalewski W., Orczyk W., Gasparis S., Nadolska-Orczyk A. (2012) HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes. BMC Plant Biology, 12: 206.

L8.4

Expression of three diadinoxanthin de-epoxidase genes of Pheodacylum tricornutum in Escherichia coli Origami b strainMonika Olchawa-Pajor1, Monika Bojko1, Wojciech Strzałka2, Paulina Kuczyńska1, Dariusz Latowski1, Kazimierz Strzałka1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland; 2Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland

All photosynthetic organisms develop photoprotective mechanisms with carotenoids playing a fundamental role in the dissipation of excess light energy. Photoprotection is connected with de-epoxidized forms of xanthophyll pigments which are formed by enzymatic removal of ep-oxy groups under high light conditions. These reactions occur in the processes commonly known as the xantho-phyll cycles, the most common among them being the violaxanthin and diadinoxanthin cycles. In violaxanthin cycle, violaxanthin is converted to zeaxanthin via anthe-raxanthin whereas in diadinoxanthin cycle epoxy group is removed from diadinoxanthin and diatoxanthin is cre-ated. This conversion takes place e.g. in diatoms with in-volvement of the enzyme diadinoxanthin de-epoxidase. Previously, only one gene of this de-epoxidase, designed DDE, was postulated. Today in one of the diatoms, Phae-odactylum tricornutum (CCAP 1055/1 strain which all genomes are sequenced) three forms of de-epoxidases were identified but only one of them corresponded to VDE. This form of DDE is also called VDE, its gene is marked as PtVDE and might be involved in the conven-tional xantophyll cycle. While two VDE-like de-epoxi-dases (designated as PtVDL1 and PtVDL2) may be more specialized in the chromist-specific diadinoxanthin cycle.The purpose of our research was to obtain the PtVDE, Pt-VDL1 and PtVDL2 genes of Phaeodactylum tricornutum (UTEX 646 strain) and constructing expression vectors pET-15b/VDE subsequently used for production of these enzymes with polyhistidine tag.Total RNA was prepared from 5 day old culture of Ph. tricornutum using GeneJET Plant RNA Purification kit. cDNA was synthesized using M-Mul V Reverse tran-scriptase and Random primer. Primer pairs and cDNA were used as a template for amplification of PtVDE, Pt-VDL1 and PtVDL2. Sense primer contained the initia-tion Met, codons of amino acid from the mature protein N terminus and Nde I site. Antisense primers contained a stop codon, codons from C terminus and BamH I site. PCR products were visualized and confirmed by agarose gel electrophoresis, then digested and inserted to digested by Nde I and BamH I – pet-15b vector using ligation kit. Plasmid was named pet-15b/PtVDE, pet-15b/PtVDL1 and pet-15b/PtVDL2.

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The sequences of PtVDE, PtVDL1 and PtVDL2 were compared with sequences of these gene of Ph. tricornu-tum CCAP 1055/1 strain.Pet-15b/PtVDE, and pet-15b/PtVDL2 plasmids (similar sequences to the respective Ph. tricornutum gene) were used to transform E. coli Origami b and BL21 strain cells. After IPTG induction, the presence of VDE and VDL2 proteins was confirmed by western-Blot analysis. The sta-ble expression of enzymes was observed in both tested E. coli strains (Origami b, BL21). Obtained VDE and VDL2 with molecular weight about 49 kDa and 60 kDa respectively, exhibited ability to de-epoxidise violaxan-thin.

L8.5

The role of ubiquitins in cucumber (C. sativus L.) flower morphogenesisMagdalena Pawełkowicz, Cezary Kowalczuk, Paweł Osipowski, Michał Wojcieszek, Grzegorz Kojder, Katarzyna Kosik, Zbigniew Przybecki

Department of Plant Genetics, Breeding & Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences- SGGW, Nowoursynowska 166 St., 02-776, Warsaw, Poland

Sex determination and flower morphogenesis are a very broad and complex processes controlled at many levels. We isolated four clones from cucumber transcriptomes connected with ubiquitination processes and checked if the expression of the genes corresponding to those clones is different among vegetative and generative tissues (leaf, shoot apex, flower buds 1–2 mm) in monoecious and gynoecious cucumber lines. Therefore, to determine to the extent the role of sequences in flower morphogenesis a  comprehensive analysis was performed using compu-tational studies to acquire insight into characteristic fea-tures in genes structure, upstream regulatory elements and protein motifs. We conclude that the cucumber generative organs have different sensitivity to plant hor-mones due to distinct signal transduction mediated by ubiquitins in male and female organs in floral buds and shoot apex. Ubiquitins could be correlated with alterna-tive way of hormones signal transduction in flowers of opposite sex, taking part in inhibition of unwanted gen-erative organs causing the development of unisex flower.AcknowledgementsThis research was supported by grants from the Polish Ministry of Science and Higher Education (MNiSW) – N302 3633 33 and National Science Center – 2011/01/B/NZ2/01631.

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Posters

P8.1

Comparative analysis of Gal4 and split-ubiquitin-based yeast two-hybrid systems for Arabidopsis thaliana Proliferating Cell Nuclear Antigen studiesAgata Jakubowska, Olga Sztatelman, Wojciech Strzałka

Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-378 Crakow, Poland

The PCNA (proliferating cell nuclear antigen) protein is well–conserved across Eukaryota both in structure and function. It plays an essential role in DNA replica-tion, DNA repair and cell cycle regulation. Human and yeast PCNA has been shown to form complexes with many different nuclear proteins. Multiprotein complex-es are increasingly recognized as the molecular basis of a substantial amount of biological processes. Mapping of protein interaction networks in such complexes is of prime importance. It allows to characterize the physio-logical functions of the component proteins and usually results in better understanding of multifactorial-depen-dent processes. One of the most common methods of studying protein-protein interactions in vivo is the yeast two hybrid (Y2H) system. Since its first introduction by Fields in 1989 various modifications have appeared. Two features determine the Y2H system as the first choice for interaction studies: simplicity and high-throughput ca-pacity, which enables screening for interactions on a ge-nome-wide scale.The aim of this study was to determine which Y2H sys-tem is potentially better for screening proteins interact-ing with Arabidopsis thaliana PCNA1 and PCNA2. We compared two commercially available Y2H systems: the classic one based on the reconstitution of the Gal4 tran-scription factor (Invitrogen) and a split-ubiquitin system based on the reconstitution of ubiquitin (MoBiTech), both adapted to Gateway technology. Since A. thaliana PCNA1 and PCNA2 were shown to form homo- and hetero-trimeric ring structures, PCNA1/1, PCNA1/2, PCNA2/2 and PCNA2/1 interactions were studied to test the selected Y2H systems. The investigated interactions could only be detected with the split-ubiquitin Y2H sys-tem. This observation suggests that this system might be more effective in screening for proteins interacting with plant PCNA.

P8.2

Efficient Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explantsMagdalena Klimek-Chodacka, Iwona Zapała, Rafal Barański

Unit of Genetics, Plant Breeding and Seed Science, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland

Hairy root cultures obtained after Agrobacterium rhizo-genes-mediated genetic transformation can serve as model system for studying plant metabolism and physiology or can be utilized for the production of secondary metabo-lites. So far sugar beet hairy roots were used mainly for studying resistance to BNYVV, Heterodera schachtii or Tetanops myopaeformis, but no efficient protocol of hairy root production has been publically released. Usually ex-plants of diploid or tetraploid donor sugar beet plants were used for transformation however, the use of haploids could be more desirable. Diploidization of genetically modified haploid hairy roots could result in fast development of tis-sue that is homozygous at the transgene locus.Here we report an efficient system for sugar beet genetic transformation leading to the development of hairy roots. The study included two A. rhizogenes strains (A4T and LBA1334) carrying a binary vector pBIN-m-gfp5-ER or pCAMBIA1301 possessing gfp and uidA reporter genes, re-spectively. Petiole and midrib explants of two haploid and two diploid sugar beet genotypes were inoculated in five treatment combinations including time of explants exposure to ultrasound, application of bacteria suspension before or after sonication and time of bacteria-explant co-culture.The formation of hairy roots was observed as early as two weeks after inoculation at the site of tissue wounding. Hairy roots appeared on 0% to 36% explants depend-ing on the treatment combination. The highest frequen-cy was achieved when explants of a haploid genotype No. 169 were sonicated for 15 s in the A4T pCAMBIA1301 inoculum of OD600 = 0.5 followed by a 3-day co-culture. Using the same treatment combination the explants of the remaining genotypes developed hairy roots with the frequency ranging from 10% to 30%.The T-DNA presence in hairy roots was confirmed by PCR and the copy number of transgenes was assessed by South-ern hybridization and Real-Time PCR and determined as one to several copies depending on the transformation event. Expression of the transgenes in hairy roots was con-firmed visually i.e., beta-glucuronidase activity was veri-fied by histochemical staining and the synthesis of green fluorescent protein by its fluorescence in UV light. 54% of the used hairy root cultures showed transgene expression. The results indicate that the developed protocol allows pro-duction of 42 hairy roots per one hundred explants.AcknowledgementsThis work was supported by the Polish Ministry of Agriculture and Rural Development (Decision No. HOR hn-801-10/13).

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P8.3

DesC desaturase of cyanobacterium Synechococcus vulcanus expression in canola plants does not improve the low positive temperature growthMariia Slyvets1, 2, Liudmyla Sakhno1, Yuri Sheludko1

1Institute of Cell Biology and Genetic Engineering NAS of Ukraine; 2National Technical University of Ukraine “Kyiv Polytechnic Institute”

One of the plant adaptation mechanisms to cold is the in-crease in the unsaturation of fatty acid residues in cellular membranes, sustaining the required membrane fluidity. The important role in this process is attributed to fatty acid desaturases, catalyzing the transformation of a single bond between carbon atoms in acyl chains (C–C) into the double bond (C=C).Earlier we have obtained spring canola (Brassica napus L., cv Obreey) lines bearing hybrid gene of acyl-lipid ∆9-de-saturase (desС) from cyanobacterium Synechococcus vul-canus translationally fused with termostable lichenase from Clostridium thermocellum (licBM3) reporter gene in their nuclear genome using Agrobacterium tumefa-ciens-mediated leaf disk transformation. Desaturase gene expression was estimate qualitatively (lichenase plate test) and quantitatively (photometry) on the basis of ac-tivity of licBM3 gene product as a part of hybrid protein. Gas-chromatographic data revealed that cyanobacterium DesC expression did not lead to qualitative changes in canola leaf fatty acid composition but resulted in increase in the lipid content (up 48%) and in the quantitative alter-ation in the fatty acid composition. Trienoic (С16:3 and С18:3) fatty acid content was increased up 33% and sat-urated fatty acid content (С16:0) was decreased up 16%. These alterations allows suppose that desC canola lines might be more resistant to chilling damage in compari-son with control ones.The present work was aimed at investigation of transgen-ic line growth under low positive temperature (+4°C). During experiments control and desС expressing canola lines (primary transformants 18а, 18в and plants of first generation Bn18а/6, Bn18в/25) were analysed after prop-agation by cutting under aseptic conditions. Plants were grown during four weeks in termal room (16/8 photo-period, +23°С) in Sigma 25×150 мм (Sigmawave™) tubes containing 15 ml agar solidified MS medium without hormones. Then they were transferred to chamber (16/8 photoperiod, +4°С) for 5 days. After growth under low temperature plants were returned under initial condi-tions for four weeks. Stress effect on plant growth was evaluated using measurement of fresh weight, total sol-uble protein content and superoxide dismutase activity. We documented that desC canola plants did not differ significantly from control under all tested temperature regimes. It seems trienoic fatty acid content increase in our canola due to DesC expression was not sufficient for

cold resistance improved. Perhaps, desC activity was not effective in cytosol. Earlier it was reported that tobacco plants expressing native desC gene from S.vulcanus had enchanced chilling resistance though information about chloroplast targeting of introduced gene was absent. We plan to modify our genetic construction by inclusion of chloroplast signal peptide to obtain canola with cold re-sistance improvement.AcknowledgementsThe work was supported with the grant of National Academy of Science of Ukraine № 0110U006062.

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P8.4

A method of epoxides determination in bacterial cellsPaulina Kuczynska1, Sylwia Leskiewicz1, 2, Wojciech Strzalka3, Monika Olchawa-Pajor1, Monika Bojko1, Dariusz Latowski1, Kazimierz Strzalka1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University; 2Faculty of Chemistry, Jagiellonian University; 3Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University

Organic compounds containing a ternary cyclic complex consisting of an oxygen linked to two carbon atoms are named epoxides. They arise from oxidative metabolism of compounds derived from the cell and the environment via chemical and enzymatic processes and have high chemical reactivity, genotoxicity, cytotoxicity. Our stud-ies deal with determination of epoxides in bacterial cells overexpressing zeaxanthin epoxidase (ZEP). This enzyme catalyzes conversion of zeaxanthin (an epoxy-free xan-thophyll) into violaxanthin (di-epoxide xanthophyll) in the violaxanthin cycle. It has been assumed that the lack of ZEP substrate may cause nonspecific reactions lead-ing to production of toxic epoxides. We noticed a cor-relation between ZEP expression in Escherichia coli and increase in amount of epoxides. A spectrofluorometric method has been used for the determination of total ep-oxides according to Sano and Takitani (1985). As a result of standardizing the method the best conditions to car-ry out the experiment using bacteria cells were worked out. Two steps reaction was performed; the first with or without sodium sulfide and the second with taurine and o-phthalaldehyde reagents. The studies were conducted on E. coli strain Origami B (DE3) transformed with Ara-bidopsis thaliana ZEP open reading frame cloned into pDEST17 vector. Increase in amount of epoxides was observed in transformed cells during the time of growth. Moreover, it was also correlated with lower growth rate of bacteria what was probably caused by toxic nonspecific products of epoxidation. Higher epoxides content during the time of ZEP overexpression induction shows that this enzyme is responsible for the epoxide formation. This study shows that described method can be used for deter-mination of total epoxides in cells thus it might be useful for studies on proteins engaged in epoxide formation or degradation.

P8.5

Comparison of expression and activity of violaxanthin and diadinoxanthin de-epoxidases in Escherichia coli Origami b strain Monika Olchawa-Pajor1, Monika Bojko1, Wojciech Strzałka2, Paulina Kuczyńska1, Dariusz Latowski1, Kazimierz Strzałka1

1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland; 2Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland

Photoprotection in the most of photoautotrophs is strongly connected with de-epoxidized forms of xan-thophyll pigments. These compounds are formed in the processes commonly known as xanthophyll cycles. The most widespread in the nature is the violaxanthin cycle, in which epoxy groups are removed from violaxanthin by enzyme violaxantin deepoxidase (VDE) forming tran-siently antheraxanthin and then zeaxanthin as the final product. In another type of xanthophyll cycle, under high light conditions monoepoxide, diadinoxanthin, is de-epoxidised into diatoxanthin. This conversion takes place e.g. in diatoms with involvement of the enzyme di-adinoxanthin de-epoxidase and this xanthophyll cycle is called diadinoxanthin cycle. Previously, only one gene of this de-epoxidase, designed DDE, was postulated. Since 2008 it has been known that in one of the diatoms, Phae-odactylum tricornutum three forms of de-epoxidase are present. One of them corresponds to known VDE oc-curing in higher plants. This form of DDE is also called VDE and its gene is marked as PtVDE. The purpose of our research was to obtain the VDE gene from Arabi-dopsis thaliana (WT Columbia strain) (AtVDE) and VDE gene of Phaeodactylum tricornutum (CCAP 1055/1 strain) (PtVDE) and constructing expression vectors pET-15b/VDE subsequently used for production of these enzymes with polyhistidine tag. Finally we compared the effects of one step purification and activity of the en-zymes. Total DNA prepared from 5 day old culture of Ph. tri-cornutum was used as a template for PtVDE and primer pairs. Sense primer contained the initiation Met, codons of amino acid from the mature protein N terminus and Nde I site. Antisense primers contained a stop codon, co-dons from C terminus and BamH I site. PCR products were visualized and confirmed by agarose gel electropho-resis, then digested and inserted to digested by Nde I and BamH I – pet-15b vector using ligation kit. Plasmid was named pet-15b/PtVDE. The wild type AtVDE gene uses rare codons with a high frequency. Moreover, it contains several negatively cis-acting motifs which might hamper expression in pro-karyota. Therefore, AtVDE gene was obtained by chemi-cal synthesis adapting the codons from A. thaliana to the

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codons bias of Escherichia coli gene. During the optimi-zation process the following cis-acting sequence motifs were omitted: internal TATA-boxes, chi-sites and ribo-somal entry sites; AT-rich or GC-rich sequence stretch-es; repeat sequences and RNA secondary structures. The optimized gene was inserted to pet-15b vector using li-gation kit. Purified plasmid containing AtVDE named pet-15b/AtVDE was received. Both plasmids were used to transform E. coli Origami b strain cells. PtVDE and AtVDE effective expression after IPTG induction were evidenced by SDS-Page electropho-resis and Western-Blot methods. VDE proteins were pu-rified using TALON super flow Metal Affinity resin. The stable expression in E. coli (Origami b strain) provided enzymes with molecular weight about 42 kDa for VDE from A. thaliana and 49 kDa for VDE from Ph. tricornu-tum. Both obtained enzymes were active relative to Vx although AtVDE exhibited higher activity than PtVDE.

P8.6

Identification and analysys of WUSCHEL (WUS/WOX) and CLAVATA genes in cucumber (Cucumis sativus L.)Michał Wojcieszek1, Magdalena Pawełkowicz1, Marcin Olszak1, Agata Jędrzejuk2, Zbigniew Przybecki1, Wojciech Burza1

1Department of Plant Genetics, Breeding & Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences- SGGW, Nowoursynowska 166 St., 02-776, Warsaw, Poland; 2Department of Ornamental Plants, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences- SGGW, Nowoursynowska 166 St., 02-776, Warsaw, Poland

The genes WUSCHEL (WUS), WUSCHEL-RELATED HOMEOBOX (WOX) and CLAVATA (CLV) play key roles in many life processes in plants. In Arabidopsis the homeobox gene WUS encodes a homeodomain tran-scription factor that has been shown to be a regulator of a pool of pluripotent stem cells in the shoot apical meri-stem (SAM). In SAM, an essential mechanism for meri-stem organization and maintenance is a regulatory feed-back loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous man-ner. This intercellular signaling network coordinates the development of the organization center, organ boundar-ies and distant organs. In root apical meristem (RAM) gene WOX5 can be considered analogous to WUS that acts to maintain stem cells of the shoot apex. The studies of other authors showed that induced overexpression of the PGA6 gene (identical to WUS) caused high-frequen-cy somatic embryo formation in all tissues and organs tested, without any external plant hormones.In our research we performed broad in silico analysis of WUS and CLV genes from C. sativus genomes – Pol-ish B10 and Chinese 9930. Upstream regions examina-tion, protein function determination and collation with A. thaliana analogues were the main procedures utilized in this work. The results obtained, especially for line B10 are the first step to a better understanding and control of the plant morphogenesis processes under in vitro and field conditions. This outcome will be useful for our in-vestigations concerning plant pluripotent and totipotent induced stem cells.AcknowledgementsThis research was partially supported by grants from the Polish Ministry of Science and Higher Education (MNiSW) – N302 3633 33 and National Science Center – 2011/01/B/NZ2/01631.

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P8.7

Protoplasts of Lathyrus sp. – optimization of isolation procedureBarbara Piwowarczyk1, Wojciech Rybiński2

1Department of Botany and Plant Physiology, Faculty of Horticulture, University of Agriculture in Crakow, Poland; 2Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland

Interspecific hybridization between various species of ge-nus Lathyrus is difficult. Conventional methods of cross-ing have been successful only in several instances. This obstructs obtaining of hybrids and introduction of new desirable features to crops. Application of biotechnologi-cal methods seems to be the only solution of this obstacle. Currently, among the available biotechnology techniques, protoplast fusion is often used for obtaining somatic hy-brids. Besides, protoplasts are excellent explants for ge-netic transformation due to the absence of barrier that is cell wall. Prerequisite to use these methods is devel-opment of efficient procedures of protoplasts isolation, their cultivation and plant regeneration. Lathyrus species belong to large-seeded Fabaceae plants that are very sen-sitive to in vitro manipulations and very recalcitrant to regeneration.In the study, protoplasts were isolated from mesophyll of two Lathyrus species: L. tingitanus and L. cicera. Leaves, originating from either 3 week-old ex vitro seedlings or 2 month-old plants growing in the field conditions, were used as explants. Different enzymatic mixtures (com-posed of Cellulase, Pectinase, Macerozyme and osmotic agent (sorbitol) in different concentration) as well as vari-ous time of tissue incubation were investigated. Effective-ness of applied isolation conditions was evaluated on the basis of the number of protoplasts per 1 g plant tissue, protoplasts viability and the rate of cell wall digestion.These studies will determine the optimal conditions for efficient isolation of viable protoplasts that will be a con-tributive research to perform the fusion between proto-plasts of Lathyrus species in the future.AcknowledgementsThis work was supported by the Ministry of Science and Higher Education of Republic of Poland (BM 4541).

P8.8

Processing of plant tissue bearing S-HBsAg for an oral vaccine against hepatitis BMarcin Czyż1, Tomasz Pniewski1, Józef Kapusta2, Radosław Dembczyński3, Roman Marecik3, Andrzej Płucienniczak2

1Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland; 2Institute of Biotechnology and Antibiotics, Warszawa, Poland; 3Poznan Life Science University, Poland

Potential plant-derived oral vaccine against Hepatitis B Virus must have a formula which would enable effi-cacious and convenient administration under defined regime in addition to long-term stability of borne anti-gen, here the small surface antigen (S-HBsAg) formed into highly immunogenic Virus-Like Particles (VLPs). Freeze-drying was a chosen technique that could allow to fulfil above-mentioned requirements.The aim of this project was to investigate and optimise the processing of previously obtained transgenic lettuce plants expressing S-HBsAg to prepare an oral vaccine formula. Conditions of lyophilisation and storage of ob-tained preparation were investigated regarding the stabil-ity of the antigen.Several physical freeze-drying parameters were tested. That included freezing method of an initial plant mate-rial, in-process shelf temperatures and primary and sec-ondary drying times. Established conditions were used in the second stage of research on effect of added lyopro-tectants. A number of substances and their concentrations were studied, in addition to parameters of tissue satura-tion. Subsequently, antigen preservation after prolonged storage was evaluated. Freeze-dried and powdered plant tissue was stored at various temperatures, with optional presence of silica gel desiccator and neutral nitrogen at-mosphere. Impact of lyoprotectants, applied process and storage parameters were assessed.Based on obtained results the most effective profile of freeze-drying process, and the most potent lyoprotectant were determined. Under the selected conditions 80% of VLPs-assembled S-HBsAg was retained after freeze-dry-ing. Lyophilised tissue was successfully used for mouse oral immunisation and also could be converted into a  formula for possible human vaccination. However, long-term storage of that semi-product was successful only at lowered temperatures. Therefore, unrestricted long-term storage of lyophilised formula still requires some elaboration.So far obtained results indicate that it is possible to ef-ficiently preserve S-HBsAg in immunogenic VLP form in lyophilised stored plant tissue, providing rationale for further research on formulation of plant-derived an-ti-HBV oral vaccine.AcknowledgementsThis work was supported by grant No. N N302 157837 from Polish Ministry of Science and Higher Education.

Animal biotechnology in biomedicine

Lectures

L9.1

Stress preconditioning of cells for improved output in cloning, IVF and stem cell researchCsaba Pribenszky

Szent Istvan University Budapest, Hungary

In vitro procedures with gametes and embryos, including their culture, manipulation or storage, has always been hampered by their inherent sensitivity to environmental changes and external interventions. Consequently, most of the strategies to increase success rates in embryo pro-duction, cryopreservation, cloning and other procedures focus on reducing the inevitable damage caused by the manipulation, and, whenever it is applicable, imitating the natural physiology. In contrast to this principle, the efficiency of various procedures could be improved by preconditioning gametes and embryos with precisely ad-justed and applied sublethal stress treatment (reviewed in Callesen 2010, Pribenszky et al. 2010a, 2012). Precisely adjusted stress preconditioning in the form of hydrostatic pressure (HP) (at 600 times the physiological value) has been demonstrated to improve the cryosurvival of blas-tocysts in a mouse model (Pribenszky et al. 2005). Ad-vantageous effect on porcine, bovine and ovine embryos, oocytes and spermatozoa, embryonic stem cells and hu-man chord blood in combination with different in vitro techniques such as slow freezing, vitrification, in vitro fertilization and somatic cell nuclear transfer has also been documented (reviewed in Pribenszky et al. 2010a, Pribenszky & Vajta 2010). Studies showed, that DNA and membrane integrity has also been improved together with the reduced number of apoptotic cells and nuclei. Hydro-static pressure treatment of porcine oocytes prior to vitri-fication resulted in significantly increased blastocyst rates and higher cell numbers after warming, parthenogenetic activation and in vitro culture (Pribenszky et al. 2008a, b, Du et al. 2008, Lin et al. 2010). These results were con-firmed in studies on activated human oocytes, where dis-carded in vitro matured or non-fertilized oocytes showed increased survival and cleavage rates following HP treat-ment (Pribenszky et al. 2010b, Matyas et al. 2010).Despite these observations of a biological effect of the HP technique, a fundamental understanding of its mech-anism of action at the molecular level is still far from being complete. Different groups have shown that the HP-increased cryotolerance of embryos or spermatozoa

is concomitant with the altered expression of growth ar-rest-, oxidative stress-, cell pluripotency-, apoptosis- and lipid metabolism-related genes (Bock et al. 2010, Sique-ira Filho et al. 2011, Bogliolo et al. 2011). Huang et al. 2009a showed HP related changes in the proteome of treated porcine spermatozoa that was detected after the HP treatment, but remained throughout the cryopreser-vation procedure and after thawing as well, and might be associated with improved survival and fertilizing capacity resulting in higher pregnancy rate and litter size upon in-semination. Studies indicate that HP treatment results in significant changes of gene expression and highlight the need for a comprehensive transcriptome analysis in mam-malian cells. Recently, global gene expression profiling of HP-treated porcine oocytes, and the parthenogenetically activated or cloned embryos developed from these oo-cytes, identified several HP-responsive genes. Preconditioning gametes and embryos with a predefined, controlled sublethal stress (hydrostatic pressure) seems to enhance significantly cell survival and function after vari-ous in vitro procedures such as cryopreservation, in vitro culture or cloning.ReferencesBogliolo L., Ariu F., Leoni G., Uccheddu S. & Bebbere D. (2011) High hydrostatic pressure treatment improves the quality of in vitro-produ-ced ovine blastocysts. Reproduction, Fertility, and Development 23, 809–817.Callesen H. (2010) Challenge testing of gametes to enhance their viabili-ty. Reproduction, Fertility, and Development 22, 40–46.Du Y., Pribenszky C., Molnar M., Zhang X., Yang H., Kuwayama M., Pedersen A.M., Villemoes K., Bolund L. & Vajta G. (2008) High hydro-static pressure: a new way to improve in vitro developmental compe-tence of porcine matured oocytes after vitrification. Reproduction 135, 13–17.Huang S.Y., Pribenszky C., Kuo Y.H., Teng S.H., Chen Y.H., Chung M.T. & Chiu Y.F. (2009a) Hydrostatic pressure pre-treatment affects the pro-tein profile of boar sperm before and after freezing-thawing. Animal Reproduction Science 112, 136–149.Lin L., Luo Y., Sørensen P., Prætorius H., Vajta G., Callesen H., Priben-szky C., Bolund L. & Kristensen T.N. (2013) Effects of high hydrostatic pressure on genomic expression profiling of porcine parthenogenetic ac-tivated and cloned embryos. Reproduction, Fertility, and Development [in press] (http://dx.doi.org/10.1071/RD13037).Lin L., Pribenszky C., Molnar M., Kragh P.M., Du Y., Zhang X., Yang H., Bolund L., Callesen H., Machaty Z. et al. (2010) High hydrostatic pressu-re treatment of porcine oocytes induces parthenogenetic activation. Cel-lular Reprogramming 12, 475–480.Matyas S., Pribenszky C., Kovacs P., Rajczy K., Molnar K., Losonczi E., Molnar M., Kaali S.G. & Vajta G. (2010) Preconditioning oocytes by sublethal hydrostatic pressure stress in order to improve cryosurvival – animal models and human application. Proceedings of The 1st Inter-national Congress on Controversies in Cryopreservation of Stem Cells, Reproductive Cells, Tissue and Organs Valencia, Spain.Pribenszky C. & Vajta G. (2010) Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes’ and embryos’ performance? Reproduction, Fertility, and Development 23, 48–55.Pribenszky C., Du Y., Molnar M. & Vajta G. (2008b) Sublethal stress on porcine oocytes enhances the efficacy of ART procedures. Human Reproduction 23, 161.

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Pribenszky C., Du Y., Molnar M., Harnos A. & Vajta G. (2008a) In-creased stress tolerance of matured pig oocytes after high hydrostatic pressure treatment. Animal Reproduction Science 106, 200–207.Pribenszky C., Lin L., Du Y., Losonczi E., Dinnyes A. & Vajta G. (2012) Controlled stress improves oocyte performance-cell preconditioning in assisted reproduction. Reproduction in Domestic Animals 47, 197–206.Pribenszky C., Matyas S., Losonczi E., Stanca C., Bock I. & Vajta G. (2010b) Stress for stress tolerance: improving cell survival by sublethal stress treatment of eggs before vitrification – pilot study. Fertility and Sterility 9, S32.Pribenszky C., Molnar M., Cseh S. & Solti L. (2005) Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressu-re challenge. Animal Reproduction Science 87, 143–150.Pribenszky C., Vajta G., Molnar M., Du Y., Lin L., Bolund L. & Yovich J. (2010a) Stress for stress tolerance? A fundamentally new approach in mammalian embryology. Biology of Reproduction 83, 690–697.Siqueira Filho E., Caixeta E.S., Pribenszky C., Molnar M., Horvath A., Harnos A., Franco M.M. & Rumpf R. (2011) Vitrification of bovine bla-stocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression. Reproduction, Fertility, and Development 23, 585–590.

L9.2

Large animal models in biomedical researchMarlena Szalata1, 2, Daniel Lipiński1, 2, Joanna Zeyland1, Magdalena Boksa1, Hanna Przystałowska1, Bartosz Grześkowiak1, 3, Karol Tuśnio1, 3, Marzena Skrzypczak-Zielińska2, Ryszard Słomski1, 2, 3

1Department of Biochemistry and Biotechnology, Poznan University of Life Science, Poland; 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; 3NanoBioMedical Centre (NBMC) at Adam Mickiewicz University in Poznan, Poland

Large animal models has become a common expression in biomedicine and refers to livestock (pigs, goats, sheep, cattle, horses, donkeys), cats, dogs, non-human primates and sometimes rabbits. Large animal models are not as widely used as mice, flies, or nematodes, often require different research infrastructure than small rodents and higher funding. Historically, large animals have made many contributions to greater understanding of human health.Large animal research opens up the possibility to take frequent biopsies and samples, non-invasive monitoring by ultrasound without having to sacrifice the animal. The similarity in organ size in some animal models allows for better models of surgery, hemodynamics and possible fu-ture xenotransplantation. Large animals can be used for “pharming” to produce large quantities of useful proteins in milk of transgenic cows, pigs, sheep or goats. Using large animal models in biomedical research may also benefits in advantages for animal agriculture.We are currently involved in generating and analyzing genetically modified pig models for xenotransplantation and using of transgenic rabbits and goats as bioreactors. Another field of our research covers the assessment of closely related pigs.AcknowledgementsThe studies were financially supported by the projects N R12 0036 06/2009, N R13 0075 06/2009 and UDA/POIG.01.04.00-24-002/11-00.

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L9.3

Risk of viral infections in xenotransplantation and prevention strategiesUrszula Mazurek, Joanna Gola

Department of Molecular Biology, Medical University of Silesia, 1 Narcyzow St., 41-100 Sosnowiec, Poland

The shortage of organs and other tissues for use in human transplantation procedures is increasingly being supplied with material taken from pigs. Among several types of vi-rus found in pigs which might be transmitted to humans as a result of xenotransplantation, one of the greatest problems is presented by porcine endogenous retrovirus-es. Of particular concern in this respect are porcine en-dogenous retroviruses (PERVs). Three subtypes of PERV have been identified, of which both PERV-A and PERV-B have the ability to infect human cells in vitro. While the third subtype, PERV-C, does not show this ability, recom-binant PERV-A/C forms have demonstrated infectivity in human cell culture. PERVs have been shown to be able to infect human cells in vitro. However, this phenome-non has not been confirmed in vivo. In view of the risk presented by these observations, the International Xeno-transplantation Association recently recommended four strategies to prevent transmission of PERVs during xe-notransplantation. Screening xenotransplant recipients for PERV transmission can be done in a number of ways. Those include detection of PERV DNA, RNA, reverse transcriptase (RT) activity assessment and proteins as well as anti-PERV antibodies evaluation. In summary, de-tection of PERVs in xenotransplantations should be car-ried out with the necessary caution at all stages to elim-inate the possibility of transmission of PERV infection.

L9.4

Novel strategies in the somatic cell cloning of mammalsMarcin Samiec, Maria Skrzyszowska

National Research Institute of Animal Production, Department of Biotechnology of Animal Reproduction, Balice near Crakow, Poland

Nuclear transfer (NT)-derived offspring have already been obtained not only in livestock species or infertile crossbreds/bastards (cattle, sheep, goats, Chinese swamp buffalos, rabbits, horses and mules), but also in labo-ratory animals (mice, rats), companion animals (cats, dogs, domestic ferrets, horses), as well as unthreatened or endangered species living in the wild (gaur, mouflon, red deer, dromedary camel, African wild cats, sand cats, Euro-Asian gray wolves) and even extinct species (bucar-do mountain wild goat - Pyrenean ibex; a subspecies of Spanish/Iberian ibex). The propagation of mammalian specients by somatic cell nuclear transfer (SCNT) has important economical implications in biotechnology and biomedicine as so far has been demonstrated by genera-tion of transgenic cloned animals with either the ability to produce valuable recombinant human proteins in their body fluids (milk, urine, seminal plasma, blood) or the re-sistance to interspecies transmissible and heritable diseas-es. Pig embryo engineering (transgenesis combined with somatic cell cloning) is also a particularly important re-search field of assisted reproductive technologies (ARTs) due to increasing role of the porcine tissues and organs in xenotransplantology or creation of animal bioreactors synthesizing and secreting biopharmaceuticals. In spite of tremendous improvement in somatic cell cloning effi-ciency of mammals, high early-, mid- and late-gestation mortality rates of nuclear-transferred (NT) embryos/foe-tuses, cumulative occurrence of stillbirth, increased in-cidence rates of both sudden perinatal death and serious neonatal morbidity as well as numerous malformations (i.e., various congenital histo- and anatomopathological abnormalities) of cloned newborn offspring still often appear. Therefore, the effectiveness of SCNT technology in mammals remains unsatisfactory. Generally, the main cause of low somatic cell cloning efficiency in different mammalian species, with many severe developmental anomalies (anatomo-histological disorders in foetal and extrafoetal/placental tissues, immune dysfuntions) lead-ing to high pregnancy losses and neonatal deaths, may be an incomplete epigenomically-conditioned reprogram-ming of transcriptional activity for donor cell-descend-ed genes. The results of somatic cell cloning-associated experiments indicate that NT embryos of farm livestock species (pigs, rabbits, horses, sheep, goats, cattle) and laboratory animal species (mice, rats) rarely reach full pre- and/or postimplantation development, especially in relation to the number of reconstructed oocytes and/or to the number of embryos suitable for surgical transfer (reimplantation) into the reproductive tracts of surrogate

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recipient females. That is why, the attempts to increase the efficacy of SCNT in mammals have been made by many investigators lately. For these purposes, they have devel-oped the original and interesting methods or innovative solutions in this research field of genomic/genetic em-bryo engineering. Their efforts have been aimed at evolv-ing, adopting and optimizing the new systems for prepa-ration of nuclear donor cells (somatic cells) and nuclear recipient cells (oocytes) at different stages prior to and throughout the SCNT. These latter involve among others: 1) epigenomic transformation of cultured donor somat-ic cells and/or 2) in vitro maturing recipient oocytes as well as 3) activated nuclear-transferred oocytes using ei-ther non-specific histone deacetylase (HDAC) inhibitors (e.g., trichostatin A, scriptaid, valproic acid, oxamflatin, sodium butyrate) or non-specific DNA methyltransfere-ase (DNMT) inhibitors (e.g., 5-aza-2’-deoxycytidine, zebularine, S-adenosylhomocysteine). Moreover, these novel approaches involve the generation of NT-derived embryos applying such inventive systems of artificial stimulation for developmental program of reconstructed oocytes as: 1) pseudophysiological transcomplementary activation mediated by zygote-descended cytoplasts (i.e., zygoplasts) and 2) sequential physicochemical activation based on combination of various agents (electric pulses, calcium ionophore antibiotics, protein synthesis block-ers, selective and non-selective inhibitors of cyclin-de-pendent protein kinases; CDKs). Other approaches en-compass the production of cloned embryos with the use of completely new sources of either: 1) nuclear recipient cells (zygotes, single blastomeres of two cell-stage embry-os) or 2) nuclear donor cells [bone marrow- and periph-eral blood-derived multipotent mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs)]. All the above-mentioned strategies are applied to facilitate and accelerate the epigenomic reprogrammability for gene expression of donor cell nuclei that had been transplant-ed into cytoplasmic microenvironment of recipient cells and subsequently underwent the dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during the pre- and/or post-implantation development of cloned embryos. On the one hand, a better understanding of the molecular mech-anisms underlying epigenetic transcriptional reprogram-ming of the donor nuclear genome can contribute to im-provements in the efficiency of this process. On the other hand, it can give rise to enhancement of the developmen-tal potential of NT embryos. This will be helpful in solv-ing the problems resulting from mammalian somatic cell cloning and may open up new possibilities for common utilization of this technology in human biomedicine.AcknowledgementsThis study was supported by the Polish Ministry of Science and Higher Education as the statutory activity No. 02-1.00.1, which is realized from 2012 to 2014.

L9.5

Retroviral restriction factor gene expression in normal human dermal fibroblasts after porcine endogenous retrovirus infectionMałgorzata Kimsa, Magdalena Kimsa, Barbara Strzałka-Mrozik, Joanna Gola, Jolanta Adamska, Urszula Mazurek

Department of Molecular Biology, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland

Xenotransplantation can offer a potential solution for the shortage of allogeneic human organs, tissues and cells. However, possibility of pathogen interspecies transmis-sion from xenografts into humans should also be consid-ered. In pigs, one of the greatest problems is presented by porcine endogenous retroviruses (PERVs). Replication of the retroviruses can depend on the balance between cel-lular cofactors and host antiviral restriction factors, e.g. tripartite motif (TRIM) protein family, apolipoprotein B mRNA-editing catalytic polypeptides (APOBEC), bone marrow stromal cell antigen 2 (BST-2, tetherin) or zinc finger antiviral protein (ZAP). Many studies have shown that these factors may play an important role in safety of xenotransplantation, but it is still unclear whether PERVs may be inhibited by these factors.The present study focused on retroviral restriction factor gene expression in normal human dermal fibroblasts af-ter porcine endogenous retroviruses infection.The PERV infectivity was analyzed using a co-culture sys-tem of normal human dermal fibroblasts (NHDFs) and porcine kidney epithelial cells (PK15 cell line). Total RNA was extracted from cells using TRIzol reagent. Detection of the copy number of PERV A, PERV B DNA and PERV A, PERV B RNA was performed using real-time Q-PCR and QRT-PCR. The expression of retroviral restriction factor genes was compared between PERV-infected and uninfected NHDF cells using commercially available oli-gonucleotide microarrays HG-U133A 2.0. Validation of microarray data was performed by QRT-PCR.PERV A DNA was detected in NHDF cells after co-cul-tures with PK15 cell line, whereas PERV B DNA was not found. In turn, both PERV A and PERV B RNA were ob-served in NHDF cells. Typing of differentiation genes was performed in a panel of selected 95 retroviral restriction factor transcripts for 58 genes. The up-regulated tran-scripts were recorded for three genes: TRIM1, TRIM16, TRIM48.Our results suggest that the TRIM family may play an im-portant role in innate immunity to PERV infection.AcknowledgementsThis study was supported by the grant No. KNW-2-031/D/3/N from Medical University of Silesia, Katowice, Poland.

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Posters

P9.1

Infection of Human Cell Lines with porcine endogenous retrovirusesSabina Gałka, Dagna Sołtysik, Daria Matczyńska, Daniel Sypniewski, Tomasz Loch, Ewa Nowak, Ilona Bednarek

Department of Biotechnology and Genetic Engineering, Medical University of Silesia in Katowice, Narcyzow 1, 41-200 Sosnowiec, Poland

An origin of most human transplants is allotransplatation. Allografts are limited by graft donor deficiency. Under those circumstances xenotransplantation, being interspe-cies transplantation, may serve as an alternative solution. A domestic swine has been identified as the most suitable donor animal. However all pigs carry a viral particles called porcine endogenous retroviruses (PERVs). Gene sequenc-es of PERVs have been integrated into the genomes of all pig. It is well known that retroviral infection may involve significant changes in the human genes which are import-ant for the tumor growth and progression. Therefore, it’s really important to investigate, whether in vitro human cell lines are prone to PERVs infection.The aim of the study was to investigate the long-term ef-fects of PERVs infection on human cells and estimation of PERVs integration, stability in human genome and replication of new virions.Three different cell lines were used in our experiments: HeLa cells [ATCC nr: CCL-2], (human cervical cancer line); the human embryonic kidney cell line HEK 293 [ATCC: CRL-1573] and the porcine kidney cell line PK-15 [ATCC: CCL33 ], producing porcine endogenous retroviruses. All cells were maintained under standard condition (37°C, 5% CO2), in a medium containing 10% FBS. The culture supernatant of the PK-15 cells was fil-trated to remove cellular debris, subsequently was used as the inoculum to infect two other human cell lines. After exposure to PERVs, after each of cultures passage, during about 6 weeks, DNA and RNA were extracted, respective-ly: from cells and from supernatant. Presence of PERVs integrated in cells human genome was verified by PCR and virus replication was confirmed in cells’supernatant by RealTime™ RT-PCR with primers complementary to four different PERVs’ genes (env A, env B, gag, pol).Our results showed positive infection of both human cell lines – the sequences of PERVs genes were detected after cells infection, however, successive loss of PERV genes took place. Moreover, ability to produce and release ac-tive PERV particles was confirmed only in human em-bryonic kidney cell line. We assumed, that efficient PERV infection of human cells is possible in vitro, but is not sta-ble. Moreover, porcine endogenous retrovirus infects but does not replicate in HeLa cancer human cell line.AcknowledgementsThis work was supported by research grants of Polish Ministry of Sciences No. N R12 0036 06/7/2009; DOP-D/138/09.

P9.2

The influence of porcine endogenous retrovirus infection on the expression of toll-like receptor genes in human dermal fibroblasts Magdalena Kimsa1, Sławomir Dudek2, Małgorzata Kimsa1, Barbara Strzałka-Mrozik1, Joanna Gola1, Celina Kruszniewska-Rajs1, Jolanta Adamska1, Urszula Mazurek1

1Department of Molecular Biology, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland; 2Department of Pharmacognosy and Phytochemistry, Medical University of Silesia, Jagiellonska 4, 41-200 Sosnowiec, Poland

The therapeutic usage of porcine material in xeno-transplantation is not confirmed to be completely safe because of the presence of various pathogens such as porcine endogenous retroviruses (PERVs). Numerous studies have demonstrated that PERVs are transmitted to different human cell lines but it is still necessary to conduct research to explain the interaction between PERVs and human cells and to search for diagnostic markers of infection.Toll like receptors (TLRs) are specific immunologic markers. The understanding of the molecular details of effector immune responses and signal transduction activated by TLRs may provide guidelines for the devel-opment of novel diagnostic and therapeutic strategies in the aspect of PERV infection during xenotransplanta-tion. The present study focuses on the identification the effect of porcine endogenous retroviruses on human dermal fibroblasts by investigating the changes of the aspect of the expression of TLR and TLR-dependent genes. The PERV infectivity was analyzed in the co-culture sys-tem. Normal human dermal fibroblasts (NHDF cell line) were co-cultured for 5 days with normal porcine kidney epithelial cells (PK15 cell line). Genomic DNA was iso-lated from harvested cells using the salting out extraction method. Total RNA was extracted from cells using TRIzol reagent. The infectivity of PERVs was determined using real-time Q-PCR and QRT-PCR assay. The analysis of the expression profile of TLR and TLR-dependent genes was performed using HG-U133A 2.0 oligonucleotide mi-croarrays. The copy number of PERV A and PERV B RNA in NHDF cells (637.30±363.0; 77.00±58.0, respectively) were re-vealed after co-culture. However, only the copy number of PERV A DNA was observed (10.14±7.6). The study indicated that after PERV infection, NHDF cells showed a statistically significant increase of expression of TLR3 (p = 0.008, FC = 1.46). Among TLR3-dependent genes, 45 genes were differentially regulated with a fold change above 1.1 and pOf these 45 genes, 22 were up-regulated, 23 were down-regulated and 6 were found to be regulated by more than 1.5-fold.

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These findings contribute to the clarification of molecular mechanisms involved in the interactions between por-cine endogenous retroviruses and Toll like receptors of human cells. The expression changes in TLR-dependent genes may lead to further insights into their role in the PERV infection during xenotransplantation.AcknowledgementsThis study was supported by the project No. KNW-2-032/D/3/N from Medical University of Silesia, Katowice, Poland.

P9.3

Cyclosporine A and expression of genes associated with cell cycle in normal human dermal fibroblasts cultured in vitroGrzegorz Hibner1, Adam Wilczok1, Tomasz Janikowski2, Urszula Mazurek2

1Department of Biopharmacy, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland; 2Department of Molecular Biology, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland

The cell cycle or cell division cycle is a series of events that occur in eukaryotic cell, leading to its division. General-ly, these events can be divided into two, usually not very long, periods: interphase and division phase M. During interphase the cell grows, accumulating nutrients need-ed for cytokinesis and division of its genetic material (karyokinesis). Within the M phase the cell divides into two separate ones. Regulation of cell cycle contains the key elements, including the detection of DNA lesions and genetic material repair and various restriction points to prevent uncontrolled cell division. Molecular processes that control the cell cycle are ordered and oriented, which means that each process occurs in a sequential manner, and it is impossible to “reverse” the cycle. The object of our study was to assess the impact of cyclosporine A – a peptide with immunosuppressive activity widely used in transplant patients - on expression of genes associated with the cell cycle in normal human dermal fibroblasts (NHDF).Using oligonucleotide microarray technique HG–U133A 2.0 (Affymetrix) we compared transcriptional activi-ty of genes associated with the cell cycle in NHDF cells exposed to cyclosporine A (C = 100 ng/ml; t = 8 h) in relation to control cells. GeneSpring GX fluorescence signals analysis of 7845 probes (T test, asymptotic p-val-ue computation with Benjamini-Hochberg correction), which represented the expression of 5726 genes selected from the NetAffx Analysis Center database, demonstrat-ed the inhibited expression of 16 genes, including ASPM, BUB1B, CDC45, CDK1, FOS, RRM2, and TOP2A (p4.0).Understanding the molecular patterns, which charac-terize the impact of cyclosporine A on the cell cycle is important for determining the drug action mechanisms and resistance formation pathways. Such knowledge also enables the design of new, more effective drugs and in-creases the effectiveness and safety of post-transplant pa-tients treatment. Keywords: cyclosporine, cell cycle, NHDF, oligonucleotide microarray.AcknowledgementsThis study was supported by the grant No. KNW-1-047/D/1/0 from Medical University of Silesia, Katowice, Poland.

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P9.4

Expression of wnt/β-catenin signalling pathway target genes in endometrial cancerTomasz Janikowski1, Grzegorz Cwynar2, Agnieszka Jęda2, Grzegorz Hibner3, Joanna Orchel1, Andrzej Witek2, Urszula Mazurek1

1Department of Molecular Biology, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec, Poland; 2Department and Clinic of Obstetric and Gynecology, Medical University of Silesia, Medykow 16 St., 40-752 Katowice; 3Department of Biopharmacy, Medical University of Silesia, Narcyzow 1, 41-200 Sosnowiec

The wnt/β-catenin signalling pathway regulates multiple key cell processes. The influence of β-catenin on cell me-tabolism, differentiation and death is well known. Its sta-bilization is regulated by a multiple protein complex con-sisting of two enzymes: GSK3, CK1 and scaffold proteins: APC, Axin. When this complex is ineffective it result in uncontrollable accumulation of β-catenin in the cell and its entering to the nucleus as well as activating transcrip-tion factor TCF/LEF. This aberration can lead to cancer genesis by stimulating angiogenesis, progression and me-tastasis. The crucial role of β-catenin is well documented in various cancer types, but its role in endometrial cancer progression is still not fully explained.The analyses was made on endometrial cancer samples collected from patients during surgery and afterwards histopathologically classified accordingly to WHO stand-ards. From all samples total RNA was extracted using TRIZOL® reagent. Next was performed HGU 133A mi-croarrays (Affymetrix) analysis. There were obtained 22283 ID mRNA from which were selected 823 associated with wnt/β-catenin signalling pathway and for the statistical analysis GeneSpring GX was employed. Our results suggest that in endometrial cancer wnt signalling is down-regulated, but biological-ly significant genes in proliferation and angiogenesis are up-regulated by other pathways.

P9.5

Changes in the transcriptome of genes related to TNF induced pathways PERV infected cellsJoanna Gola, Barbara Strzałka-Mrozik, Magdalena Kimsa, Małgorzata Kimsa, Jolanta Adamska, Celina Kruszniewska- -Rajs, Urszula Mazurek

Department of Molecular Biology, Medical University of Silesia, 1 Narcyzow St., 41-200 Sosnowiec, Poland

Background. The interaction between PERVs and hu-man cells in inflammatory conditions is still unclear. The key factors of the immune response in the presence of in-fectious agents are many cytokines, including tumour ne-crosis factor (TNF). TNF acts through receptors TNFR1 and TNFR2, inducing MAPK, NFkB and caspase signal-ling cascades. Aim. The aim of this work was to assess the changes in the transcriptome of genes related to TNF signal trans-duction pathways infected with PERVs with and without lipopolysaccharide stimulation.Methods. Human fibroblasts (NHDF) were co-cultured with normal epithelial porcine kidney cells (PK15 cell line) in the presence of lipopolysaccharide (LPS). Total RNA was extracted with the use of phenol-chlorophorm method. The expression profile of genes related to the TNF signal transduction pathways was appointed with the use of oligonucleotide microarrays HG-U133A 2.0 (Affymetrix). Data analysis was performed with the use of GeneSpring 12.0 platform (Agilent Technologies). Genes were considered differentiating when p£0,001 and FC³3,0 (fold change).Results. In PERV infected fibroblasts in the pres-ence of lipopolysaccharide six genes were differentiat-ing: MAP3K7, MAP3K8, MAP4K3, BIRC5 (survivin), MEF2C and TANK, comparing to control. The only downregulated gene was BIRC5 – survivin (FC = 4.09). Conclusion. PERV infection and the presence of inflam-matory factor lead to significant changes in the mRNA level of survivin and MAPK genes.

141Eurobiotech 2013

P9.6

Abundance of blastocysts developed in vitro from porcine cloned embryos reconstructed with cell nuclei of adult bone marrow-descended mesenchymal stem cellsMarcin Samiec, Jolanta Opiela, Jurij Koseniuk

National Research Institute of Animal Production, Department of Biotechnology of Animal Reproduction, Balice near Crakow, Poland

The present study was carried out to explore the effect of adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) that were used as nuclear donor cells on the in vitro developmental potential of cloned pig em-bryos. In the somatic cell nuclear transfer (SCNT), oo-cytes that had reached the metaphase II stage following in vitro meiotic maturation (IVM) provided the source of nuclear recipient cells. The cumulus-oocyte complex-es (COCs) were matured in vitro for 20 to 22 h in TC 199 medium enriched with 10% foetal bovine serum (FBS), 10% porcine follicular fluid (pFF), 5 ng mL–1 recombi-nant human basic fibroblast growth factor (rh-bFGF), 10 ng mL–1 recombinant human epidermal growth fac-tor (rhEGF), 0.6 mM L-cysteine, 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP), 0.1 IU mL–1 human menopausal gonadotropin (hMG) and 5 mIU mL–1 porcine follicle-stimulating hormone (pFSH). The COCs were subsequently cultured for a further 22 to 24 h in the fresh IVM medium depleted of db-cAMP, hMG and pFSH. To form the ooplast-nuclear donor cell complexes, the previously enucleated oocytes were subjected to microinjection of either fully confluent/trypsinised ABM-MSCs (Group I) or foetal fibroblast cells (FFCs; Group II) under their zonae pellucidae. The ooplasts were then underwent simultaneous fusion and electrical activation (SF-EA). The electroactivated nu-clear-ooplasmic hybrids (clonal cybrids) were exposed to 5 µg mL-1 cytochalasin B (CB) for 2 h, followed by in vitro culture to morula and blastocyst stages in 0.4% bovine serum albumin (BSA)- and 10% FBS-supple-mented NCSU-23 medium for 6 to 7 days. A total of 293 and 234 enucleated oocytes that were electrically fused with either ABM-MSC nuclei or FFC nuclei were simul-taneously activated in Groups I and II, respectively. In Groups I and II, 275/293 (93.9%) and 208/234 (88.9%) oocytes were successfully fused/activated and selected for in vitro culture, respectively (P ≥ 0.05; χ2 test). Out of 275 and 208 cultured SCNT-derived embryos allot-ted into Groups I and II, 262 (95.3%) and 164 (78.8%) were able to divide, respectively (P2 test). The percentag-es of embryos that completed their development to the morula and blastocyst stages were 216/275 (78.5%) and 125/275 (45.5%) or 123/208 (59.1%) and 67/208 (32.2%) in Groups I or II, respectively (P2 test). In conclusion, porcine cloned embryos reconstituted with ABM-MSC

nuclei displayed significantly higher frequencies of both cleavage divisions and morula/blastocyst formation as compared to those reconstituted with FFC nuclei. AcknowledgementsThe research project was funded by the Polish National Science Centre resources allocated on the basis of decision number DEC-2011/03/D/NZ9/05537.

142 Eurobiotech 2013

P9.7

Scriptaid-induced epigenetic modulation of adult dermal fibroblast cells before their use for somatic cell nuclear transfer in pigsMarcin Samiec, Maria Skrzyszowska

National Research Institute of Animal Production, Department of Biotechnology of Animal Reproduction, Balice near Crakow, Poland

The current study was conducted to examine the in vitro developmental competences of porcine cloned embry-os reconstructed with oocytes receiving the cell nuclei of adult cutaneous fibroblast cells that had been epige-netically transformed by treatment with new-generation non-specific inhibitor of histone deacetylases, known as scriptaid. Cumulus-oocyte complexes (COCs) were matured in vitro for 20 h in Tissue Culture Medium 199 (TCM 199) enriched with 1 mM L–1 dibutyryl cyclic ad-enosine monophosphate (db-cAMP), 10 IU mL–1 equine chorionic gonadotropin (eCG), 10 IU mL–1 human chorionic gonadotropin (hCG), 10% porcine follicular fluid (pFF), 10 ng mL–1 recombinant human epidermal growth factor (rhEGF), 5 ng mL–1 recombinant human basic fibroblast growth factor (rh-bFGF) and 0.6 mM L–1 L-cysteine. Afterwards, the COCs were cultured for an additional 22 to 24 h in the db-cAMP- and eCG+h-CG-deprived medium. Prior to use for somatic cell clon-ing, the permanent fibroblast cell lines (between passages 1 and 3) that had been established from the primary cul-tures derived from ear skin biopsies of a prepubertal gilt were exposed to 350 nM L–1 scriptaid (6-(1,3-dioxo-1H, 3H- benzo[de] isoquinolin- 2- yl) - hexanoic acid hydroxy-amide) during 24-h serum starvation. Reconstruction of enucleated oocytes was accomplished by their electrofu-sion with epigenetically modulated fibroblast cells. Fu-sion of ooplast-somatic cell couplets was triggered using two consecutive DC pulses of 1.2 kV cm–1 for 60 μs. The same electric pulses that evoked the fusion of ooplast-nu-clear donor cell complexes were simultaneously applied to initiate the activation of reconstituted oocytes (clonal cybrids). Immediately after electrofusion/electroactiva-tion, clonal cybrids were incubated in North Carolina State University-23 (NCSU-23) medium supplemented with 5 µg mL-1 cytochalasin B for 1 to 2 h, followed by in vitro culture up to morula and blastocyst stages in NCSU-23/BSA/FBS medium for 144 to 168 h. The rates of divid-ing embryos (99/131; 75.6%A), morulae (73/131; 55.7%C) and blastocysts (38/131; 29.0%C) that originated from nu-clear-transferred oocytes reconstituted with adult dermal fibroblast cells undergoing scriptaid treatment were sig-nificantly higher than in the scriptaid-unexposed group (68/116; 58.6%B, 52/116; 44.8%D and 21/116; 18.1%D, re-spectively) [A,B PC,D P2 test]. Altogether, the improvements in not only cleavage activity of cloned pig embryos, but also their morula/blastocyst yields seem to arise from en-

hanced abilities for promotion of faithful and complete epigenetic reprogramming of scriptaid-treated adult cu-taneous fibroblast cell nuclei in a cytoplasm of reconsti-tuted oocytes.AcknowledgementsThis study was supported by the Polish Ministry of Science and Higher Education as the statutory activity No. 02-1.00.1, which is realized from 2012 to 2014.

143Eurobiotech 2013

P9.8

The effects of the alloferon and its analogues on the immune responses and heart beating in Tenebrio molitor beetleArkadiusz Urbański1, Elżbieta Czarniewska2, Szymon Chowański2, Jan Lubawy2, Mariola Kuczer3, Edward Baraniak1, Grzegorz Rosiński2

1Department of Systematical Zoology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland; 2Department of Animal Physiology and Development, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland; 3Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland

Alloferon for the first time was isolated from the blowfly Calliphora vicina larvae. This compound was character-ized by a strong antibacterial activity which protected blow fly larvae against adverse environmental conditions. In the last few years, increased an interest in using allofer-on in biomedicine. This is caused by discovery of anti-tumor activities and inhibition of Human herpesvirus, Papillomaviruses and Coxsackieviruses replication by alloferon. Furthermore, this peptide stimulated cytotoxic activity of natural killer cells, haemocytes apoptosis and interferon synthesis. For many biological activities, the most important is discovering the active structures which will affect the immune and circulatory system.In presented research were used alloferon and its 10 structural analogues, which were characterized by the highest proapoptotic activities. The significant differences in the actions of alloferon analogues on cellular and hu-moral responses were observed, but only two analogues decreased both immune responses. These compounds had structural modification in position 1 where native amino acid histidine was changed on synthetic aromat-ic amino acid Phe(p-Cl) or Phe(p-OMe). Both structural modifications in position 1 probably increase proteolytic stability of alloferon analogues. In addition, long-term immunomodulation effect may suggest a long half-life of these peptides in the haemolymph of T. molitor. Allofer-on and its analogues didn’t affect the heart beat frequen-cy. This result indicates a high specify activity of these compounds in T. molitor. Presented results enhance the knowledge about relationship between bioactivity and structure of alloferon. The discovery of alloferon active core is necessary for the design and synthesis of peptid-omimetics, which can be used for pest control and new antitumor therapy.

P9.9

Can we use the beetle heart as a model of cardiac ageing?Karolina Walkowiak, Monika Szymczak, Joanna Pacholska-Bogalska, Grzegorz Rosiński

Department of Animal Physiology and Developmental Biology, Faculty of Biology, Adam Mickiewicz University

Over the past several years insects have gained inter-est as model organisms used in studies of fundamental physiological processes. Some beneficial features, such as short life cycle, large number of offspring and low costs of rearing, but also genetic simplicity and less complex metabolism make insects promising alternative to mam-malian models in studying mechanisms of many human diseases.The aim of this study was to analyze the relationships in the heart performance during ageing between beetle Ten-ebrio molitor L. and humans.In vitro bioassays of a semi-isolated myocardium and video microscopic techniques combined with dynamic image analysis were used to trace mechanic and hemo-dynamic parameters of ageing beetle heart. We detected that parameters, such as ejection fraction, stroke volume and cardiac output of myocardium of ageing beetles un-dergo changes similar to the decline of cardiac function in elderly people with heart failure.Our results suggest that beetle myocardium seems to be an appropriate candidate as a model to study cardiac per-formance decline with age and disease and can be used e.g. in pharmacological or sport medicine researches.

144 Eurobiotech 2013

P.9.10

Identification of 77 novel SNPs within entire intron A of the pregnancy-associated glycoprotein 2-like gene subfamily (PAG2-L) in cross-breed pigs*Martyna Bieniek, Grzegorz Panasiewicz, Aleksandra Zamojska, Bozena Szafranska

Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn-Kortowo, Poland

Pregnancy-associated glycoprotein gene family (PAGs) encodes multiple chorionic polypeptide precursors that start expression in trophoblast cells during peri-im-plantation (Szafranska et al. 2006), the period of the highest embryonic mortality in many eutherians, in-cluding the domestic pig (Cetartiodactyla). Previous analysis of genomic DNA (gDNA) revealed conserved structure of PAG genes (approx. 9.5 kbp), consisting of 5’-flanking promoter sequence, 9 exons and 8 introns (A-H), within identified bovine PAG1 and PAG2 (Xie et al. 1995; Telugu et al. 2009) and porcine PAG2 – pPAG2 (Szafranska et al. 2001), so far. Previously in the pig genome, there were identified all 9 exonic and only 3 short intronic (C, G and H) sequences of the pPAG2-like (pPAG2-L) gene subfamily, however 5 still missing long introns (A, B, D, E & F) were identified more re-cently and their sequences deposited in the GenBank database (Bieniek et al. 2013).The aim of this study was to identify single nucleotide polymorphism (SNP) within 1093 bp novel sequence of intron A of the pPAG2-L gene subfamily, which was de-posited in the GenBank under accession no. KF471015 (Bieniek et al. 2013). Frequency (px) of homozygous and heterozygous genotypes (alleles) was calculated for each SNP. The gDNA templates were isolated from leukocytes of cross-breed pigs (N = 30) and then amplified paral-lel to positive control templates of Bacterial Artificial Chromosome clones: CH242-60C13 and CH242-294016 (BACPAC Resources, USA; http://bacpac.chori.org), con-taining the pPAG3 and pPAG6 or pPAG3 gene sequences, respectively. Amplicons were produced by PCR with the use of sensATG/Ex2as or IntrAseSr/IntrBasdo2 primers (~1050, 1150, 1250 bp or ~654 bp, respectively). Electro-phoreticaly separated pPAG2-L amplicons were cut out from agarose gel, purified and then sequenced in both sense and antisense directions (3130 Genetic Analyzer, Applied Biosystems). Automatically generated sequenc-es were verified qualitatively on the chromatograms with the use of Finch TV program (Geospiza, Inc., USA) and SNP variations were encoded according to IUPAC (In-ternational Union of Pure and Applied Chemistry). All sequences were compared by DNASIS v.3.0 software (Hi-tachi Software Engineering Co. Ltd., Japan).Among 77 novel SNPs (iA.8–iA.1042; intron A num-bering), 59 SNPs were identified within the 5’-donor flanking region of intron A (iA.8–iA.449), including one

insertion (iA.157/158ins c, px = 0.37), 32 transitions, 24 transversions and 2 SNPs, in which occurred both tran-sitions and transversions (iA.118 / iA.167). Within the 3’-acceptor flanking region (iA.558–iA.1042), 18 SNPs were identified, including 13 transitions and 5 transver-sions. Amongst all 77 SNPs, 70 SNPs were identified as dominant homozygous genotype (px = 0.57–0.96), 6 SNPs as dominant heterozygous genotype (px = 0.529–0.913) and 1 SNP (iA.92a>g) was equivalently frequent for ho-mozygous and heterozygous genotypes (px = 0.5). This is the first study that describes identification of 77 nov-el SNPs within the intronic A sequence of the pPAG2-L gene subfamily. Our data extend the current knowledge about domestic pig genome and polymorphism of the pPAG multigene family in cross-breed pigs.AcknowledgementsSupport MNiSW/NN311/066237.

145Eurobiotech 2013

P9.11

Identification of 5 novel SNPs within intron E & F of porcine Pregnancy-Associated Glycoprotein 2-like subfamily (pPAG2-L) in two hybrid lines*Grzegorz Panasiewicz1, Aleksandra Zamojska1, Martyna Bieniek1, Roman Jędryczko2, Bozena Szafranska1

1Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn-Kortowo; 2Veterinary Diagnostic Laboratory, 11-036 Gietrzwald

Multiple PAG gene family (PAGs) codes large group of secretory products identified in some Eutherians (95 cloned cDNAs; and 15 predicted gDNAs). So far, some PAG cDNAs have been already patented (USA), mainly for the Ruminantia or Carnivora taxa. However, structur-al exon-intron organizations of 3 genes have been iden-tified only: bovine PAG-1 & -2 (Xie et al. 1995; Telugu et al. 2009); and pPAG2 (Szafranska et al. 2001). Lately, still missing 5 intron sequences (A, B, D, E & F) of the pPAG2-L subfamily have been identified in cross-breed pigs (Bieniek et al. 2013).The aim of this study was to identify SNPs, mainly within intron E (between exon 5 & 6) and the largest intron F (between exon 6 & 7), amongst organizational structure of eight introns between nine exons of the pPAG2-L sub-family (Szafranska et al. 2001), in two swine hybrid lines (Hirschmann – HM; and Naima Per Ar Lan – PAL). All gDNA templates of pigs (N = 13) were isolated from leu-kocytes of the HM (n=7) or PAL (n=6), then subjected for modified PCR with the use of primer pair (IntrEsensd6 and IntrFasGap), amplifying internal region (1483 bp) of the pPAG2-L genes encompassing (288 bp of 3’-flanking acceptor end of intron E, 117 bp of entire exon 6, and 1078 bp of 5’-flanking donor of intron F; GenBank Acc. Nos.: KF527576/927, U41424/117 and KF537535/1455 bp, respectively). Obtained amplicons of the hybrid pigs, were examined parallel to commercial BAC clone CH242-60C13, as positive control template. Separated pPAG2-L amplicons were visualized by UV, cut out from agarose gels, purified, labeled (Big Dye® Terminator v.3.1) and sequenced in both sense and antisense directions (3130 Genetic Analyzer, Applied Biosystems). Amplicon sequences were in silico analyzed (Megablast or Blastn, NCBI) for homology discovery, due to the entire intron E & F sequences of the pPAG2-L (GenBank). Finally, fre-quency (px) of homozygous or heterozygous genotypes was calculated for each SNP.Within intron E, among 8 SNPs previously found in cross-breed pigs (CBP), presently we identified only 2 SNPs (E numbering; IU code): transitions iE.867g>a (R) and iE.872g>a (R) in HM, while iE.867R in PAL only. Three iE.867R genotypes (gg/ga/aa) were identified in both hybrid lines, similarly as in CBP, with dominant heterozygous (ga) genotype (HM px = 0.71 and PAL px = 0.5). The second SNP (iE.872R), was identified only

in HM hybrids with two genotypes only (ga/px = 0.43 and dominant gg/px = 0.57), in contrast to three (gg/ga/ad-ditional gt) in CBP. Within the entire exon 6 sequence, SNPs were not identified in both porcine hybrid lines, in contrast to 4 SNPs previously found in CBP.Within intron F, among 6 identified SNPs, 5 were novel in hybrids (F numbering; IU code); and only one transition iF.831c>t (Y) was identical, among 17 SNPs in CBP. This single SNP was dominant genotype (cc) in PAL (px = 0.6) and in HM (px = 0.7), in contrast to two genotypes (tt/tc) in CBP. The first novel transition iF.492t>c (Y), with dominant heterozygous (tc) genotype was identified in PAL (px = 0.8) and HM (px = 1). Second SNP, trans-version iF.493t>g/a (K/W), with genotype gg (px = 0.2) and dominant ga (px = 0.8) was identified in PAL; while only genotype ga (px = 1) in HM. Third, transversion iF.515a>c (M) was with three genotypes in PAL (aa/px = 0.4; ac/px = 0.4; cc/px = 0.2), and dominant (ac) in HM only. The last two SNPs, transition iF.516a>g (R) and trans-version iF.970g>t (K), with heterozygous genotypes (ag/px = 0.2 and gt/px = 0.2) were identified in PAL only.This is the first study describing initial discovery of nov-el SNPs within intron E & F of the pPAG2-L gene sub-family, presently identified in porcine hybrid lines. Our preliminary results provide novel data concerning poly-morphism of the pPAG multigene family, and increase current knowledge extending domestic pig genome data. Further study will allow to define some correlations between SNP-genotypes with important reproductive QTLs, then use as pre-selection markers of piglets for their economic growth required for breeding of the best progenitors among hybrid lines.AcknowledgementsSupported by NN311/066237.

146 Eurobiotech 2013

P9.12

Novel glycoprotein family within human placenta detected with polyclonals against recombinant or native porcine Pregnancy-Associated Glycoproteins*Marta Majewska1, aleksandra Zamojska2, Grzegorz Panasiewicz2, Martyna Bieniek2, Zbigniew Łaganowski3, Bozena Szafranska2

1Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, 10-082 Olsztyn; 2Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn-Kortowo; 3The Woman’s Health Clinic, 10-684 Olsztyn, Poland

Multiple PAG family (PAGs) has been previously iden-tified in various domestic and wild species (Szafranska et al. 2006). So far, we detected the PAGs in the pig (Ma-jewska et al. 2005, 2006), the European bison (Majewska et al. 2008), and three Camelidae species: the alpaca, C. bactrianus and C. dromedarius (Majewska et al. 2009, 2011, 2013). The PAGs belong to aspartic proteinase superfamily (AP), which includes different proteolyt-ic and lysosomal enzymes (i.e. pepsins or cathepsins). All AP members possess a two-bilobe structure with a cleft capable for short peptide binding. As a role of the PAGs is still unclear, it is however involved in proper embryo-maternal interaction and placenta development (Szafranska et al. 2007; Panasiewicz et al. 2007). Thus abnormal PAG expression reflects placental disorders. Therefore, the PAGs are routinely used as prenatal diag-nostic markers for various pregnancy tests (similarly to hCG-tests), based on the PAG concentration in periph-eral maternal blood or milk of ruminant species (US Patents; USA).The aim of this study was to identify PAG-like expression within hemochorial human placenta by heterologous de-tection with the use of two different rabbit polyclonals raised against recombinant porcine PAG2 antigen (an-ti-RpPAG2) or polyvalent polyclonals raised against na-tive pPAG-like (pPAG-L). Late placental tissues of three healthy women were collected after caesarean section and immediately stored at –70oC. The PAG-like immunore-activity was examined by immunohistochemistry (IHC) and Western blotting. Total proteins were extracted from homogenized placental explants and concentrated by ultrafiltration (MWCO >10 kDa). Extracted placental proteins were directly used for Western dot-blotting or separated by SDS-PAGE. Proteins were semi-dry trans-ferred onto nitrocellulose membranes and used for Western. Primary rabbit polyvalent anti-pPAG-L (1:300) or anti-RpPAG2 (1:50) polyclonals, characterised pre-viously (Szafranska et al. 2002), were used to identify molecular mass of human PAG-like proteins. The immu-no-complexes were detected with mouse anti-rabbit IgG monoclonals (1:100 000)–conjugated with alkaline phos-phatase and then visualized with BCIP/NBT substrates.

Cryosectioned human placental tissues were fixed, dehy-drated and used for heterologous dual fluorescent IHC (htdF-IHC) with the same polyclonals as for Western blotting. The PAG immuno-complexes within human placental sections were visualized with secondary goat anti-rabbit immunoglobulins (1:1000)–conjugated with Alexa 488 dye (495ext/519em nm), as the visualizing fluorochrome (green). Finally placental sections were counterstained with propidium iodide (red) to visualize nuclei.This is the first study identifying cellular localization of the PAGs in human placental proteome. The human PAG family (hPAGs) expression was found to be restricted to various trophectoderm (chorionic epithelium) cells only. The separated human placental proteins weakly reacted with polyvalent anti-pPAG-L polyclonals, but strongly with anti-recombinant pPAG2 polyclonals. Western blot-ting revealed dominant approx. 60 kDa hPAG isoform. The cross-reactivity of the anti-RpPAG2 polyclonals with the hPAGs revealed structural epitope similarities of the PAG isoforms in human placenta and other eutherian species.AcknowledgementsSupported by UWM (WBiB#528-0206-806 and WNM#1501.801).

147Eurobiotech 2013

P9.13

Antibacterial peptides from WPC hydrolisatePaulina Worsztynowicz, Dagmara Leśniak, Włodzimierz Grajek

Poznan University of Life Sciences

Introduction. The bioactive peptides derived from food proteins show antibacterial activity. Great interest among researchers antimicrobial peptides has milk protein. The reason for this is the fact that milk contains antimicrobial proteins: lactoperoxidase and lactoferrin but the hydroly-sis of whey protein could increase their spectrum antimi-crobial effects. It has been shown that the antimicrobial peptides have several advantages: quick kill the bacterial cells, and ex-hibit a broad spectrum of activity, including some resis-tant nosocomial pathogens [Rizzello et al. 2005]. Include demonstrated that peptides generated during proteolysis of lactoferrin have activity against both Gram (+) and Gram (–) example: Escherichia, Helicobacter, Listeria, Salmonella, Staphylococcus, yeast and filamentous fungi [Korhonen and Pihlanto. 2006]. AIM. The aim of this study was to investigate the antibac-terial activity of the whey protein hydrolyzate obtained in the process of enzymatic hydrolysis carried out with the participation of protease isolated from lactic acid bacte-ria.Materials and methods. Materials. A commercial whey protein concentrate (WPC80) from dairy cows was ob-tained from Spomlek. Lactococcus lactis purchased from Poznan University of Life Sciences, Department of Bio-technology and Food Microbiology.Enzyme production. In the isolation of bacterial prote-ases was used modification the method of Requena et al. (1993) with a slight modification. The resulting superna-tants were the source of proteases.Preparation of WPC hydrolysate. WPC was dissolved in 0,1M Tris-HCl, pH 8,0 at a concentration of 4% (w/v) and heated in 65°C for 30 min. The enzymatic hydrolysis was carried out at 40°C with an enzyme to substrate ratio (E/S) of 5:1. Samples were collected at different hydrolysis times (0 h, 8 h, 16 h, 22 h, 28 h, 40 h) and immediately heated in 100°C water bath for 5 min to inactivate the protease and stop the hydrolysis reaction. After the hy-drolysate solution was centrifuged at 4500x g for10 min in 4°C to remove non-hydrolysed protein. The superna-tant was collected and lyophilised.Determination of antibacterial effects. The antibacterial effect of WPC hydrolysate against E. coli, L. monocyto-genes and S. enteritidis was examined according to the method of Nonnecke and Smith (1984) with a slight modification. Briefly, 0.5 ml working inoculums of bac-terial cultures ware added to 40 mL nutrient broths and incubated in oscillation incubator for 12 h at 37°C. The cultures ware then diluted with physiological saline to a final concentration of approximately 1x106 cfu/ml and

used for the antibacterial assay. The growth medium con-sisted of (w/v) 1% peptone, 0.5% yeas extract and 0.5% sodium chloride. Each tube was filled with 4 ml growth medium, 0.5 ml bacterial inoculum and 0.5 ml WPC hy-drolysate (10 mg/ml). WPC hydrolysate was dissolved in physiological saline which was sterilised by filtration through a 0.22 mm membrane. Control assays contained all components except WPC hydrolysate. The mixtures were incubated at for 37°C 12 h. Viable bacterial counts of E. coli were obtained using the plate count method after being cultured on nutrient agar. Plates were incu-bated at 37°C for 24 h. The antibacterial effect of WPC hydrolysate and its fraction was defined as antibacterial rate again E. coli, L. monocytogenes and S. enteritidis was calculated by the following equation: Antibacterial rated (%) = (Nc – Ns)/Nc x 100 where Nc and Ns are the bac-teria numbers of the control group and the sample group, respectively.SEM analysis. Scanning electron microscopy (SEM) was used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides.Results. It has been shown that the studied strain L. lactis produced three kinds of proteases: cell wall-asso-ciated proteinase, intracellular and extracellular protein-ase. The antimicrobial potential of WPC hydrolyzed on E. coli, L. monocytogenes and S. enteritidis were de-termined. While hydrolysates for 8 h, 16 h, 22 h and 28 h did not show antibacterial activity, whey proteins hydrolyzed for 40 h by cell wall-associated proteinase, intracellular and extracellular proteinase exhibited an-tibacterial activity.

Animal biotechnology in agriculture

Lectures

L10.1

Leptin and leptin antagonists in research and biotechnologyArieh Gertler

Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, 76100, Israel

Superactive leptin antagonists (D23L/L39A/D40A/F41A, mutants) of mouse, human, rat or ovine leptins were de-veloped in our lab, expressed in E. coli, refolded and pu-rified to homogeneity as monomeric proteins. Pegylation of leptin antagonists resulted in a potent and effective long-acting reagents suitable for in vivo studies. In the present report we summarize the possible use of leptin antagonists as (a) research reagents in the study of bone development, autoimmune diseases, metabolic syndrome and T2DM with particular emphasis on creation of a nov-el, fast and reversible model of metabolic syndrome and T2DM in mice and (b) possible leptin blockers in various human pathologies such as uremic cachexia, inflammato-ry and autoimmune diseases, and cancer. In conclusion, recognition and mutagenesis of D23L of previously de-veloped leptin antagonists (L39A/D40A/F41A) enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. The possible use of leptin in agriculture was explored using small weight new born piglets comparable to human ba-bies with intra-uterine growth restriction (IUGR) as it is well established that low birth weight resulting from in-trauterine growth retardation (IUGR) is a risk factor for further development of metabolic diseases. It was found that (1) IUGR piglets present altered postnatal growth and increased adiposity; (2) IUGR piglets exhibit abnor-mal hypothalamic distribution of leptin receptors that may be linked to further disturbance in food-intake be-havior; and (3) postnatal leptin administration can par-tially reverse the IUGR phenotype by correcting growth rate, body composition, and development of several or-gans involved in metabolic regulation. Early postnatal 6-days leptin treatment induced also an increase in body weight and the relative weights of the liver, spleen, pan-creas, kidneys, and small intestine without any changes in triglycerides, glucose and cholesterol levels. This study highlights a new role for leptin in general developmental processes and may provide new insight into IUGR pathol-ogy and show that leptin supplementation can partially reverse the IUGR phenotype. Long-term follow-up study

is required to conclude whether short-term post-natal treatment of IUGR piglets may be beneficial to agricul-ture. Furthermore as pig appears to reproduce nearly all of the phenotypic pathological consequences of human IUGR and is likely to be more relevant than rodents in studies of neonatal development in humans

149Eurobiotech 2013

L10.2

Development of non-antibiotic treatment to prevent animal digestive tract from bacterial infectionsStefan Kwiatkowski1, Karl Dawson1, Maciej Gryszel2

1Alltech Biotechnology Center USA, Alltech Inc.; 2Faculty of Chemistry Warsaw University of Technology

Virulent strains of Escherichia coli and Salmonella cause serious infections of human and animal digestive and uri-nary tracts. These bacteria are present in feces that can contaminate water sources or feed if not proper housing of animals is in place. Antibiotics have been used as feed additives to prevent infections. Their vide use in food production caused bacterial resistance to antibiotics and their use was banned. Therefore new, effective, non anti-biotic feed additives that will not promote bacterial resis-tance are needed. The infection starts from bacterial cell adherence to ep-ithelial cells lining of animal or human digestive tract. E.coli uses type 1 pili which have mannose binding site (FimH protein) located at its tip. The FimH protein forms extended complex with α-D-mannose reach glycosyl groups of the epithelial cells proteins. The complex be-tween FimH protein and Man-α-(1→3)-(Man-β-(1→6)-Man-β-(1→4)-GlcNAc-β-(1→4)-GlcNAc have been crys-tallized and its 3D structure showed that only three: Man-α-(1→3)-Man-β-(1→4)-GlcNAc out of five monosaccha-rides are involved in complex interactions [1]. Aqueous solutions of α-D-Mannose and methylman-noside can partially inhibit infections [2] but only at very high concentrations (2.5%w/v). Alltech products: BioMos and Actigen which are reach in mannose poli- and oligosaccharides are also able to bind to these bac-teria [3] but they are not as effective as antibiotics. Man-α-(1→3)-Man-β-(1→4)-GlcNAc trisaccharide, which has the highest, known inhibitory activity [2] is not commercially available. Published, multi step syntheses of this compound are able to supply only minute quan-tities of it, and thus the product has never been proved to work in animals challenged with bacterial infections. Our goal is to develop feasible method for a large scale production of this material and use it in animal trials. We assume that irreversible binding of bacteria by this water soluble trisaccharide will prevent digestive tract coloniza-tion without causing bacterial resistance. Two monosaccharides: α-D-mannose (from yeast cell wall) and 2-acetamido-2-deoxy-D-glucose (from lobster shells) are used as starting materials. They are convert-ed into Man-α-(1→3)-Man-1-OH heptaacetate and Glc-NAc-1,3,6-triacetate in simple and high yield transforma-tions. Condensation of the mannose disaccharide using trichloroacetonitrille with the GlcNAc derivative yields fully protected with acetate groups: Man-α-(1→3)-Man-β-(1→4)-GlcNAc trisaccharide, which is than transester-ified (with methanol) to yield the desired trisaccharide.

References[1] Wellens A. et al., PLOS one 3(4):e2040 (2008).[2] Neeser J-R. et al., Infection and Immunity 52(2): 433.[3] Parks C.W. et al., Poultry Science 84(12):1967–73.

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L10.3

ESC technology in farm animals – current state of knowledge and prospects for the futureZofia Madej

University of Life Sciences Poznan, Department of Genetics and Animal Breeding

The establishment of stable embryonic stem cell lines (ESC) from farm animals creates a potent resource for both agricultural and biomedical research. ESC technol-ogy could greatly improve the efficiency of transgenic an-imal production. The benefits would be both agricultural (animals resistant to certain diseases or producing thera-peutic proteins) and medical (creating animal models of human diseases or testing the efficiency of gene therapy). However, despite many years of intensive studies, true ESC lines have been derived only from mouse (Evans, 1981), rat (Buehr, 2008) and human (Thomson, 1998) em-bryos. In case of bovine and other animal species, such as pigs, sheep, goats, rabbits, horses, hamsters, minks, dogs and cats only ESC-like cell lines have been obtained. This is mainly due to the limited knowledge about the signal-ling pathways and species specific factors responsible for maintaining pluripotency in these species. Existing cell lines show several major deficiencies raging from short life in culture to the lack of controlled pluripotency. It is now evident that although there are some shared mech-anism regulating pluripotency and self-renewal in the ESC, mammalian species differ in morphology, surface marker and gene expression patterns. Presently scientif-ic efforts concentrate on (1) establishing the optimal de-velopmental stage (timing) to isolate primary embryonic cell lines, (2) finding the appropriate markers that would enable the identification of true ESC and (3) identifying the right set of conditions to propagate and sustain these lines in culture.AcknowledgementsResearch supported by: Foundation for Polish Science (FNP) Programme Homing HOM/2009/B and The Ministry of Science and Higher Education research grant: MNiSzW:4429/B/P01/2010/39.

L10.4

Animals as a tool for metabolic processes modelingEwa Ocłoń, Krystyna Pierzchała-Koziec, Joanna Zubel-Łojek, Anna Latacz

University of Agriculture in Crakow, Department of Animal Physiology and Endocrinology, al. Mickiewicza 24/28, 30-059 Crakow, Poland

Animal models represent important tools for investigat-ing the physiological and pathological mechanisms of metabolic processes. The main reason of using animal models for regulation of metabolism is that the results can be extrapolated of the humans. Traditionally, an-imal models can be grouped into the following catego-ries: spontaneous models; genetically modified models; induced or experimental models and negative models [3]. Continuous development of genomics, proteomics, biotechnology and bioinformatics changed trends in ap-plying of animal models. For instance, transgenic tech-nique allows to study the role of specific gene products involved in the physiological regulation, development or pathogenesis [5].Most of the available models are based on rodents because of their small size, short generation interval, easy avail-ability and economic considerations [1]. Unfortunately, rodents models differ from human metabolic processes in many aspects. For example, normal mouse lipoprotein profiles have essentially atheroprotective HDL, whereas human lipoprotein profiles contain originally atherogen-ic low-density lipoproteins (LDL) [7]. For modelling the metabolic disorder, obese mice combined with dyslip-idemia, hypertension and/or elevated glucose levels has been determined. In recent years, large number of new genetically modified animal models including transgenic, generalized knock-out and tissue-specific knockout mice or rat have been engineered for the study of metabolic processes and disorders (e.g. mice IRS2-KO, AG4KO, MG4KO, AMG4KO, IR/IRS-1) [2]. Unfortunately, none rodents model can exactly mimic all aspects of human metabolism [7].The selection of an animal model is difficult and might lead to misinterpretation of data or even to the wrong conclusions. Therefore, non-rodent models of metabolic diseases are urgently needed as a valuable supplement to rodents for improving an extrapolation of study results. The pig has many similarities in structure and function to humans, including size, feeding patterns, digestive phys-iology, dietary habits, propensity to obesity and social behaviours [6]. Additionally, pig cardiovascular anato-my and physiology, in combination with the porcine re-sponse to atherogenic diets, have made them a universal-ly standard model for the study of atherosclerosis. Their gastrointestinal anatomy has some significant differenc-es from that of humans; however, the physiology of the digestive processes has made them a valuable model for studying disorders. Therefore, the Gottingen, Yucatan,

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and Ossabaw pigs breeds have been used widely for in-vestigations of adipose tissue activities, glucose and lipids metabolism, obesity or cardiovascular diseases [4].Undoubtedly, there are some limitations like expensive-ness, practical difficulties and ethical considerations as-sociated with the use of large animal species (pigs, dogs, monkey). However, in order to better understanding the regulation of metabolism mechanisms; discover new tar-gets for the treatment of metabolic diseases in humans, these models are required [5].AcknowledgementsSupported by DS 3243/KFiEZ/13.References[1] Artinamo A., Castro M. (2009) Experimental rat models to study the metabolic syndrome. Br. J. Nutr. 102(9): 1246–53.[2] Gilliam L.A., Neufer P.D. (2012) Transgenic mouse models resistant to diet-induced metabolic disease: is energy balance the key? Pharmacol. Exp. Ther. 342(3): 631–6.[3] McMurray F., Moir L., Cox R.D. (2012) From mice to humans. Curr. Diab. Rep. 12(6): 651–8.[4] Neeb Z.P., Edwards J.M., Allosh M., Long X., Mokelke E.A., Sturek M. (2010) Metabolic syndrome and coronary artery disease in Ossabaw compared with Yucatan swine. Comp. Med. 60(4): 300–15.[5] Speakman J., Hambly C., Mitchell S., Krol E. (2008) The contribution of animal models to the study of obesity. Lab. Anim. 42(4): 413–32.[6] Spurlock M.E., Gabler N.K. (2008) The development of porcine models of obesity and metabolic syndrome. J. Nutr. 138(2): 397–402.[7] Tschop M., Heiman M.L. (2001) Rodent obesity models: an overview. Exp. Clin. Endocrinol. Diab. 109(6): 307–19.

Posters

P10.1

Cytogenetic mapping of the HSPB genes in the domestic BovidsBarbara Danielak-Czech, Katarzyna Kruczek, Anna Kozubska-Sobocińka, Barbara Rejduch, Agnieszka Bąk

Department of Animal Cytogenetics and Molecular Genetics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice near Crakow, Poland

The stress response involving up-regulation of small heat shock proteins is an important mechanism to deal with harmful conditions such as hyperthermia, hypoxia or oxidative stress. The sHSPs expressed in the nervous system exhibits a clear neuroprotective potential, where-as the mutations of genes encoding these proteins lead to cell dysfunction associated with myopathies, motor neuropathies and neurodegenerative disorders. The aim of this study was the cytogenetic mapping of five HSPB genes encoding small heat shock proteins involved in neurodegenerative processes in the domestic Bovids: cat-tle, sheep and goat. Clones, containing sequences of the HspB1, HspB2, HspB5, HspB6 and HspB8 genes, were de-rived from the CHORI-240 Bovine BAC library (http://bacpac.chori.org/librariesphp) and used as probes which facilitated the successful assignment of these genes to the bovine (BTA), ovine (OAR) and caprine (CHI) chromo-somes by FISH technique. Because it was not possible to select separate BAC clones for the HspB2 and HspB5 genes (due to their close proximity in the mammali-an genome), thus the same clone containing sequences of both these genes was used. In this study we present localizations of the above mentioned five HSPB genes, respectively, in BTA and CHI genome regions: 25q22, 15q14-q21, 18q24 and 17q24-q25 as well as OAR 24q22, 15q21-q21, 14q24 and 17q24-q25 corresponding regions. The HspB2 and HspB5, two small heat shock genes lo-cated adjacently in the vertebrate genome, were mapped to the same chromosome band – 15q14-q21. Similarly in humans, these two related genes, composed of two and three exons, respectively, are located in HSA11q22-q23 genome region, arranged in a head-to-head manner (with an intergenic sequence about 1 kb) and transcribed in the opposite direction. All the localizations presented in this report were in agreement with their physical positions in the human genome, based on the human-cattle compara-tive chromosome-painting map (ZOO-FISH). In order to better understand the function of the HSPB gene family in livestock mammals, further research should be under-taken, especially in animals exhibiting a neuropathic and neurodegenerative disorders.AcknowledgementsResearch work financed from National Science Centre funds, author’s project no. N N311 082540.

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P10.2

Chromosomal assignment of the small heat shock proteins genes in the pig genomeBarbara Danielak-Czech, Katarzyna Kruczek, Anna Kozubska-Sobocińska, Barbara Rejduch, Agnieszka Bąk

Department of Animal Cytogenetics and Molecular Genetics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice near Crakow, Poland

The small heat shock proteins (sHSPs) exert housekeep-ing role in normal cell metabolism as well as protective functions under stress conditions such as exposure to elevated temperature or toxic environmental and met-abolic products. Developmentally regulated expression of sHSPs is required for differentiation of nervous cells whereas the mutations of genes encoding these proteins are responsible for desmin-related myopathies or neuro-degenerative and autoimmune diseases. The aim of this study was the chromosomal localization of five sHSPs genes involved in neurodegenerative processes in the pig genome. Clones, containing sequences of the HspB1, HspB2, HspB5, HspB6 and HspB8 genes, were obtained from the CHORI-242 Porcine BAC library (http://bacpac.chori.org/librariesphp). The clones, selected based on in-formation about BAC-end sequences (BES) (http://www.sanger.ac.uk/Projects/S_scrofa/BES.shtml), were used as probes in the FISH experiments. It was not possible to select separate BAC clones for the HspB2 and HspB5 genes due to their close proximity in the pig genome, thus the same clone containing sequences of both these genes was used. The genes were assigned to the following pig chromosome (SSC) regions: HspB1 (SSC3p15), HspB2 (SSC9p21), HspB5 (SSC9p21), HspB6 (SSC6q12) and HspB8 (SSC14q21). The HspB2 and HspB5 loci, clustered at the distance of above 0.6 kb in the pig genome (http://www.ncbi.nlm.nih.gov/gene/), were identified in the same SSC9p21 chromosome band. Similarly in humans, these two related genes are located in HSA11q22-q23 genome region and arranged in a head-to-head manner with an intergenic sequence of less than 1 kb, raising a possibility of shared regulatory elements for their expression. The human HspB2 and HspB5 genes (composed of two and three exons, respectively) are transcribed in the opposite direction and expressed in most cells under physiological conditions. However, recent studies point out that they both are dispensable for cardiac function and mainte-nance of myocardial integrity as well as suggest their im-portant neuronal function under stress conditions. The experiments involving genomic context and structure of porcine sHSPs genes provide a compelling rationale of fu-ture biomedical studies to define the complete interaction of small heat shock proteins in cardiac and neuronal cells under both basal and stress conditions.AcknowledgementsResearch work financed from National Science Centre funds, author’s project No. N N311 082540.

P10.3

Comparative mapping of UCP2 and UCP3 genes in the pig genomeAnna Kozubska-Sobocińska, Agnieszka Bąk, Barbara Danielak-Czech, Barbara Rejduch, Małgorzata Miszczak

Department of Animal Cytogenetics and Molecular Genetics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice near Crakow, Poland

Uncoupling proteins 2 and 3 (UCP2 and UCP3) belong to a family of mitochondrial transmembrane carriers, which participate in processes of thermoregulation and energy metabolism. UCP2 gene is expressed in almost all mam-malian tissues, which suggests that it plays a functional role in global energy metabolism in the body, whereas UCP3 gene is predominantly expressed in mammalian skeletal muscle and brown adipose tissue. In humans UCP2 and UCP3 genes form a cluster localized in the dis-tal q13-14 segment of chromosome 11. Studies of UCP2 and UCP3 genes on the level of nucleotide sequences re-vealed the relatively high conservation among different species. Comparative analyses of pig and human UCP2 genes showed 86% identical nucleotide sequences, so ge-netic conservation of these genes made it possible to use human molecular probes in interspecies in situ hybridiza-tions to ascertain physical localization of UCP2 and UCP3 genes on pig chromosomes. For this purpose, cross-spe-cies in situ hybridizations (FISH) with human, commer-cial, fluorescent labelled probe (R110 probe, Q-BIOgene – UK) specific for human genome region HSA11q13-14 containing loci of UCP2 and UCP3 genes were carried out on pig metaphase chromosomes. Fluorescence signals obtained were observed in Axio Imager.D2 (Zeiss) flu-orescence microscope equipped with Axio Vision com-puter-assisted image analysis system Axio Vision. In an effect of ZOO-FISH technique red fluorescence signals in SSC9p21-24 pig chromosome region were obtained, which enabled to assign localization of UCP2 and UCP3 genes on physical pig genome map. Cytogenetic local-ization of UCP2 and UCP3 genes on pig chromosomes confirmed results of previous physical mapping with ap-plication of somatic cells hybrid (RH) panels as well as ge-netic mapping on the basis of linkage analysis. Cross-spe-cies in situ hybridizations carried out confirmed con-served nature of the linkage group containing UCP2 and UCP3 genes as well as homology of HSA11q13-14 and SSC9p21-24 chromosome regions in which these genes were localized in humans and pigs. The results obtained may contribute precise pointing out of evolutional rear-rangements concerning chromosomes complied in com-parative mapping.AcknowledgementsThis study was conducted as part of NRIAP statutory activity, projects No. 04-2.01.1, 04-6.03.1.

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P10.4

Visfatin as a bioindicator of endothelial cells dysfunctionEwa Ocłoń, Krystyna Pierzchała-Koziec, Joanna Zubel-Łojek, Anna Latacz

University of Agriculture in Crakow, Department of Animal Physiology and Endocrinology, al. Mickiewicza 24/28, 30-059 Crakow, Poland

Visfatin is a protein with several suggested functions: cy-tokine, enzyme as well as adipokine. This adipokine, also known as extracellular pre-B-cell colony-enhancing fac-tor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is peptide whose circulating levels are enhanced during metabolic disorders [1]. The study has demon-strated that visfatin may exert direct destructive actions on the cardiovascular system, including cell proliferation, monocyte/macrophage activation, vascular inflamma-tion and remodeling, all of them leading to the develop-ment of atherosclerosis [2].Thus, the aim of the study was to determine the synthe-sis and secretion of visfatin from endothelial cells in re-sponse to hyperglycemia. Additionally, the adipokine was estimated as an indicator of endothelium dysfunction.Human aortic endothelial cells (HAEC) were cultured in a standard medium (M 199) supplemented with LSGS kit (Invitrogen). The experiment was performed two times on fourth passage of HAEC culture. The cells were incubated for 24 hours with glucose in concentrations 22.2 mmol/l. After completing the experiment, total RNA was isolated from cells using Ambion® Cells-to-CT™ Kits (Life Technologies). Reverse transcription was undertak-en using High Capacity Reverse Transcription Kit (Life Technologies). Quantitative PCR analysis was performed using StepOnePlus Real-Time PCR System (Life Tech-nologies) with the Universal Master Mix and TaqMan chemistry (Life Technologies). Visfatin concentration was measured using a commercial ELISA kit (BioVendor R&D), according to the manufacturer’s protocol.The activity of aortic endothelial cell negatively changed in response to hyperglycemia. The cells proliferation was decreased by 36,9% compare to control group (pAcknowledgementsSupported by DS 3243/KFiEZ/13.References[1] Fukuhara A., Matsuda M., Nishizawa M. (2005) Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science. 307(5708): 426–430.[2] Stam F., van Guldener C., Schalkwijk C.G. (2003) Impaired renal function is associated with markers of endothelial dysfunction and increased inflammatory activity. Nephrology Dialysis Transplantation 18(5): 892–898.

P10.5

An universal method for the quantitative determination of cattle and horse components contaminating raw and processed pork meatMałgorzata Natonek-Wiśniewska, Piotr Krzyścin, Katarzyna Kruczek

Department of Animal Cytogenetics and Molecular Genetics, National Research Institute of Animal Production, Balice, Poland

Species identification of pork meat adulterated with beef and horse meat is an important factor in checking the reliability of food product information provided by the producer. Samples of pork adulterated with 0.1% to 80% beef were analysed. DNA was isolated using the Ax Food kit (A&A Biotechnology). This method allows for quanti-tative determination of identified species using standard curve and TaqMan MGB probes by real-time PCR. A test that uses short amplicons (69 bp) in the gene encoding ATPase6 was developed to identify bovine components. The primer and probe sequences were as follows:F/Bov(ATPase6)

5’-CTGTGAGCAGGAGCCGTAATT

R/Bov(ATPase6)5’-TGGTAAGAAATGGGCAAGTGATG

P/Bov(ATPase6)5’-Vic ATTCCGCAATAAAAC

The primers and the probe that detect horse components were taken from TANABE et al. (2007), Biosci. Biotech-nol. Biochem., 71; 3131-3135. A 75-bp cytB fragment of horse mtDNA was amplified. The limit of quantitation (LOQ) for the beef and horse component in pork meat is 0.2%.The small size of the amplicon used in the above analyses is useful for detecting species composition of processed food products with degraded DNA. For this reason, the method is applied to analyse adulterations in both raw and processed meat.

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P10.6

A real-time quantitative PCR detection method for beef, pork, chicken and mutton components in foodsMałgorzata Natonek-Wiśniewska, Piotr Krzyścin, Katarzyna Kruczek

Department of Animal Cytogenetics and Molecular Genetics, National Research Institute of Animal Production, Balice, Poland

In order to determine species composition of food prod-ucts for humans as well as pet food, a method for rap-id and sensitive identification of their components was developed. Species specific primers and TaqMan probes were designed on mitochondrial DNA: ATPase6 for cat-tle, cox1 for sheep and pigs, and 16SrRNA for chickens. In the case of chicken and sheep identification, the pro-posed primers amplify the DNA of the species for which they were designed. When identifying cattle and pigs, a cross-reaction was found between these species during the 35th reaction cycle. However, this did not hamper the analysis of the results because during this cycle a reaction takes place for the identified product at a level of 0.05% and at 100% for the second species.The limit of quantitation for chickens and sheep has not been determined, but it is lower than 0.1%. The limit of quantitation for cattle and pigs, due to the cross-reaction, is 0.1%.The developed method is suitable for species identifi-cation of raw meat, processed meat and components of meat-and-bone meals subjected to thermobaric treat-ment at a temperature of 133°C and pressure of 3 bar for 20 minutes.

P10.7

New polymorphisms in regulatory regions of CAPN3 gene in broiler chickensKatarzyna Piórkowska2, Joanna Doktor3, Katarzyna Połtowicz3, Katarzyna Ropka-Molik2

1Laboratory of Genomics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland; 2Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland

The calpains are the enzymes belong to calcium-depen-dent, non-lysosomal cysteine proteases expressed in mammals and many other organisms. The CAPN3 gene encodes a major intracellular protease, which is muscle specific. In chickens it is localized on chromosome 5. The calpains have a high capacity degradation of cytoskeletal and muscle fibers proteins. Therefore they play an im-portant role in fusion of mioblasts, proliferation, growing and migration of cells. The CAPN3 has been chosen as candidate gene associated with meat quality in chickens. Consequently the aim of our study was to identified new polymorphisms in regulatory region of CAPN3 gene and examine their impact on CAPN3 transcript abundance in breast muscles. In experiment used broilers of two genet-ic lines: fast and slow-growing. As a screening method the High Resolution Melting (HRM) was used. The poly-morphisms were identified with on Beckman Coulter se-quencer based on the Sanger method. The CAPN3 gene expression was estimated on Real Time 7500 Applied Bio-systems using succinate dehydrogenase complex; subunit A (SDHA) and 60S ribosomal protein L4 (RPL4) genes as endogenous control. Statistical and linkage analyses were performed using SAS Enterprise. Four new poly-morphisms were found. One in promoter region c. –450 G > A and three in 3`UTR (c. 176* C>T, c. 137*_147* del., c. 144* G > C) region. One of them in 3`UTR region was deletion of 11 nucleotides (CAGCCCTGCTT). New poly-morphisms were identified by used restriction enzymes ScrFI, BslI, AcuI, HpyHV, respectively. The frequency of polymorphisms found in 3`UTR region was similar in both lines. Nevertheless, in fast growing line appeared more genotypes than in slow growing according to c. C > T 176* and c. G > C 144* polymorphisms. Moreover, both broiler lines were in Hardy-Weinberg disequilibri-um of c. –450 G > A polymorphism, although this poly-morphism does not change any transcription binding site. Analysis of effect of new polymorphisms on CAPN3 gene expression showed, that in fast growing lines the chickens with GG genotype according to c. –450 G > A polymorphism characterized with the highest CAPN3 gene expression. Other polymorphisms in 3`UTR region seem not effect on CAPN3 gene expression.AcknowledgementsThis work was supported from National Research Institute of Animal Production statutory activity, research project No. 01-4.02.1

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P10.8

Different culture media affect growth rate and morphology of porcine bone marrow-derived mesenchymal stromal cells- the preliminary results of media screening suitable for both porcine MSCs and bovine embryo in vitro cultureJolanta Opiela, Michał Bochenek, Joanna Romanek, Zdzisław Smorąg

Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice near Crakow, Poland

The presented results are the part of the experiment which aims to study the co-culture of bovine embryos with por-cine mesenchymal stem cells. We assume that the stem, multipotencial nature of the MSCs used for co-culture systems would significantly improve the bovine embryo in vitro development. Therefore, as a result the significant enhancement of in vitro embryo production efficiency is expected. So far, the in vitro culture of bovine embry-os along with MSC as a feeder layer was applied in two media (MENEZO B2 + 2,5% fetal calf serum (FCS) and SOF + 10% estrus calf serum (ECS), however the em-bryo development was blocked at the morula stage. The quality of MSC was noticeably decreased which indicated the suboptimal culture conditions. This prompted us to explore for the medium  suitable for both porcine MSCs and bovine embryo in vitro culture.MSCs were isolated from porcine bone marrow and expanded in DMEM supplemented with 10% FCS. Selected MSC surface markers (CD31, CD34, CD90) were analyzed by flow cytometry to confirm their mesenchymal characteristics. Then, positively verified MSCs were cultured in nine established cell culture media: TCM 199 + 10% FCS, NCSU-23 + 10% ECS, TCM 199 + 2,5% FCS, NCSU-23 + 2,5% FCS, MENEZO B2 + 10% FCS and MENEZO B2 + 2,5% FCS, SOF + 10% ECS, G2 + 2,5% FCS, DMEM/F12 + 2,5% FCS. The MSC were cultured in two systems: in a dish with 3 ml of medium and the 4-well dish in 0.5 ml of medium under mineral oil. MSC morphology and proliferation rate were observed on Days 3 and 7 of in vitro culture in all of analyzed media. The cells in each analysed media were given a score from 1 to 5 subjectively where 1 indicates poor or low and 5 indicates excellent and high.The flow cytometry of MSC markers proved their mes-enchymal origin. The positive expression of the CD90 was observed in 99.8% of analyzed cells and the negative expression of CD31 and CD34 antigens was observed in 3.8% of analyzed cells. Cell morphology and proliferation rate varied importantly between the media. The best cell morphological quality and proliferation rate were ob-served in TCM 199 and NCSU-23. Interestingly, the least suitable media for MSC culture turned out to be Menezo

B2 and SOF, which are the media routinely used for bo-vine embryo culture.The choice of MSCs expansion medium can have a sig-nificant influence on both growth and cell morphology which is of fundamental importance for their imple-mentation in other biotechnological procedures like e.g. embryo in vitro culture. The in vitro co-culture system of embryos with MSCs requires further research.AcknowledgementsThis research was supported by the Statutory Activity of National Research Institute of Animal Production 02-4.03.1 and by the Polish National Science Centre resources allocated on the basis of decision number DEC-2011/03/D/NZ9/05537.

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P10.9

The impact of long-term in vitro culture and differentiation of porcine mesenchymal stem cells (pMSC) on the cell cycle distribution and nuclear DNA profileJolanta Opiela, Marcin Samiec, Michał Bochenek, Joanna Romanek

Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice near Crakow, Poland

The aim of experiment was to determine the impact of long-term in vitro culture and differentiation of porcine mesenchymal stem cells (pMSC) on the cell cycle dis-tribution and nuclear DNA profile. This determination could be helpful to confirm or exclude the suitability of physico-chemical culture conditions for the purposes of both the maintenance of an undifferentiated state and to promote differentiation in pMSCs.Flow cytometry was applied to analyse the cell cycle and occurrence of aneuploidy/diploidy. The χ2 test was used to compare the total rates of G0/G1-, S-, and G2/M-phase cell fractions with diploid and aneuploid DNA and the DNA index ratios between three experimental groups of pMSCs.Five weeks of in vitro culture under differentiating condi-tions resulted in a considerable reduction of DNA stabili-ty and a remarkable increase in the rate of cells exhibiting an aneuploid DNA stemline; however, a similar depen-dence was not found in the non-differentiated MSCs.To sum up, we propose cytokinetic diagnostics using flow cytometry as an objective and useful method for screen-ing the tumour-forming capacity and malignancy poten-tial of both in vitro long-term cultured MSCs and MSCs subjected to ectopic differentiation.AcknowledgementsThis research was supported by the Statutory Activity of National Research Institute of Animal Production 02-4.03.1.

P10.10

RNA-seq analysis of differentially expressed genes in muscle tissue of Pietrain and Puławska pigsKatarzyna Ropka-Molik1, Kacper Żukowski2, Robert Eckert2, Artur Gurgul1, Katarzyna Piórkowska1

1Laboratory of Genomics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland; 2Department of Animal Genetics and Breeding, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland

Next Generation Sequencing RNA-seq technolo-gy is a  powerful tool which creates new possibilities in whole-transcriptome analysis. The RNA analysis using high-throughput sequencing method allows to measure gene expression on a genome-wide scale. In our study, RNA-seq method was applied to analyze global changes in transcriptome form the muscle tissue (m. semimem-branosus) in two pig breeds (Pietrain – 5 pigs, Puławska – 7 pigs). The total RNA wasisolated using TRI-Reagent. The quantity and quality of RNA were evaluated with the NanoDrop 2000 and by 2% agarose gel electrophoresis. cDNA libraries were prepared by using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to stan-dard protocol. Sequencing – by –synthesis of the libraries was performed on a HiScanSQ System with 50 single-end cycles using TruSeq SBS Kit v 3 – HS chemistry (Illumina).Using two different approaches by using DESeq, egdeR freewares and considered the most restrictive criteria we identified 184 differentially expressed genes between Pietrain and Puławska pigs. In both breeds, the most abundant were: transcripts encoding ribosomal proteins, genes associated with metabolic processes, which play a key role in respiratory electron transport chain in mi-tochondrion, intercellular or cation transport (NDUFS6, SYPL2, CHCHD3, ATP5A1, DLD).In Puławska pigs we indicated up-regulation of several genes that play a variety of roles crucial for metabolic processes – metabolism of polysaccharides an carbohy-drates (CHID1, HS3ST5, MFSD10), amino acid and pro-tein metabolic processes (NUBP2, ALDH16A1, OTUB1). Furthermore, when compare to Pietrain pigs, the signifi-cantly higher expression was obtained for genes encod-ing proteins associated with cell adhesion and cell cycle (EMILIN1, EMILIN3, GADD45G, H1FX). In Pietrain breed, only 37 genes were over-expressed and majority of them play an important role in cell developmental pro-cesses i.a. regulation of apoptosis, mitosis, cell-cell sig-naling and adhesion (SGPP1, HTRA2,SLITRK2, MAFB, ACTL6B), as well as in the muscle contraction and cat-ion/anion transport (GLRB, SLC9A7). The different ex-pression profile of selected genes obtained for two pig breeds may be the result of long-term selection to im-prove the meat content in carcasses conducted especially in Pietrain breed.AcknowledgementsThis study was supported by the National Research Institute of Animal Production statutory activity, Research Project No. 01-5.03.1.

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P10.11

An increase of piglets survivability after “biolactin” per os administrationBarbara Gajda1, Barbara Szczęśniak-Fabiańczyk1, Katarzyna Poniedziałek-Kempny1, Izabela Mandryk1, Florian Ryszka2, Barbara Dolińska2, Lucyna Leszczyńska2, Zdzisław Smorąg1

1Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice near Crakow, Poland; 2Pharmaceutical Research and Production Plant (FZNP)

Prolactin (PRL) is a protein hormone synthesized in and secreted predominantly by lactotroph cells of the ante-rior pituitary gland. It has been found to stimulate the immune system in animals as well. Because prolactin se-creted in milk by the mother’s body is often insufficient, the administration of exogenous prolactin may signifi-cantly contribute to improving the survivability of piglets. The objective of this study was to determine the effect of 0.1 mg PRL per kg of body weight of piglets administered per os to newborn piglets.Prolactin (“Biolactin”, Biochefa, Poland) was obtained from freeze-dried pig hypophyses. An experimental group (63 litters / 757 piglets) received per os 0.1 mg PRL per kg of body weight and a control group (30 litters / 358 piglets) 1.0 mL of 0.9% physiological saline solution. The number of piglets born alive at birth and on 21, 28 and 70 days of age was monitored. Based on this the mortal-ity of piglets in the experimental and control groups was estimated.At birth the average litter size was 12.0 and 11.9 piglets in experimental (after per os “Biolactin” administration) and control groups respectively. At 21 and 28 days (wean-ing) after farrowing the number of piglets, the average lit-ters were 10.8 and 10.7 piglets in experimental and 10.5 and 10.3 piglets in control litters respectively. An average of 10.6 and 10.0 piglets were left from experimental and control litters respectively at 70 days of age. The reduc-tion of mortality in the experimental group (9.9%, 11.1% and 12.0%) in comparison with the control group (11.7%, 14.0% and 15.9%) during the first three, four and ten weeks, respectively was observed.In conclusion, the results demonstrated an increase in the survivability of piglets that received “Biolactin”.AcknowledgementsSupported by grant No. NR12-0057-10 of NCBiR, Poland.

P10.12

Association of calpastatin gene polymorphisms and meat quality traits in pigAnna Bereta2, Katarzyna Ropka-Molik1, Mirosław Tyra2, Marian Różycki2

1Laboratory of Genomics National Research Institute of Animal Production National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland; 2Department of Animal Genetics and Breeding National Research Institute of Animal Production National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland

Calpastatin is an endogenous inhibitor specific for the calpains (calcium-dependent cysteine protease). Due to the regulation of proteolysis processes in cells, the activi-ty of calpastatin is related with several meat quality traits such as tenderness, drip loss, water holding capacity, conductivity or color. Calpastatin is also associated with the rate of degradation of muscle structural proteins post mortem via controlling of calpain activity. Furthermore, the calpastatin protein is thought to influence the expres-sion levels of genes encoding structural or regulatory pro-teins. Based on above function, calpastatin gene (CAST) is considered to be a candidate gene for the meat quality in many domestic animals including pig.The aim of the study was to determine the effect of CAST gene polymorphisms on selected meat quality traits in different pig breed used as maternal component: Polish Landrace (PL)(n = 157) and Polish Large White (PLW) (n = 224), paternal component Pietrain (n = 94), Duroc (n = 93) and in the Puławska, which is included in con-servative breeds (n = 110). Animals were maintained in the Pig Station of the National Research Institute of An-imal Production in Pawłowice according SKURTH pro-cedure. All animals were genotyped for three polymor-phisms using PCR-RFLP method (CAST/HinfI; CAST/HpaII; CAST/RasI) according to Ernest et al. (1998).The results obtained showed that two among 3 analyzed polymorphisms were significantly associated with meat pH and color. Due to the CAST/HinfI and CAST/HpaII polymorphisms, homozygotes BB and DD (respectively) were characterized by the highest pH measured 45 min-utes and 24 hours after slaughter in both ham and loin. The significant differences in red and yellow color intense of meat between CAST/HinfI polymorphism were also observed. The BB genotype had the highest red intensity (a*) and the lowest yellow intensity (b*). Moreover, the FF genotype (CAST/RsaI) significantly affects water-holding capacity (PCAST gene polymorphisms can be potential use as a marker for meat quality traits in pig.AcknowledgementsThe study was supported by the Polish Ministry of Science and Higher Education (project No. NN311349139).ReferencesErnst C.W., Robic A., Yerle M., Wang L., Rothschild M.F. (1998) Mapping of calpastatin and three microsatellites to porcine chromosome 2q2.1±q2.4. Animal Genetics, 29, 212–215.

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P10.13

Genetic characterization of Hucul horse populations based on microsatellite polymorphismAgnieszka Fornal, Anna Radko, Agata Piestrzyńska-Kajtoch

National Research Institute of Animal Production, Department of Cytogenetics and Molecular Genetics of Animals, Balice near Crakow, Poland

Short tandem repeat (STR) loci, i.e. microsatellites are a class of genetic markers commonly used for population studies and parentage control. This study determined the usefulness of microsatellite markers recommended by In-ternational Society for Animal Genetics (ISAG) for iden-tification and pedigree analysis in horses based on an ex-ample of Hucul horses populations (Equus caballus). The set of 17th STRs was tested (AHT4, AHT5, ASB2, HMS2, HMS3, HMS6, HMS7, HTG10, HTG4, HTG6, HTG7, VHL20, ASB17, ASB23, CA425, HMS1, LEX3) for three Hucul horses populations. 216 individuals were geno-typed and mean number of alleles per locus was estimat-ed as 6.039. The low diversity between each population was observed. Means of observed (Ho) and expected (He) heterozygosity calculated for all populations were 0.735 and 0.669, respectively. Ho was similar in all populations analyzed (respectively: 0.692; 0.625; 0.689). The average polymorphism information content (PIC) for seventeen microsatellite markers indicates the usefulness of this set of markers for Hucul horses parentage testing.

P10.14

The impact of varied values of High Hydrostatic Pressure applied on porcine Mesenchymal Stem Cells on their survival rate and early apoptosis after cryopreservationJoanna Romanek, Jolanta Opiela, Zdzisław Smorąg

Department of Biotechnology of Animal Reproduction National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland

High Hydrostatic Pressure (HHP) by stimulating the de-fensive reaction to stress increases cells resistance to an-other adverse factor. Cells after HHP treatment overex-press resistance proteins, therefore they can quicker adapt to next adverse factor like cryopreservation. As a result, a higher survival rates after cryopreservation can be ob-tained. As a beneficial effect of HHP on sperm, oocytes and embryos cryotolerance is already documented, we assume that the HHP treatment may also have a positive effect on the Mesenchymal Stem Cells (MSCs) quality af-ter cryopreservation.The aim of this study was to examine the influence of varied HHP values on porcine MSCs to increase survival rate and quality after cryopreservation. The survival rate and phosphatidylserine (PS) exposure in external surface of the cells were analyzed to indicate the most optimal HHP value for further experiments. In normal cells PS is located on the cytosolic side of cell membranes but during the early stages of apoptosis PS becomes exposed on the surface of the cell. Annexin V has the highest af-finity for PS, therefore it is used as a probe to detect cells that have expressed PS.The MSCs were isolated from bone morrow. Aspirate was diluted with PBS and subjected to centrifugation with Ficoll. The mononuclear cells were isolated from the in-terface, washed twice in PBS and resuspended in DMEM supplemented with 10% FCS. The MSCs were cultured for about 2 weeks until 80% confluence was reached. Then, MSCs were detached with 0.25% trypsin/EDTA, centri-fuged at 300 g and aspirated into 0.5 ml straws with M199 Hepes modification supplemented with 2% FCS. Such prepared straws were subjected to the five different HHP treatments (20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa) for 1 h in 24°C. After HHP treatment, MSCs were sus-pended in 10% DMSO and stored in liquid nitrogen for at least 24 h. Thawed MSCs were used for survival rate and early apoptosis detection by trypan blue staining and Annexin V-Biotin Apoptosis Detection Kit, respectively.Regarding MSCs viability, the high significant difference (P < 0.001) was noted between MSCs subjected to HHP ranging for 30 MPa to 60 MPa and control MSCs. The significant difference (P < 0.05) was noted between MSC subjected to 20 MPa HHP and control. No significant dif-ference was observed in PS exposure in any of analyzed experimental groups.

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According to the presented result, HHP increases MSC survival rate regardless the applied HHP value. Moreover, HHP does not have any negative impact on cells quality as measured by early apoptotic marker – PS. The further experiments will be performed to indicate the most op-timal HHP treatment for increasing the MSCs tolerance after cryopreservation.AcknowledgementsThis research was supported by the Statutory Activity of National Research Institute of Animal Production 02-4.03.1 and by the Polish National Science Centre resources allocated on the basis of decision number DEC-2011/03/D/NZ9/05537.

P10.15

Analysis of the changes in the expression profile of liver transcriptome by RNA-seq in pigs fed with various diet supplementsMaria Oczkowicz1, Katarzyna Ropka-Molik1, Małgorzata Świątkiewicz2, Kacper Żukowski3, Artur Gurgul1

1National Research Institute of Animal Production, Laboratory of Genomics; 2National Research Institute of Animal Production, Department of Animal Nutrition And Feed Science; 3National Research Institute of Animal Production, Department of Animal Breeding and Genetics

RNA sequencing is a new, cutting edge technology which allows for quantification of transcripts, identification of a new SNPs and a new splicing variants. In our study we aimed to compare the liver transcriptome of pigs fed with different diet supplements. All animals were kept in the same housing conditions and fed with standard feed mix-ture according to their nutritional requirements. There were three experimental groups, differing with type of used fat: beef tallow (n = 4), coconut oil (n = 4) and rape-seed oil (n = 4) and one group with addition of rapeseed fat but no addition of DGGS (Dried Destilled Grains with Soluble) in contrast to the other groups. When the animals reached the weight of 118 Kg were slaughtered. Samples of liver were collected and frozen in a liquid ni-trogen immediately. Next, RNA was isolated from the samples using Trizol reagent. Quality and concentration of RNA were evaluated using Nanodrop 2000 and by 2% gel electrophoresis. cDNA library was prepared with 400  ng of good quality RNA and TruSeq RNA Sample Preparation Kit v2 (Illumina). Clustering was performed on cBot (Illumina) and Sequencing by Synthesis was per-formed on a Hi Scan SQ System with 50 single-end cycles using TruSeqSBS Kit v 3 HS chemistry (Illumina). After data analysis, one sample was excluded from the experi-ment because of low quality of mapping. Transcrpits ex-pression differences were analyzed with Cuffdiff freeware. In total, 40 genes differed in the expression level when all experimental groups were compared. The highest num-ber of differentially expressed genes (n = 15) were pres-ent between animals supplemented with coconut oil and pigs without DGGS in the feed stuff. Some of the iden-tified genes are responsible for lipid transport (ABCD3, APOA4), metabolism and cholesterol metabolic process-es (HMGCS1). These genes will be further analyzed by Real-Time PCR.AcknowledgementsThis work was supported by National Research institute of Animal Production.

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P10.16

Regulation of Tenebrio molitor heart beating by monoamines and tropane alkaloidsSzymon Chowański, Jan Lubawy, Arkadiusz Urbański, Marta Spochacz, Grzegorz Smykalla, Grzegorz Rosiński

Department of Animal Physiology and Development, Faculty of Biology, Adam Mickiewicz University in Poznan

Insects generally possess pumping structures and various diaphragms to ensure that haemolymph flows through-out the haemocel. The primary pump for moving haemo-lymph is a dorsal vessel also named as heart. The myocar-dium in all insects is spontaneously active. This type of heart is termed myogenic because the electrical activity underlying contractions a rises in the myocardium itself. This is in contrast to a neurogenic heart present in, for ex-ample, crustaceans. The heart is innervated by paired lat-eral nerves from segmental ventral ganglia or by cardiac nerve running along the heart and coming out from the brain. Furthermore, the heart contractility is regulated in neurohormonal way by neurotransmitters and neurohor-mone peptides for example by proctolin and crustacean cardioactive peptide.Monoamines function as a neurotransmters, neuromod-ulators and neurohormones in nervous system both in vertebrates as in invertebrates. In case of vertebrate, the catecholamines (epinephrine, dopamine and serotonin) have the main role in nervous system regulation and in case of invertebrates it is trace amines (octopamine and tyramine). In insects, this compounds influent on many different physiological processes. They modulate central nervous system activity, control contractility activity and metabolism of muscles.Tropane alkaloids, atropine and scopolamine, are an an-tagonist of cholinergic muscarinic receptors (mAChRs). In vertebrates, acting of atropine and scopolamine on mAChRs leads to increased heart rate. These receptors are also found in insects. The available data suggest that they are involved in behaviour related to locomotion, chewing and pharyngeal movements and the behaviour associated with singing and also in regulation of wings and antennae movements. Furthermore, activation of muscarinic receptors may affect the changes in gene ex-pression in neurons as well as regulation of neuro-endo-crinic processes. However, there is not information, how agonists or antagonists of mAChR affect insect myocar-dium. In case of vertebrates, atropine and scopolamine cause an increase of heart rate and abolition bradycardia and asystoles.The aim of study was to determine the influence of tropane alkaloids and monoamines on the myocardial activity in the beetle Tenebrio molitor. The results showed that compounds tested have diversed effect on the myocardium contractile activity in T. molitor. Tropane alkaloids did not cause changes in the action of semi-isolated heart in the whole concentration range

(10–10–10–3 M). But the in vivo experiment carried out on 1-day old pupae T. molitor showed, that these compounds lead to decrease in heart rate what was observed as negative chronotropic effect both in ortodromic and antidromic phase of contractility between 4th and 10th hour after application of scopolamine and atropine.Epinephrine did not cause changes in the heart contractility both in vivo and in vitro experiments. Dopamine had an opposed effect. In vitro it leads to a slight increase of heart rate (only at highest tested concentration – 10–3 M) but in vivo it acts as a myoinhibitor. Tyramine and octopamine both in vivo and in vitro caused negative chronotropic effect but only between 6th and 10th hour after application.Obtained data may suggest indirectly participation of scopolamine and atropine in regulation of heart beating. There is probable that these compounds affect neuro-se-cretory structures, which are involved in the myocardium activity regulation. No changes in the heart action under influence of tropane alkaloids can be a result of lack of mAChRs in the heart or presence of receptors insensi-tive on these antagonists. Trace amines (octopamine and tyramine) influenced on myocardium both in vivo and in vitro what indicaste, that these compounds take part directly in regulation of myocardium activity.

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P10.17

Genetic polymorphisms of lep and lepr genes in relation with production and reproduction traits in cattleAnna Trakovická, Nina Moravčíková, Radovan Kasarda

Slovak University of Agriculture in Nitra, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, The Slovak Republic

Leptin and leptin receptor genes are considered as mark-ers of production traits in dairy or beef cattle. The aim of this study was to verify the associations of polymor-phisms in bovine LEP and LEPR genes with production and reproduction traits in Slovak Spotted and Pinzgau cows. Evaluated were long – life production: milk, pro-tein, and fat yield and reproduction traits: age at first calving, calving interval, days open, and insemination in-terval. In total 296 blood samples of Slovak Spotted and 85 hair roots samples of Pinzgau cows were analyzed. In order to detect LEP/Sau3AI (BTA 4, inron 2) and LEPR/T945M (BTA 3, exon 20) genotypes PCR-RFLP method was used. In Slovak Spotted and Pinzgau cows were the allele frequencies 0.838/0.162 and 0.694/0.306 for A and B LEP variants, and 0.954/0.046 and 0.912/0.088 for C and T LEPR variants, respectively. For testing the asso-ciations between SNPs LEP/Sau3AI and LEPR/T945M and evaluated traits the General Linear Model (GLM) procedure in SAS Software was used. Statistical analysis showed that the SNP LEP/Sau3AI significant affected milk, protein and fat yield (PAcknowledgementsThis work was supported by the Slovak Research and Development Agency under the contract No. APVV-0636-11.

P10.18

Prp4 kinase is required for proper segregation of chromosomes during meiosis in Schizosaccharomyces pombeMiroslava Požgajová1, Ľuboš Čipák2, Anna Trakovická1

1Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra, Slovakia; 2Max F. Perutz Laboratories, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria

Chromosome segregation during meiosis is a complex process which leads to production of four haploid gam-etes from two precursor cells. Reversible phosphoryla-tion of proteins plays a crucial role in this process. The Schizosaccharomyces pombe Prp4 is an essential serine/threonine kinase which belongs to the Clk/Sty family. To study the role of Prp4 in meiosis, we analysed chro-mosome segregation in a strain carrying analog-sensitive allele of Prp4 (prp4-as2). Our data show, that Prp4 plays important role in chromosome segregation during mei-osis, as revealed by enhanced missegregation of chromo-somes in prp4-as2 mutant cells.AcknowledgementsThis work was supported by the Slovak Research and Development Agency under the Contract No. APVV-0636-11.

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P10.19

Genetic diversity in populations of Slovak Spotted cattle based on snps analysesNina Moravčíková, Anna Trakovická, Alica Navrátilová

Slovak University of Agriculture in Nitra, Department of Animal Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, The Slovak Republic

The aim of this study was identification of SNPs in leptin (LEP), leptin receptor (LEPR) and growth hormone (GH) genes in order to analyze genetic diversity of Slovak Spot-ted cattle. The total numbers of blood samples were tak-en from 353 Slovak Spotted cows originating from four farms. Genomic DNA was isolated by phenol-chloroform extraction method and analyzed by PCR-RFLP method. After digestion with restriction enzymes were detect-ed in whole population of cows alleles with frequency: LEP/Sau3AI A 0.83 and B 0.17 (±0.0141); LEPR/BseGI C 0.96 and T 0.04 (±0.0076) and GH/AluI L 0.69 and V 0.31 (±0.0173). Based on the observed vs. expected genotypes frequencies populations across loci were in Hardy-Wein-berg equilibrium (P > 0.05). Predominant for SNP LEP/Sau3AI was AA genotype (0.69), for SNP LEPR/T945M CC genotype (0.92), and for SNP GH/AluI was dominant LV genotype (0.47). The observed heterozygosity of SNPs across populations was also transferred to the low or me-dian polymorphic information content 0.25 (He 0.28), 0.07 (He 0.08) and 0.33 (He 0.42) for LEP, LEPR and GH genes, respectively. Within genetic variability estimating negative values of fixation indexes FIS (–0.09 – –0.05) and FIT (–0.07 – –0.03) indicating heterozygote excess were observed. The value of FST indexes (0.018-0.023) shows very low levels of genetic differentiation in allele frequen-cies of loci among evaluated subpopulations. The low val-ues of genetic distances (0.0018–0.0159) indicated high genetic relatedness among animals in subpopulations probably caused of common ancestry used in breeding program of farms.AcknowledgementsThis work was supported by the Slovak Research and Development Agency under the contract No. APVV-0636-11.

P10.20

Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human healthMartina Miluchová, Michal Gábor, Anna Trakovická

Slovak University of Agriculture in Nitra, Department of Genetics and Breeding Biology, Tr. A Hlinku, 2, 949 76 Nitra, Slovakia

The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta ca-sein can result in the production of the bioactive beta casomorphin-7 (BCM-7), this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascu-lar and immune systems. The material involved 111 Slo-vak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit and used in order to estimate beta – casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozy-gote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37  animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2 – 0.5405 were the most frequent, while homozy-gotes A1A1 – 0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7 and thus is safe for human con-sumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of pos-sible variability realization (41.80%), which corresponds to the level of polymorphic locus (1.7071).AcknowledgementsThis work has been supported by the grants: The Slovak Research and Development Agency under the contract No. LPP-0220-09 and No. APVV-0636-11.

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P10.21

Analysis of CAPN1 and CAST gene polymorphisms in native dual purpose breeds of cattle in SlovakiaMichal Gábor, Martina Miluchová, Anna Trakovická

Slovak University of Agriculture in Nitra, Department of Genetics and Breeding Biology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia

The aim of this work was to optimize fast, reliable and effi-cient modification of the PCR methods for identification of polymorphism for candidate genes calpain 1 (CAPN1) and calpastatin (CAST) and to analyze the genetic struc-ture in population of 145 animals of Slovak Spotted breed and 137 animals of Slovak Pinzgau breed. Genetic structure for the selected breeds was evaluaeted by using three markers of CAPN1 gene (CAPN316, CAPN4685, CAPN4751) and three markers of CAST gene (CAST-T1, CAST-UoG, CAST-WSU). Genomic DNA was isolated from whole blood, sperm and hairs by using commercial column kit. The study of CAPN1 and CAST gene poly-morphisms was done by using molecular-genetics meth-ods such as PCR-RFLP, ARM-PCR and HRMA. Genetic analysis confirmed a higher frequency of preferred alleles (C; C; A; C) and genotypes (CC; CC; AA; CC) of mark-ers CAPN316, CAPN4751, CAST-T1 and CAST-UoG as-sociated with meat tenderness, in Slovak Pinzgau breed unlike Slovak Spotted breed, which was characterized by the lowest frequence of preferred alleles and genotypes of these markers. For marker CAPN4685 associated with a higher lean share in valuable cuts was detected the prev-alence of favourable allele T and genotype TT in Slovak Pinzgau breed too. Our primary results suggest, that the Slovak Pinzgau breed has favourable genetic base for im-provement of quality meat such as tenderness in compare with the Slovak Spotted breed, which has markedly preva-lency of favourable allele C for marker CAST-WSU which is associated with longevity and reproduction traits.AcknowledgementsThis work has been supported by the projects APVV No. LPP-0220-09 and APVV-0636-11.

P10. 22

Allatostatins as potential bioinsecticides?Jan Lubawy1, Arkadiusz Urbański2, Szymon Chowański1, Ewelina Paluch3, Mariola Kuczer4, Grzegorz Rosiński1

1Department of Animal Physiology and Development, Adam Mickiewicz University in Poznan; 2Department of Systematic Zoology, Adam Mickiewicz University in Poznan; 3Department of Plant Physiology, Adam Mickiewicz University in Poznan; 4Faculty of Chemistry, University of Wroclaw

Neurohormones synthesized by neuro-endocrine system are importnt factors regulating processes of growth and development, reproduction and behavior in insects. The quick development of analytical techniques has lead to discovery of more than 30 families of peptide neurohor-mones. In recent years research is conducted on usage of these compounds in agriculture and medicine. There are being designed, on the basis of naturally occurring neu-rohormones, new biostable pseudopetides and peptido-mimetics which can be used as environmentally friendly pesticides. One of vast group of insect neurohormones are allatostatins, which show pleiotropic activities influ-encing a number of physiological processes in insects, such as inhibition of the synthesis of juvenile hormone in corpora allata of insects, regulation of visceral muscle contraction, synthesis and release of vitellogenins from fat body or synthesis of digestive enzymes. Such features made them interested for scientists and peptide analogs based on them are now tested on fighting with mosqui-toes or aphids.In this paper we, present the effects of synthetic alla-tostatin Grybi-AS B1 (GWQDLNGGWa) on haemocytes morphology, phenoloxidase (PO) activity and oviduct contractions of Tenebrio moitor beetle, a synanthrope which is a pest of cereal. The studies show that activity of PO has increased after application of 500 pM and 50 nM of peptide. Simultaneously the peptide caused morpho-logical changes in cytoskeleton structure of haemocytes of the beetle. Bioassays with oviduct have shown bio-modal myotropic activity of Grybi-AS B1, at lower con-centrations (10–12–10–9 M)it increases endogenic muscle contractions and at the higher concentrations (10–6 and 10–5 M) it nhibits contractions of oviduct.

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P10.23

Relationships between type traits and functional productive life in Slovak Holstein CowsEva Strapáková, Peter Strapák, Juraj Candrák

Slovak University of Agriculture in Nitra

The relationships between type traits and functional pro-ductive life were analyzed in 60208 Slovak Holstein cows first calved from 1996 to 2010. Functional productive life was defined as the number of days from first calving to death, culling or censoring. All cows were scored for conformation during the first lactation. Weibull models were fitted to analyze the data. The strongest relationship between estimated breeding values for survival and type traits were found for rear legs side view, rump width, an-gularity, fore udder attachment, dairy strenght, body con-dition and udder depth. Analyses were done one at a time for each of those 7 type traits. Based on analyses was found that cows with shallower udder, moderate fore ud-der attachment, marked angularity, correct rear legs side wiev or narrower rump achieved longer productive life. Evaluation of body condition confirmed that very thin or very fat cows reached higher involuntary risk of culling.AcknowledgementsFinancial support was provided by the project APVV-0636-11 and KEGA 027SPU-4/2012.

Legislation for biotechnologists

Lectures

L11.1

Recent developments in gene and stem cell patenting in EuropeRainer Friedrich, Elisabeth Greiner

df-mp Patents Trademarks Designs

The presentation will introduce you to requirements and limitations regarding the protection of your biotechnolo-gical inventions in Europe.

L11.2

Genetically modified soybean with altered fatty acid profile and direct consumer benefitsAndrew Tommey

DuPont Pioneer, Avenue des Arts 44, 1040 Brussels, Belgium

Commodity soybean oil has been widely used in the food industry for many years and is often hydrogenated by oil processors in order to increase its stability and shelf life. Whilst this results in a functionally stable and longer lasting partially hydrogenated soybean oil, the hy-drogenation process also leads to an increase in trans fats in the final product and this can impact on cholesterol levels thereby increasing the risk of heart disease. These issues have been addressed through genetic modification of the fatty acid profile in soybean (GM soybean event DP-3Ø5423-1) and the resultant oil from the high oleic soybeans is zero trans fat providing direct benefits to the final consumer, in addition to benefits for the farmer and food industry. Food producers have continued to test this product and interest is high due to the improved oil profile of these soybeans allowing food companies to bring enhanced, healthier products to the market.This GM soybean product has now been approved by re-gulatory authorities for either cultivation and/or import, food and feed uses in all major parts of the world with the exception of the European Union. The EU risk assessment and regulatory approval processes are exceedingly strin-gent and timelines are lengthy given the quantity and quality of safety data requested as well as the time taken for review by the EU authorities. However, the European Food Safety Authority (EFSA) is now close to completing its risk assessment and Member States in the EU should soon be voting as to the formal approval of this healthy high oleic soybean product.

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L11.3

Genetically modified plants in Polish agricultureSławomir Sowa, Janusz Zimny

Plant Breeding and Acclimatization Institute National Research Institute, Biotechnology and Cytogenetic Department, GMO Controlling Laboratory

Green biotechnology plays an important role in bio-economy strategies in both developed and developing countries. According to the “Strategy Europe 2020” bio-economy is a key element for smart and green growth in Europe. Now it is obvious that Europe needs new approaches to production and consumption in order to meet global environmental and economical challenges. In 2012 genetically modified plants were cultivated in 28 countries worldwide and its role in food/feed production is increasing steadily in the last years. On the other hand Europe is very reluctant in adoption of GM plants but remains big GM feed importer. Poland is one of the few EU countries where GM plants have been cultivated in the past years on commercial scale. In South of Poland where infestation with the European corn borer (Ostrin-ia nubilalis) pose severe damages to maize production Polish farmers used to grow authorized for cultivation in EU genetically modified maize varieties. As from 28th of January 2013 regulation of Polish Council of Ministers prohibited the use of MON810 maize seeds and Amflora potato – the only GM events approved for cultivation in EU. Poland along with Austria, France, Hungary become one of the European countries where GM plants are not being cultivated. Wide adoption of genetically modified plants in European and Polish agriculture could be im-portant not only to ensure food security, reduce depen-dence on non-renewable resources but also to create jobs and maintain competitiveness on the world market. Cur-rently GM plant varieties are not available to Polish farm-ers but recent advancements in innovative plant breeding techniques brings new insight to application of genetic engineering in Polish and European agriculture.

L11.4

Biotechnology regulation and society, Anno Domini 2013Tomasz Twardowski

Institute of Bioorganic Chemistry PAN, Poznan, Poland

It is too late to close the door on GM products: biomate-rials, bioenergy, biopharmaceuticals as well as food and feed. The political economy of biotechnology policies depends on consumers. In the United Europe the con-sumers (= the voters) said no to the GM farming giants in several states, however that didn’t stop millions of tones of GM soya and corn entering the European food chain. Legislation including IPR, innovative technology, public acceptance and many more factors are critical for the fur-ther development of biotechnology.

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L11.4

European patents for human embryonic stem cellsEwa Waszkowska

Patent Office of the Republic of Poland

Human embryonic stem cells hold great promise for ad-vancing human health. However, patents for the use of human embryonic stem cells have been the subjects of extensive public discussion and are regarded as extremely problematical. The ethical and legal problems are asso-ciated.In October 2011, The Court of Justice of the Europe-an Union issued its decision in Brüstle v Greenpeace (C-34/10), which relates to the patentability of technolo-gy based on the use of human embryonic stem cells.The European court ruled that German scientists could not patent a technique based on human embryonic stem cells because it involved the destruction of something “capable of commencing the process of development of a human being” – in other words a human embryo.The definition of a human embryo used by the court in-cluded types of artificially created “embryos” covering these that are not capable of developing into a fetus.Is this definition too broad?The initial aim of patents is to promote innovation and reward scientific efforts.The ban on patenting human stem cell research could harm its future development in Europe?

Posters

P11.1

Conjoint duty for biotechnologistsSebastian Kwiatkowski1, 2, 3

1University of Warsaw; 2Faculty of Biology; 3Students Scientific Association of Genetics and Epigenetics UW

First of all, when we are talking about biotechnology as interdisciplinary science which could solve global famine problem, we have to emphasize the importance of GMOs together with the attitude towards public opinion this technology and, finally, legislation which concerns all co-lours of biotechnology [1].Everybody knows how controversial all kind of genetic modifications of: bacteria, plants and animals could be. All genetic modifications, including genes coding Bt tox-ins and EPSP synthase, are not acceptable by public opin-ion because of the minimal knowledge about biology and biotechnology. The different cause is simply the fear as a result of the unknown. Other reason of that state is lack of trust in science and scientists [2].To conclude, fear of unknown and lack of trust are the reasons of unfavorable legislation in several countries in European Union (e.g. in Poland). On the other hand there are countries (e.g. Great Britain or Czech Republic) in which attitude towards public opinion and legislation are more favorable for biotechnology [3].In my opinion one of the most important duties for all biotechnologists is taking care of improvement opinion about biotechnology. Especially nowadays, when some organisations are showing scientists (above all biotech-nologists) as corrupt and danger for society.References[1] Twardowski T. (ed.) (2012) Aspekty Społeczne i Prawne Biotechnolo-gii. Polska Akademia Nauk.[2] Aerni P. (2013) Resistance to agricultural biotechnology: The importance of distinguishing between weak and strong public attitudes. Biotechnology Journal. doi: 10.1002/biot.201300188.[3] USDA – Foreign Agricultural Service (2013) Agricultural Biotech-nology Annual. Global Agricultural Information Network.

Induced pluripotent stem cells: a future of biomedicine

Lectures

L1.1

Hypoxia and oxidative stress in reprogramming and differentiationAlicja Jozkowicz

Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow

Human induced pluripotent stem cells (hiPS) are generat-ed by epigenetic reprogramming of somatic cells through the exogenous expression of four transcription factors (Oct4, Klf4, Sox2, c-Myc). These cells has the all char-acteristic features of human embryonic stem cells (hES) as self-renewal and ability to differentiate to cells of all three germ layers. Growing body of published data clearly shows that that epigenetic mechanisms are involved in the process of reprogramming of somatic cells into pluripo-tent state. TRIM28/KAP1 protein is one of the epigene-tic regulators that regulate heterochromatin formation through histone modifications and DNA methylation. Over 500 unique zinc finger proteins containing KRAB domain recruit TRIM28/KAP1 protein to genomic DNA in sequence-specific fashion thus leading to transcrip-tional repression of neighboring genes. Here, we showed that TRIM28/KAP1 protein controls self-renewal of hES and hiPS cells. Stable knockdown of TRIM28/KAP1 gene expression in both hES and hiPS cells achieved using len-tiviral vectors carrying specific shRNAs resulted in pro-gressive loss of pluripotent phenotype as confirmed by analysis of surface embryonic markers expression using immunofluorescence and FACS analyses. Moreover, we have documented progressive loss of expression of select-ed embryonic transcription factors in hES and hiPS cells depleted of TRIM28/KAP1 exkpression. Our ongoing ex-periments are focused on detailed analysis of the molecu-lar mechanisms that are involved in the observed pheno-type and may shed new light on epigenetic mechanisms that preserve self renewal of hES and hiPS cells.

L1.2

There and back again: short history of reprogramming. From a somatic cell to an inducible pluripotent cellMaria Anna Ciemeryc

Department of Cytology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw

Embryonic stem cells (ESCs), derived from prelimplan-tation embryos at the blastocyst stage, are unique in un-limited self-renew ability and pluripotency allowing their differentiation into any cell type. Until the beginning of XXI century ESCs were considered as the only cell lines which pluripotency was proved both in vitro and in vivo. In 2006 first paper describing reprogramming of fibro-blasts into sa called induced pluripotent cells (iPSCs) was published. The basis of this spectacular achievement done by Yamanaka and his collaborators was establised in 50s and 60s of the XX century. The short history of the cellular reprogramming and pluripotent stem cells research that led to the Nobel Prize for Yamanaka and Gurdon will be a topic of this talk.

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L1.3

Epigenetic mechanisms of human induced pluripotent stem cells (iPS)Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Induced pluripotent stem cells (iPS) cells are generat-ed by dedifferentiation of adult cells through the forced expression of few embryonic transcription factors. The reprogramming process involves ordered and global epigenetic alterations including histone modifications and DNA methylation that progressively silence expres-sion of lineage-specific genes whilst enabling transcrip-tion of embryonic factors genes. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor TRIM28/KAP1 during the reprogram-ming process. The results of our work strongly suggest that knockdown of TRIM28/KAP1 expression facilitate de-differentiation of human primary fibroblasts towards the iPS cells. In the other hand the inhibition of TRIM28/KAP1 function in established human pluripotent cells re-sults in progressive loss of their self-renewal potential in contrast the the wild-type human iPS cells. Additionally, our parallel lines of research clearly demonstrated that binding of TRIM28/KAP1 through KRAB-containing transcriptional repressors results in specific methylation of the cellular promoters. Finally, we have revealed that epigenetic factors recruited by TRIM28/KAP1 controls retrotransposition events during the reprogramming of human somatic cells to pluripotency. Taken together, our results demonstrated novel role for TRIM28/KAP1 pro-tein in the reprogramming process and self-renewal of human iPS cell as well as their involvement in modeling landscape of genomic DNA methylation during that pro-cess. In-depth understanding of epigenetic mechanisms involved in the cellular reprogramming and self-renew-al of human iPS cells will have implications for basic re-search and may pave ways to novel therapies.

L1.4

The importance of peeing earnest: urine derived cells as model systems in biology and biomedicineTing Zhou3, Christina Benda3, Sarah Dunzinger2, Matthias Wieser2, Heinz Redl5, Johannes Grillari1, Miguel A Esteban3, Regina Grillari2

1Evercyte GmbH, Vienna, Austria; 2Department of Biotechnology, BOKU Vienna, Austria 3Key Laboratory of Regenerative Medicine, Chinese Academy of Sciences, Guangzhou, China; 4Guangdong Stem Cell and Regenerative Medicine Research Centre, University of Hong Kong, China; 5Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Vienna, Austria, 6Austrian Cluster for Tissue Regeneration, Vienna, Austria

Human cell cultures are of ever increasing importance in many scientific areas ranging from basic biology, biotech-nology to medicine. In all these fields cells find applica-tion as model systems, products and producers. Especial-ly their use as model systems to understand cellular be-haviour or pathogenic conditions relies on the availability of sufficient quantities of cells.We have therefore established several cell lines that close-ly resemble their primary counterparts in key character-istics. Among these cells range endothelial cells, adult stem cells and renal proximal tubular epithelial cells (RPTECs). During these studies, we realized that RPTEC like cells are exfoliated into urine. These cells can be converted into iPS cells with high efficiency, and can be re-differentiated into neuronal cells, hepatocyte-like cells and cardiomyocytes.As these cells are derived from a non-invasive source that is not exposed to DNA damaging UV radiation like skin, we propose urine to become the preferred source for gen-erating iPSCs in many instances. The ease of this meth-od may facilitate the standardization of iPSC technology, will boost the generation of cell based disease model sys-tems and is also an advance in the direction of clinical use of iPSCs.

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L1.5

Stem cells in drug development and toxicity testing: implementation of emerging technologiesLeonora Buzanska

Stem Cell Bioengineering Laboratory, NeuroRepair Department Mossakowski Medical Research Centre, 02-106 Warsaw, Poland

Developing reliable human-based in vitro systems to study drug toxicity and their mode of action is a major challenge for establishing new and safe therapies. The emerging technologies involved in the creation of such systems combine advancement in stem cell research with bioengineering of the stem cell niche. This allows creat-ing microscale biomimetic microenvironments on high content/throughput platforms to test response of cells representing relevant human disease models with specific genetic background.The generation of disease and patient-specific cell lines become possible during the last few years due to devel-opment of induced pluripotent stem cells (iPSC) technol-ogy. iPSCs can be obtained from any tissue of the body by ectopicly delivered transgenes of specific sets of tran-scription factors, thus circumventing both ethical and immunological limitations implicated by the use of hu-man embryonic stem cells. The iPSC-based disease mod-els tested on microengineered drug discovery platforms have been already shown to be useful in filling the gap between animal testing and clinical trials.The important role of human embryonic and somatic tissue-derived stem cell models for drug discovery and toxicity testing, especially in the aspect of developmen-tal biology, was widely proven during the last decade and such examples, including the results of our group, will be presented. However the iPSC technology provides a gi-ant advancement step for pharmacology and regenerative medicine applications, enabling personalized testing, di-agnosis and treatment.This lecture will provide the state of the art on the devel-opment of human-based in vitro systems with stem cells and micro/nano engineered cell-growth platforms, used to test a variety of compounds and possibly to correct the disease-related phenotypes.AcknowledgementsSupported by: MSHE grant No. N N302597838 and status funds of Mossakowski Medical Research Centre.

L1.6

Use of cell reprogramming technologies for neurobiological researchJacek Jaworski1, Ewa Liszewska

International Institute of Molecular and Cell Biology

Reprogramming of somatic cells made possible to study in vitro inaccessible human cells, such as different types of neurons. Almost immediate consequence of the emer-gence of this technology was the development of a num-ber of cellular models of the nervous system diseases. They are used both to explore the cellular mechanisms of these diseases and for the development of new phar-macological strategies. Reprogrammed cells are also a potential alternative to embryonic stem cells for trans-plantation. During my talk I will introduce methods used for cellular reprogramming for neurobiological research (e.g. reprogramming to iPS vs. direct reprogramming to neurons). I will present the most important achievements in the use of cell reprogramming technology in modeling of nervous system diseases such as spinal muscular atro-phy, Rett Syndrome, schizophrenia and neurodegenera-tive diseases. I will also illustrate how reprogrammed cells can be used for potential therapy of spinal cord injuries. At the same time, I will point out the limitations of the methodology and the expected directions of its devel-opment. Finally I will present preliminary results of my group in establishing cellular models of Tuberous Sclero-sis, a developmental disease caused by mutation in genes encoding either hamartin or tuberin and manifested by epilepsy and autism.AcknowledgementsAuthors express their gratitude to Prof. K. Kotulska and S. Jozwiak for sharing skin biopsies. This work is financed by ERA-NET NEURON/06/2011 “AMRePACELL” (co-financed by NCBiR) to JJ and Homing-Plus (HOMING PLUS/2012-5/6) of Polish Science Foundation to EL.

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Posters

P1.1

Role of heme oxygenase-1 in generation and differentiation of iPS cellsJacek Stepniewski, Tomasz Pacholczak, Alicja Jozkowicz, Jozef Dulak

Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow

Reprogramming of somatic cells which leads to the generation of induced pluripotent stem cells (iPSCs) is a complex process requiring many modifications at the level of gene expression profile, metabolism and chroma-tin structure. However, the initial response to the viral vectors delivering reprogramming transcription factors (Oct4, Sox2, Klf4 and c-Myc) involves the elevation in production of reactive oxygen species (ROS). This may activate Nrf2 transcription factor – master regulator of response to oxidative stress – and heme oxygenase-1 (HO-1), one of its target genes. Th e aim of our study was to evaluate the role of these two cytoprotective proteins in generation and differentiation of iPS cells. It appeared that the lack of either Nrf2 or HO-1 decreased the effi-ciency of reprogramming. Accordingly, induction of their expression or activity resulted in higher number of gener-ated iPS colonies. We also observed that the level of HO-1 was elevated in the later stages of reprogramming. Inter-estingly, stimulation of murine fibroblasts with valproic acid, which was previously demonstrated to increase the reprogramming outcome, resulted in the lower HO-1 lev-el in the cells. Accordingly, this compound decreased the reprogramming efficiency in our experiments. Obtained iPS cells were further cultured and characterized. Inter-estingly, we observed that lack of Nrf2 can increase the number of contracting bodies in spontaneously differen-tiating cells. These results indicate that Nrf2-pathway play a role in the reprogramming process and in differentia-tion of iPSC.

P1.2

Differentiation of iPS toward endothelial cellsNeli Kachamakova-Trojanowska1, Michael Beilharz3, Karolina Bukowska-Strakova1, Jacek Stępniewski 3, Antonia Chmura-Skirlińska2, Jozef Dulak3, Alicja Jozkowicz3

1Laboratory of Molecular Pharmacology of Endothelium; 2Laboratory of EPR Spectroscopy, JCET; 3Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Crakow

Induced pluripotent stem (iPS) cells can be a very useful model for studies of disease-specific endothelial dysfunctions, especially those dependent on genetic background of patients. Our aim was to elaborate a  method for differentiation of iPS toward endothelial cells.Tail-tip fibroblasts were isolated from the wild type C57Bl6 mice and then used for generation of iPS cells by delivery of four reprogramming factors (Sox2, Klf4, c-Myc and Oct4) with a single lentiviral vector. Plurip-otency of resulting cells was confirmed by staining for SSEA-1 or NANOG and by teratoma formation. Before starting the differentiation, two distinct cell populations were present in the cultures, defined by expression of either CD31 or CD34. They shifted with time towards a CD34+CD31- phenotype with a rising percentage of CD34+Tie-2+ double positive cells. However, almost no CD31 or CD105 could be detected. Tie-2 and KDR were also found up-regulated at RNA level. Nevertheless, no KDR positive cells could be observed by flow cytometry. Importantly, immunocytochemical staining revealed, that the cultured cells were positive for activated endo-thelial nitric oxide synthase (eNOS, phosphorylated at Ser1177) and positive for von Willebrand factor. More-over, eNOS functionality was confirmed by detection of nitric oxide production. Direct angiogenic capacity of the generated endothelial cells was tested by matrigel tube formation assay and by outgrowth of capillaries from spheres embedded in collagen gel. No tube formation was observed after 24 h or later time-points on matrigel, unless the iPS-derived cells were cultured together with mature endothelial cells. Also the sphere formation was delayed in comparison to endothelial cells, as the pro-cess took 6 days, instead of 1-2 days. However, after em-bedding of the spheres in collagen the pronounced out-growth of capillary network was observed, confirming the endothelial activity.To sum up, murine iPS cells can be differentiated into endothelial-like cells, with some immunophenotype fea-tures and angiogenic activities typical for endothelium. They resemble the phalanx cells (like in stable arteries), more than tip or stalk cells.

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P1.3

Application of epigenetic switch for generation of human IPS cellsJoanna Wróblewska¹, Katarzyna Kulcenty¹, Urszula Oleksiewicz1, Wiktoria Suchorska1, 2, Andersen Jannik4, Giulio Draetta4, Gustavo Mostoslavsky³, Maciej Wiznerowicz¹,²1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland; ³Boston University School of Medicine, Dept. of Medicine, Boston, MA, USA; 4Institute for Applied Cancer Science, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Human induced pluripotent stem cells (IPS) are generat-ed by reprogramming of somatic cells through enforced expression of embryonic transcription factors. However, clinical applications require that expression of introduced transgenes must be permanently switched off in the IPS cells and obtained differentiated progenies. Here, we took advantage of epigenetic switch that relies on reversible binding of tTRKRAB transrepressor to tetO element, which results in tight transcriptional repression of prox-imal promoter through heterochromatin formation. In order to apply this system for reprogramming, the tetO element was inserted into pSTEMCCA lentiviral vector carrying OCT4, SOX2, KLF4 and cMYC under control of EF-1alpha promoter. Transduction of human skin fi-broblasts with obtained pSTEMCCA-tetO allowed for expression of reprogramming factors and thus efficient generation of human iPS clones. Obtained clones were picked and further cultured until establishing stable IPS cell lines. Then cells were then transduced with lentiviral vector pLV-HK carrying tTRKRAB, in order to switch off reprogramming transgene expression.Tight repression of introduced transgenes in all human IPS clones was analyzed by RT-PCR and confirmed full functionality of our system. Obtained IPS cell lines showed no abnormalities in karyotypes. Pluripotent phe-notype of IPS cells was revealed by analysis of endoge-nous embryonic genes expression using RT-PCR and immunofluorescence staining. Analyzed cells were also able to form embryonic bodies in vitro and teratomas in immunocompromised mice, which proved their ability to differentiate into cells derived from three germ lay-ers. tTRKRAB-mediated epigenetic repression persisted through prolonged culture of obtained IPS cell lines. Im-portantly, expression of introduced transgenes remained undetectable after differentiation into embryonic bodies. In order to confirm molecular homogeneity of obtained IPS cell lines, high throughput molecular profiling in-cluding RNA-Seq and global DNA methylation analysis are currently being performed. Our results confirm that our epigenetic switch effectively prohibits re-expression of embryonic transgenes in hu-man IPS cells and their differentiated progenies paving the way for their applications in various fields of regen-erative medicine, disease modeling and drug discovery.

P1.4

Dynamics of retroelements activity during reprogramming of human fibroblast to induced pluripotent stem cells (iPSc)Katarzyna Tomczak1, 2, Urszula Oleksiewicz2, Maciej Wiznerowicz2, 3

1Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw; 2Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 3Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Retroelements constitute around 90% of all transposable elements in the human genome. These endogenous para-site sequences are able to move through the genome from one location to another contributing to genome diversity and disease, including cancer. The expression of mobile elements is regulated by epigenetic mechanisms includ-ing histone modifications and DNA methylation. Inten-sive epigenetic remodelling of the chromatin structure is important process occurring during cellular reprogram-ming and formation of induced pluripotent stem cells (iPS) through forced expression of selected transcription factors. Here, we investigated the dynamics of retroele-ment activity during generation of induced pluripotent stem cells (iPS). Our research including molecular and biochemical analysis confirmed that cellular reprogram-ming process induces activation of retroelements (such as LINE-1) in human cells. In order to obtain insight into epigenetic mechanisms of the observed phenomena we analysed the role of chosen protein in regulation of ret-roelements activity during formation of human iPS cells. Our ongoing experiments are aimed towards profiling of dynamics of retroelements activity during reprogram-ming of human cells depleted of selected epigenetic fac-tors gene expression. Our project will contribute to better understanding of the molecular mechanisms that control retroelements during cellular reprogramming and are in-volved in host defence against these genomic parasites.

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P1.5

KAP1 protein is involved in reprogramming of human induced pluripotent stem cells (iPS)Katarzyna Kulcenty1, Urszula Oleksiewicz1, Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Induced pluripotent stem cells (iPSC) are derived from human somatic cells through ectopic expression of few embryonic transcription factors. They have two unique properties-self-renewal and pluripotency. Their potential raises the possibility to develop variety of novel clinical applications in patient specific regenerative medicine. Recent studies of human iPS cells implicate, that epigen-etic mechanisms are involved in the process of repro-gramming of somatic cells into pluripotent state. Here we probe, that KAP1 protein together with transcription repressors carrying KRAB domain regulate expression of specific genes through histone modification and DNA methylation. First in our experiments, we achieved efficient knock-down of KAP1 in human fibroblast using lentiviral vec-tors carrying KAP1 specific shRNA. Then, our KAP1 knockdown cells together with wild type were repro-grammed using lentiviral vector carrying cDNA for four transcription factors: Oct4, Sox2, Klf4 and c-Myc. Plurip-otent phenotype of emerging iPS cells lines was revealed by analysis of cell surface protein markers by immuno-fluorescence and flow cytometry analysis. The effect of KAP1 knockdown on the reprogramming process was revealed by growth kinetics of the emerging iPS colonies. Our results show, that KAP1 knockdown accelerates the emergence of iPS colonies at early stages of reprogram-ming process. At this early stage, KAP1 knockout cells show higher expression of epithelial markers as well as markers characteristic for pluripotent state (alkaline phosphatase, SSEA4, Tra-1-81, Nanog). Our results im-plicate, that epigenetic mechanisms controlled by KAP1 protein are involved in the reprogramming process.

P1.6

TRIM28/KAP1 triggers DNA methylation of cellular promoters during reprogramming of human fibroblasts to induced pluripotent cellsMarta Gładych1, Urszula Oleksiewicz1, Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Induced pluripotent stem cells (iPSCs) are generated by dedifferentiation of adult cells through the forced ex-pression of few embryonic transcription factors. The reprogramming involves ordered and global epigenetic alterations including histone modifications and DNA methylation that progressively silence expression of lin-eage-specific genes whilst enabling transcription of em-bryonic factors. TRIM28/KAP1 protein is potent epigen-etic modulator that induce heterochromatin formation when recruited to DNA in sequence-specific fashion by zinc finger proteins containing KRAB domain thus leading to transcriptional repression of neighboring genes through histone modifications and HP1 recruit-ment. Exclusively during early embryonic development, TRIM28/KAP1-mediated epigenetic repression leads to DNA methylation that is largely restricted to repetitive elements thus protecting the genomes stability. Here, we have shown that trigger DNA methylation of cellular pro-moters during the reprogramming of human primary fi-broblasts to iPS cells. Conditional repression of PGK pro-moter was achieved by chimeric transrepressor (tetRKR-AB) containing KRAB domain fused with DNA binding domain of tetracycline repressor (tetR) that binds to tetO element from E. coli tetracycline operator in doxycycline (dox)-controllable manner. The tetRKRAB transgene and tetO-PGK-GFP expression cassette were stably integrated into genome of human primary fibroblasts using lentiviral vectors. Next, the engineered cells were reprogrammed to pluripotency by forced expression of Oct4, Klf4, Sox2 and Myc genes, either in presence (dox+) or absence (dox–) of doxycycline. Phenotype of obtained iPS cell lines was confirmeed by immunoflurescence analysis of expres-sion of selected embryonic markers (NANOG, SSEA4, TRA1-60 and TRA 1-81). Bisulfite sequencing of PKG promoter that was subjected to tetKRAB-mediated epi-genetic repression during reprogramming in the absence of dox revealed high level of CpG methylation (98,6%). In contrast, PGK promoter in human iPS cells obtained from the engineered fibroblasts in the presence of dox that sequestrated tetRKRAB from the tetO-PGK-GFP cassette remained largely unmethylated (1,4–2,8%). Our results strongly suggest that transcriptional repressors containing KRAB domain may be involved in changing landscape of genomic DNA methylation during cellular reprogramming.

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P1.7

Role of TRIM28/KAP1 protein in reprogramming of mouse fibroblasts to induced pluripotent stem cells (miPSCs)Marta Klimczak1, Anna Misiewicz1, Andrzej Mackiewicz1, 2, Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Mouse induced pluripotent stem cells (miPSCs) can be generated mouse somatic cells by forced over-expression of embryonic transcription factors that most commonly involve: Oct4, Sox2, Klf4 and c-Myc. miPSCs are highly similar to embryonic stem cells (ESCs) with reference to proliferation and differentiation capacity. Therefore, they provide a promising source of pluripotent cells for not only basic stem cell biology but also clinical cell-based therapies and disease modeling. The major goal of this study is to determine the role of KAP1 protein in repro-graming and self-renewal of miPSCs. TRIM28/Kap1 is a cofactor of KRAB-containing zinc finger proteins (KR-AB-ZFPs). These are known as the transcriptional repres-sors which act by formation of heterochromatin through histone modifications, HP1 binding and DNA methyla-tion. In the first step, TRIM28/Kap1 expression in pri-mary mouse embryonic fibroblasts (MEF) was silenced by a mean of lentiviral vectors carrying short hairpin RNA (shRNA) specific for TRIM28/Kap1. Next, the cells were reprogrammed to miPSC using lentiviral vector carrying Oct4, Sox2, Klf4 and c-Myc transgenes. Immu-nofluorescence analysis of embryonic markers revealed that TRIM28/Kap1 knockdown results in accelerated re-programming of mouse fibroblasts to iPS cells. Our pre-liminary results suggests that repressive chromatin state imposed by TRIM28/Kap1 prohibits the reprogramming thus helps to maintain identity of differentiated cells.

P1.8

Differentiation of induced pluripotent stem cells (iPSC) into functional neuronsSylwia Mazurek1, Joanna Wróblewska1, Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Efficient differentiation of pluripotent stem cells (iPS) to neural stem cells (NSC), and further to neurons, as-trocytes and oligodendrocytes, offers various opportu-nities including: regenerative medicine, neurological diseases modelling and neurotoxicity drug testing. iPS cells are generated by reprogramming of somatic cells to pluripotency through forced expression of embryonic transcription factors and serve as potentially unlimited source of various cell types that can be used in regener-ative medicine. In order to induce neuronal differenti-ation from human iPSC, we used Gibco® protocol with Neurobasal® Medium and Gibco® Neural Induction Sup-plement. First neural rosettes appeared after 6 days of differentiation. Immunofluorescent staining of obtained cells confirmed presence of neuronal marker (Nestin) and absence of pluripotency markers (Oct4, NANOG). The NSC differentiation was confirmed by expression analysis of selected markers for: pluripotent cells (SOX2) and neural stem cells (PAX6, SOX1, Nestin). NSC differ-entiation into neurons, astrocytes and oligodendrocytes was performed with medium consisting of Neurobasal® Medium, B27 Supplement and L-Glutamine for 14 days, additionally, during first 7 days of differentiation medi-um was supplemented with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor (IGF), ascorbic acid, dibutyryl-cAMP. Immunocytochemical staining (Nestin, Tuj) confirmed differentiation into neurons. The results of our studies can be applied for generation of function-al human neural cells from patient-specific iPS cells and pave the way for regeneration of central nervous system injury in future.

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P1.9

TRIM28/KAP1 protein controls self-renewal of human induced pluripotent stem cellsWojciech Barczak1, Katarzyna Kulcenty1, Maciej Wiznerowicz1, 2

1Gene Therapy Laboratory, Department of Cancer Immunology, Poznan University of Medical Sciences, Chair of Medical Biotechnology, Poland; 2Gene Therapy Laboratory, Department of Cancer Immunology, Greater Poland Cancer Centre, Poland

Human induced pluripotent stem cells (hiPS) are generat-ed by epigenetic reprogramming of somatic cells through the exogenous expression of four transcription factors (Oct4, Klf4, Sox2, c-Myc). These cells has the all char-acteristic features of human embryonic stem cells (hES) as self-renewal and ability to differentiate to cells of all three germ layers. Growing body of published data clearly shows that that epigenetic mechanisms are involved in the process of reprogramming of somatic cells into pluripo-tent state. TRIM28/KAP1 protein is one of the epigen-etic regulators that regulate heterochromatin formation through histone modifications and DNA methylation. Over 500 unique zinc finger proteins containing KRAB domain recruit TRIM28/KAP1 protein to genomic DNA in sequence-specific fashion thus leading to transcrip-tional repression of neighboring genes. Here, we showed that TRIM28/KAP1 protein controls self-renewal of hES and hiPS cells. Stable knockdown of TRIM28/KAP1 gene expression in both hES and hiPS cells achieved using len-tiviral vectors carrying specific shRNAs resulted in pro-gressive loss of pluripotent phenotype as confirmed by analysis of surface embryonic markers expression using immunofluorescence and FACS analyses. Moreover, we have documented progressive loss of expression of se-lected embryonic transcription factors in hES and hiPS cells depleted of TRIM28/KAP1 exkpression. Our ongo-ing experiments are focused on detailed analysis of the molecular mechanisms that are involved in the observed phenotype and may shed new light on epigenetic mech-anisms that preserve self renewal of hES and hiPS cells.