The effect of enzyme concentrations and digestion time on protoplast isolation from in vitro grown...
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S565 The effect of enzyme concentrations and digestion time on pro- toplast isolation from in vitro grown leaves of Oak (Quercus brantii) Naghi Shabanian, Mohammad Shafih Rahmani, Fatemeh Zarrinjoei Department of Forestry, College of Natural Resources, University of Kurdistan, Sanandaj, Iran Quercus brantii is one of the important species of west forests of Iran. Oaks are playing important role in ecosystem stability of these forests. In forest trees, such as Oak, owing to their heterozy- gosity in genotype and long juvenile period, classical methods of breeding is difficult and time-consuming. Therefore, parasexual breeding methods by using their protoplasts will be very beneficial. In this study, vital protoplasts were isolated from in vitro grown leaves of Quercus brantii. Different concentrations of hydrolytic enzymes were tested for protoplast isolation (0.5%, 1% and 1.5% of Cellulase Onozuka R-10 in combination with 0.1%, 0.3%, 0.5% and or 0.7% Macerozyme). Several incubation times were used (6, 10, 14, 18, and 22 h) for digestion. The highest yields were obtained when 1.5% Cellulase and 0.1% Macerozyme were used as digestion enzyme solution (2.90 × 10 5 protoplast per gram of fresh weigh). Comparative tests between different incubation times showed that digestion period of 18 h resulted in the best yield of protoplasts, in which, viability of isolated protoplasts was 75%-78%. The proto- plasts were cultured in a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 10 5 and 2 × 10 5 protoplasts ml -1 ). doi:10.1016/j.jbiotec.2010.10.033 New Microbial Fucose-Containing Polysaccharide from Glycerol byproduct: Rheological Studies Cristiana A.V. Torres 1 , Filomena Freitas 1 , Vítor D. Alves 2 , Madalena Cruz 1 , Rui Oliveira 1 , Maria A.M. Reis 1 1 REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 2 CEER - Biosystems Engineering, ISA/Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal Bacterial polysaccharides are polymeric biomaterials, which represent an interesting alternative to plant or algae polysaccha- rides. Their unique and superior rheological properties allow a wide range of industrial applications (e.g. textile, food, cosmetic and pharmaceutical industries). In this work, Enterobacter A47 DSM 23139 was grown on glyc- erol byproduct from the biodiesel industry for the production of a new fucose-containing exopolysaccharide (EPS). The EPS is a poly- disperse polymer, presenting different molecular weight fractions (between 8.0x10 5 and 1.0x10 7 ). It is composed by neutral sugars (fucose (20-35%), galactose (20-35%) and glucose (30-40%)) and acyl groups substituents (e.g. pyruvate and succinate) which confer the polymer a polyelectrolyte behaviour. In order to improve EPS production, it is necessary to keep the highly viscous broth well mixed, especially at a large scale. Thus, the study of the rheological properties of microbial culture broths is essential to improve EPS yield and productivity. The culture broth presents a Newtonian behaviour in the begin- ning and changes to pseudoplastic as the EPS was produced. The increasing in viscosity of the culture broth is not only related with the EPS concentration and molecular weight, since the broth vis- cosity continues to increase when polymer production ceased, but also with the formation of new interaction between individual EPS and other components of the culture medium. The purified EPS is able to form highly viscous aqueous solu- tions, which have a pseudoplastic fluid behaviour. The viscosity is recovered at low shear rate, after subjecting the sample to high shear rate values. The intrinsic viscosity of the EPS in 0.1 M NaCl is 11.4 dL/g, with a Huggins coefficient K H = 0.43, indicating some molecular aggregation. The described EPS characteristics demonstrate that Fucose- containing EPS has an enormous potential to be used as a viscosity enhancer in the several aforementioned industrial applications. doi:10.1016/j.jbiotec.2010.10.034 Metabolic modeling of polyhydroxyalkanoates (PHA) produc- tion from complex mixtures of competing volatile fatty acids (VFA) by mixed microbial cultures (MMC) F. Pardelha ∗ , M.G.E. Albuquerque, M.A.M. Reis, R. Oliveira, J.M.L. Dias REQUIMTE/CQFB, FCT/Universidade Nova de Lisboa, Portugal E-mail address: fi[email protected] (F. Pardelha). Abstract Polyhydroxyalkanoates (PHA) are biopolymers stored by many microbial species, having similar properties to conventional petroleum-based plastics. The use of renewable raw materials, such as fermented sugar molasses, as substrate to open mixed microbial cultures (MMC) constitutes a promising strategy to decrease PHA production costs. A metabolic model for PHA production by MMC from fermented sugar molasses is presented in this work. The model considers the simultaneous consumption of several volatile fatty acids (VFA), the regulatory mechanism associated to competition between sub- strates and limitation/inhibition factors that affected the products kinetics. This mechanism was represented mathematically by a sin- gle control parameter. The model was validated using data gathered from three PHA accumulation batch tests and enabled to elucidate the metabolic changes occurring over the culture enrichment pro- cess. During this period MMC was submitted to the feast and famine regimen in a sequencing batch reactor (SBR) using fermented sugar molasses as carbon source. Metabolic flux distribution (MFD) for each batch was calcu- lated using the proposed model for analysis of culture enrichment data. The fluxes for substrates uptake and products synthesis enhanced over the culture enrichment. Moreover, MFD also sug- gest an inhibiting effect of substrate concentration and/or other components of the fermented molasses, which was attenuated over enrichment time. The effect of the fermented molasses matrix on process performance were evaluated by performing a set of batch tests using synthetic media containing only the main four VFA present in real fermented molasses (acetate, propionate, butyrate and valerate). Moreover, the results showed that the characteris- tic P/O ratio of the MMC varies with changing reactor operating conditions, more specifically with the oxygen supply. doi:10.1016/j.jbiotec.2010.10.035
Transcript of The effect of enzyme concentrations and digestion time on protoplast isolation from in vitro grown...
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S565
The effect of enzyme concentrations and digestion time on pro-toplast isolation from in vitro grown leaves of Oak (Quercusbrantii)
Naghi Shabanian, Mohammad Shafih Rahmani, Fatemeh Zarrinjoei
Department of Forestry, College of Natural Resources, University ofKurdistan, Sanandaj, Iran
Quercus brantii is one of the important species of west forestsof Iran. Oaks are playing important role in ecosystem stability ofthese forests. In forest trees, such as Oak, owing to their heterozy-gosity in genotype and long juvenile period, classical methods ofbreeding is difficult and time-consuming. Therefore, parasexualbreeding methods by using their protoplasts will be very beneficial.In this study, vital protoplasts were isolated from in vitro grownleaves of Quercus brantii. Different concentrations of hydrolyticenzymes were tested for protoplast isolation (0.5%, 1% and 1.5%of Cellulase Onozuka R-10 in combination with 0.1%, 0.3%, 0.5% andor 0.7% Macerozyme). Several incubation times were used (6, 10,14, 18, and 22 h) for digestion. The highest yields were obtainedwhen 1.5% Cellulase and 0.1% Macerozyme were used as digestionenzyme solution (2.90 × 105 protoplast per gram of fresh weigh).Comparative tests between different incubation times showed thatdigestion period of 18 h resulted in the best yield of protoplasts, inwhich, viability of isolated protoplasts was 75%-78%. The proto-plasts were cultured in a modification of the Kao and Michaylukculture medium, using two plating densities (1 × 105 and 2 × 105
protoplasts ml-1).
doi:10.1016/j.jbiotec.2010.10.033
New Microbial Fucose-Containing Polysaccharide from Glycerolbyproduct: Rheological Studies
Cristiana A.V. Torres 1, Filomena Freitas 1, Vítor D. Alves 2,Madalena Cruz 1, Rui Oliveira 1, Maria A.M. Reis 1
1 REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova deLisboa, 2829-516 Caparica, Portugal2 CEER - Biosystems Engineering, ISA/Universidade Técnica de Lisboa,Tapada da Ajuda, 1349-017 Lisboa, Portugal
Bacterial polysaccharides are polymeric biomaterials, whichrepresent an interesting alternative to plant or algae polysaccha-rides. Their unique and superior rheological properties allow awide range of industrial applications (e.g. textile, food, cosmeticand pharmaceutical industries).
In this work, Enterobacter A47 DSM 23139 was grown on glyc-erol byproduct from the biodiesel industry for the production of anew fucose-containing exopolysaccharide (EPS). The EPS is a poly-disperse polymer, presenting different molecular weight fractions(between 8.0x105 and 1.0x107). It is composed by neutral sugars(fucose (20-35%), galactose (20-35%) and glucose (30-40%)) andacyl groups substituents (e.g. pyruvate and succinate) which conferthe polymer a polyelectrolyte behaviour.
In order to improve EPS production, it is necessary to keep thehighly viscous broth well mixed, especially at a large scale. Thus,the study of the rheological properties of microbial culture brothsis essential to improve EPS yield and productivity.
The culture broth presents a Newtonian behaviour in the begin-ning and changes to pseudoplastic as the EPS was produced. Theincreasing in viscosity of the culture broth is not only related withthe EPS concentration and molecular weight, since the broth vis-cosity continues to increase when polymer production ceased, butalso with the formation of new interaction between individual EPSand other components of the culture medium.
The purified EPS is able to form highly viscous aqueous solu-tions, which have a pseudoplastic fluid behaviour. The viscosity isrecovered at low shear rate, after subjecting the sample to highshear rate values. The intrinsic viscosity of the EPS in 0.1 M NaClis 11.4 dL/g, with a Huggins coefficient KH = 0.43, indicating somemolecular aggregation.
The described EPS characteristics demonstrate that Fucose-containing EPS has an enormous potential to be used as a viscosityenhancer in the several aforementioned industrial applications.
doi:10.1016/j.jbiotec.2010.10.034
Metabolic modeling of polyhydroxyalkanoates (PHA) produc-tion from complex mixtures of competing volatile fatty acids(VFA) by mixed microbial cultures (MMC)
F. Pardelha ∗, M.G.E. Albuquerque, M.A.M. Reis, R. Oliveira, J.M.L.Dias
REQUIMTE/CQFB, FCT/Universidade Nova de Lisboa, Portugal
E-mail address: [email protected] (F. Pardelha).
AbstractPolyhydroxyalkanoates (PHA) are biopolymers stored by many
microbial species, having similar properties to conventionalpetroleum-based plastics. The use of renewable raw materials, suchas fermented sugar molasses, as substrate to open mixed microbialcultures (MMC) constitutes a promising strategy to decrease PHAproduction costs.
A metabolic model for PHA production by MMC from fermentedsugar molasses is presented in this work. The model considersthe simultaneous consumption of several volatile fatty acids (VFA),the regulatory mechanism associated to competition between sub-strates and limitation/inhibition factors that affected the productskinetics. This mechanism was represented mathematically by a sin-gle control parameter. The model was validated using data gatheredfrom three PHA accumulation batch tests and enabled to elucidatethe metabolic changes occurring over the culture enrichment pro-cess. During this period MMC was submitted to the feast and famineregimen in a sequencing batch reactor (SBR) using fermented sugarmolasses as carbon source.
Metabolic flux distribution (MFD) for each batch was calcu-lated using the proposed model for analysis of culture enrichmentdata. The fluxes for substrates uptake and products synthesisenhanced over the culture enrichment. Moreover, MFD also sug-gest an inhibiting effect of substrate concentration and/or othercomponents of the fermented molasses, which was attenuated overenrichment time. The effect of the fermented molasses matrix onprocess performance were evaluated by performing a set of batchtests using synthetic media containing only the main four VFApresent in real fermented molasses (acetate, propionate, butyrateand valerate). Moreover, the results showed that the characteris-tic P/O ratio of the MMC varies with changing reactor operatingconditions, more specifically with the oxygen supply.
doi:10.1016/j.jbiotec.2010.10.035
