Evaluation of different digestion procedures for total metal · Total digestions revealed an...

9th Symposium on remediation in Jena

“Jenaer Sanierungskolloquium”

"Microbial impact on element mobility"

4th

-5th

October 2010

Friedrich Schiller University Jena

Wöllnitzer Str. 7, D-07749 Jena, Germany

Conference proceedings

Contents

2

Table of Contents

Page

Welcome 3

Conference Program 4

Map of the meeting place 6

Acknowledgements for financial supports 7

Abstracts of the talks 8

Session 1 - Heavy metals and radionuclides in the environment 9

Session 2 - Bacterial heavy metal resistance 14

Session 3 - Fungal impact on biogeochemical cycles 19

Session 4 - Microbial communities 24

Session 5 - Plant interactions 29

Session 6 - Soil-water system and nutrition 34

Abstracts of the posters (in alphabetical order) 39

Notes 55

Welcome

3

Welcome to

9th Symposium on remediation in Jena "Jenaer Sanierungskolloquium"

focusing on

"Microbial impact on element mobility"

The 9th Symposium on remediation is dealing with the impact of microorganisms on the

biogeochemical changes in the environment. These will include specifically the responses of both

bacteria and fungi to bioavailable fractions of metals in soil and the water phase. The molecular

mechanisms induced in cells dealing with heavy metal stress encompass intracellular and

extracellular proteins induced under these conditions. As a result of altered gene expression, the

bioavailability of metals is changed and thus a regulatory loop is formed in the environment

governed by microbial physiology. The interaction with the environment provides new conditions

which influence abiotic reactions in reactive transfer and mobility of metals, both in interaction with

soil or mineral interfaces, and with ground and surface waters.

International participants will present their research in 6 sessions with 30 talks and additional poster

presentations. Session 1 starts with heavy metals and radionuclides in the environment, with

Sessions 2 and 3 are devoted to bacterial and fungal resistance mechanisms, respectively. On

Tuesday morning, the meeting will continue with Session 4 on microbial communities, to be

followed by Session 5 on Plant interactions and a concluding session integrating latest results on the

soil-water system, nutrition and physiology. One invited speaker will open each session, to be

followed by presentations of young researchers to be selected from the applications.

PhD projects of the graduate school “Alteration and element mobility at the microbe-mineral

interface” supported by the German Research Foundation (DFG), of the Excellence Graduate

School “Jena School for Microbial Communication”, as well as research projects supported by the

German Ministry of Education and Technology are presented in this symposium. The research

focus of Bio-Geo-Interactions at the Friedrich Schiller University is also reflected by MSc programs

in Biogeosciences and Microbiology.

We welcome you warmly to the 9th remediation symposium in Jena.

Georg Büchel & Erika Kothe.

Conference Program

4

Conference Program

Monday, 4th October 2010

09:00 Start of Symposium

Opening of the meeting & Introduction: Erika Kothe

Session 1 – Heavy metals and radionuclides in the environment

Chair: Georg Büchel

10:00 Rolf Michel, Hannover, Germany

Man-made radionuclides in the environment and the radiation exposure of man

10:40 Matthias Händel, Jena, Germany

Characterization of birnessite formed on a creek sediment

11:00 Franziska Schäffner, Jena, Germany

Characterization of secondary mineral encrustations in a former uranium mining district using

mineralogical and biogeochemical methods

11:20 Anahita Pourjabbar, Jena, Germany

Groundwater and soil contamination by heavy metals in a former uranium mining site, Eastern

Thuringia, Germany

11:40 Nils Baumann, Dresden-Rossendorf, Germany

Test site Gessenwiese: Results in TRLFS investigations on surface water and pore waters, and

preliminary results on plant saps concerning uranium speciation

12:00 Lunch and Poster Session

Session 2 – Bacterial heavy metal resistance

Chair: Gerd Gleixner

14:00 Carlos Abate, Tucuman, Argentina

Heavy metal resistance in streptomycetes

14:40 Andrea Beyer, Jena, Germany

Formation of Mn-oxides by new Mn(II)-oxidizing bacterial isolates

15:00 Valerian Ciobota, Jena, Germany

Heavy metals influence on the chemical composition of Acidiphilium cryptum JF-5

15:20 Francesca Langella, Jena, Germany

Umbrella: Using microbes and plants for the remediation of heavy metal contaminated sites

15:40 Laura Emili, Viterbo, Italy

Using soil bioaugmentation with selected bacterial strains, native to the abandoned mine

dumps of Ingurtosu (Sardinia) to assess heavy metal bioavailability by adapting the fumigation-

extraction method

16:00 Coffee break

Session 3 – Fungal impact on biogeochemical cycles

Chair: Juraj Majzlan

16:30 Istvan Pocsi, Debrecen, Hungary

Interplay between the heavy metal and oxidative stress tolerances of fungi – a biotechnology-

oriented approach

17:10 Alix Günther, Dresden-Rossendorf, Germany

Interaction of U(VI) with Schizophyllum commune

17:30 Soumya Madhavan, Jena, Gemany

Characterization of genes involved in the biodegradation in Schizophyllum commune

17:50 Steffi Rothhardt, Jena, Germany

Microbial influence on the alteration of organic coatings on sand grains

18:10 Anne-Gret Seifert, Jena, Germany

Function of microorganisms in black slate degradation

19:00 Visit of the Phyletic museum and BBQ at the Institute of Microbiology

Conference Program

5

Tuesday, 5th October

Session 4 – Microbial communities

Chair: Susan Trumbore

9:00 Matthias Kästner, Leipzig, Germany

Microbial in situ degradation of contaminants in soils and sediments – chances and limitations in

complex contaminated sites

9:40 Felix Beulig, Jena, Germany

Microbial communities involved in iron oxidation at an acid mine drainage-affected creek

10:00 Wafaa Zidan, Jena, Germany

Interaction of microorganisms with clay minerals

10:20 Juliane Hopf, Bayreuth, Germany

Comparison of surface morphology and cell adhesion mechanisms of pyrrhotite in the presence of

Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

10:40 Frank Schindler, Jena, Germany

Seven years test‐site “Gessenwiese” – an intermediate result

11:00 Coffee break

Session 5 – Plant interactions

Chair: Anke Hildebrandt

11:30 Ralf Oelmüller, Jena, Germany

Metal uptake into the roots is efficiently controlled by the endophytic fungus Piriformospora indica

12:10 Thi Thu Ha Chu, Pirna, Germany

Biogas potential of plant species accumulating heavy metals

12:30 Paula Stancu, Bucharest, Romania

Testing different phytoremediation methods for the Zlatna tailing dams, Romania

12:50 Steffi Formann, Jena, Germany

Mycorrhiza - succession in petri dish

13:10 Katarzyna Turnau, Krakow, Poland

Arbuscular mycorrhizal fungi mediating differential distribution of heavy metals in plants

13:30 Lunch and Poster Session

Session 6 – Soil-water system and nutrition

Chair: Erika Kothe

15:00 Jens Wöllecke, Cottbus, Germany

First come first grow? Development of ectomycorrhiza communities

15:40 Andrei Nicoara, Bucharest, Romania

The influence of microorganism amendments on plant growth and heavy metal faith in the

rhizosphere

16:00 Felicia Gherghel, Nancy, France

Community-scale analysis of the ability of ectomycorrhizal fungi to mobilize mineral nutrients from

soil organic matter in forest ecosystems

16:20 Daniel Mirgorodsky, Jena, Germany

Heavy metal and radionuclide balances in a phytoremediation field study

16:40 Kersten Roselt, Jena, Germany

Sustainable remediation of heavy metal contaminated sites

17:00 Concluding remarks

End of the symposium

Map of the meeting place

6

Map of the meeting place

http://www.jena.de/stplan/

x: Meeting place (Wöllnitzer Str. 7)

*: BBQ at the Institute of Microbiology (Neugasse 25)

symbol PM: Phyletic museum (Vor dem Neutor 1)

Acknowledgements for financial supports

7

Acknowledgements for financial supports

Talks

8

Abstracts of the talks

Session 1 - Heavy metals and radionuclides in the environment Talks

9

Man-made radionuclides in the environment and the radiation exposure of man

Rolf Michel

Institut für Radioökologie und Strahlenschutz, Leibniz Universität Hannover

[email protected]

This contribution gives a survey about the sources of man-made environmental radioactivity. Man-

made radionuclides are brought into the environment by atmospheric explosions of nuclear

weapons, by releases of radionuclides from nuclear installations, laboratories, and industrial

enterprises during normal operation, by incidents and accidents, by clearance of waste and residues

from practices and work activities, by military use of depleted uranium, by inadequate disposal of

nuclear waste and residues, by loss and destruction of radionuclide sources, and by malicious acts.

Also radionuclides in commodities and excretion of radionuclides from released patients and out-

patients contribute to man-made environmental radioactivity. The relevance of the different sources

and of the individual radionuclides with respect to the radiation exposures is explained and the

resulting radiation exposures are discussed in the context of the natural radiation exposure of man.

Special emphasis is laid upon the consequences of the Chernobyl accident and upon the radiation

exposure due to nuclear power plants in Germany. Finally, the question of the biological

consequences of man-made environmental radioactivity and the relevance of small radiation doses

is addressed.


10

Characterization of birnessite formed on a creek sediment

Matthias Händel, Thilo Rennert, Kai Uwe Totsche

Institute of Earth Sciences, Friedrich Schiller University of Jena

[email protected]

Manganese(IV) hydrous oxides, produced by microbial activity, are highly reactive minerals in the

environment. They control redox transformations of organic and inorganic compounds and are able

to accumulate several toxic and essential elements. We studied Mn-precipitate coatings on a creek

sediment from the former manganese mining district Oehrenstock, southern Thuringia, Germany.

The objectives of this study were to characterize both the mineral and the organic composition of

the precipitates. Fourier-transform infrared (FTIR) spectroscopy and X-ray diffraction identified

birnessite as the dominant mineral phase. In all FTIR-spectra, amide bands and those of

polysaccharides were found, which are characteristic of cell biomass. Scanning electron (SE)

microscopy and reflected-light microscopy visualized incrusted bacterial cells. Energy dispersive

X-ray spectroscopy (EDX) analysis and SE-images demonstrated that the coatings of these cells

mainly comprised Mn, Ca and Ba. The Mn content is proportional to Ca and Ba as verified by both

ICP-MS/OES and EDX-analysis. This shows that Ca and Ba are incorporated in the

crystallographic structure of birnessite (Ca1.55Ba0.3Mn14O27∙9H2O (ICP-MS/OES) vs.

Ca1.62Ba0.38Mn14O27∙9H2O (EDX)). The identification of the Mn-oxidizing bacteria is still pending

and will be implemented soon. In conclusion, the coatings of the creek sediments consist of a

microbially formed calcium-barium birnessite which has accumulated large quantities of Zn (3.65

mg/g), Cu (907 µg/g), Ni (292 µg/g) and As (113 µg/g).


11

Characterization of secondary mineral encrustations in a former uranium mining district

using mineralogical and biogeochemical methods

Franziska Schäffner1, Matthias Händel

1, Andrea Beyer

3, Dirk Merten

1, Kilian Pollok

2, Denise

Akob3, Falko Langenhorst

2, Kirsten Küsel

3, Georg Büchel

1

1Friedrich Schiller University, Institute of Geosciences, Jena, Germany;

2University of Bayreuth,

Bavarian Research Institute of Experimental Geochemistry and Geophysics, Bayreuth, Germany; 3Friedrich Schiller University, Institute of Ecology, Jena, Germany

Heavy metal contamination of large areas due to uranium mining operations poses a serious long-

term environmental problem. The investigated area near Ronneburg in eastern Thuringia, Germany,

was one of the former leaching heaps where low grade uranium bearing ores (uranium content <

300 g/t) were leached from 1972 to 1990 using acid mine drainage (AMD; pH 2.7-2.8) and diluted

sulphuric acid (10 g/l).

Infiltration of the leachates into the glacial sediments underneath the heap led to the current residual

contamination of the soil with heavy metals like Cd, Co, Cr, Fe, Mn, Ni, Pb, Zn and rare earth

elements (REE) in spite of remediation. The formation of secondary minerals in the soils resulted in

an accumulation of heavy metals in certain layers.

Total digestions revealed an enrichment in the total metal content, e.g. of cadmium (up to 5.72

µg/g) and nickel (up to 311 µg/g), in these layers that is accompanied by a high manganese content

(up to 48 µg/g). Sequential extractions showed that manganese oxides, like birnessite and

todorokite, are mainly responsible for the retention of toxic elements in the soil. REE patterns

normalized to the Post-Archaean Australian Shale (PAAS) show an influence of the groundwater on

the formation of the secondary minerals due to capillary rise. A mapping of the element distribution

in the secondary minerals shows that the soil particles are encrusted first with iron oxides and later

with the manganese oxides containing the heavy metals. Biological investigations of Mn-oxidizing

bacteria (MOB) isolates obtained from a nearby-contaminated site showed that this order of

incrustation is possible when organisms are cultivated in iron and manganese containing media.

LA-ICP MS (Laser Ablation - Inductively Coupled Plasma Mass Spectrometry) of cultivated MOB

on agar plates showed that microorganisms at first utilize iron from the media before manganese is

used to form the oxides. The formation of these secondary minerals can be considered a natural

attenuation process decreasing the levels of bioavailable toxic elements.


12

Groundwater and soil contamination by heavy metals in a former uranium mining site,

Eastern Thuringia, Germany

Anahita Pourjabbar1, Tsilla Boisselet

1, Anja Grawunder

1, Dirk Merten

1, Jürgen W. Einax

2, Georg

Büchel1

1Institute of Geosciences, Friedrich Schiller University Jena, Germany;

2Inorganic and Analytical

Chemistry, Friedrich Schiller University Jena, Germany

The present study is dealing with heavy metal contamination in groundwater and soil affected by

former uranium mining activities in Eastern Thuringia, Germany. During the mining activities,

sulfide minerals were oxidized and dissolved later on during passive flooding of the underground

mine forming acid mine drainage (AMD). AMD infiltrated into the soil, and contaminated the

water-soil system with Rare Earth Elements (REE) and other heavy metals.

In this area the groundwater aquifer is relatively close to the surface, and the soil above is the main

source of groundwater contamination. To characterize the hydrogeochemistry of the groundwater,

190 groundwater samples were collected from the area in the time period 2004 to 2008.

Furthermore, 88 soil samples have been eluted with different solutions (H2O and NH4NO3). The

samples were analyzed for 40 chemical and physicochemical features including pH, and metals

including REE. To investigate the relation between REE and other analytical cluster and factor

analyses have been used. High similarities between concentrations of REE and Al, Cu, Fe, U and Y

were found. REE and the other metals concentration are inversely related to pH. The groundwater

samples are more similar to eluates formed by aqueous solution of NH4NO3 than to those of water

elutions. REE patterns from different data sets are rather similar. All samples show a MREE-

enrichment, as well as a HREE-depletion. Furthermore, all samples show a Ce positive anomaly.

However, a significant difference is a slight positive Gd anomaly. Another noticeable difference

between the samples, in particular groundwater and elutions, is the variability of the depletion of

LREE compared to HREE.

Water elution is a very simple, however not the best representative elution solution to simulate the

groundwater chemistry of a contaminated area. Water does give a qualitative impression of metals

being eluted, but underestimates the output. Salt solutions are more indicated, because they better

reflect the effects of salt already present in the soil solution, or the effect of input of surface water

influenced area.


13

Test site Gessenwiese: Results in TRLFS investigations on surface water and pore waters, and

preliminary results on plant saps concerning uranium speciation

Nils Baumann, Thuro Arnold

Forschungszentrum Dresden-Rossendorf, Institute of Radiochemistry

Plants may take up uranium from contaminated sites and thus may represent a potential risk. To

evaluate the risk of uranium being transferred from the environment into the human food chain

knowledge concerning the uranium speciation is essential since reactivity and toxicity of uranium

largely depend on its speciation.

Plants take up their nutrients from the soil in dissolved form and thus uranium may enter plants as

dissolved species. To study this process the speciation of uranium in a uranium contaminated site,

i.e. Gessenheap near Ronneburg in Saxony/Germany was studied by time-resolved laser-induced

fluorescence spectroscopy (TRLFS) in the aqueous phase, i.e. in surface water (drainage channel on

the Gessenheap) and in pore water (approximately 0.5 – 1 m below surface of test site

Gessenwiese). In both waters uranium sulfate species dominate the uranium speciation and clearly

show that uranium sulfate species are in contact with plant components.

In a second step we determined the uranium concentration in the plants obtained from the test field

Gessenheap by ICP-MS. For these analyses cell saps of birch leaves, of a birch boletus, and of

compartments of butterbur (leaves, roots, and sprouts) were obtained by ultra centrifugation. The

highest uranium concentration was found in sap samples from roots of butterbur with 39.3 ppb

uranium (approximately 1.5 10-7

M uranium); in the sap samples of birch leaves 18.1 and 16.6 ppb

uranium, respectively were detected. In all other sap samples the uranium contents were below 4

ppb uranium. The uranium content in the studied dried plant materials were less than 2.2 ppb, and

dried birch leaves contains less than 0.1 ppb uranium.

In contrast to algae cultivated in the laboratory, the above listed uranium concentrations turned out

to be too low to be successfully analyzed by TRLFS. In addition, we have to mention that plant sap

may contain substances which quench the fluorescence signal of uranium (VI), e. g. some heavy

metal ions or organic compounds. For future studies plant samples containing higher uranium

concentrations are used for TRLFS investigations, possibly mosses or lichens.

Session 2 - Bacterial heavy metal resistance Talks

14

Heavy metal resistance in streptomycetes

Carlos Abate

PROIMI, University of Tucuman, Argentina


15

Formation of Mn-oxides by new Mn(II)-oxidizing bacterial isolates

Andrea Beyer1, Franziska Schäffner

2, Matthias Händel

2, Dirk Merten

2, Georg Büchel

2, Kai Uwe

Totsche2, Denise Akob

1, Kirsten Küsel

1

1Friedrich Schiller University, Institute of Ecology, Jena, Germany;

2Friedrich Schiller University,

Institute of Geosciences, Jena, Germany

Uranium mining activities near Ronneburg, Germany resulted in widespread environmental

contamination with high concentrations of heavy metals and radionuclides. Previous work showed

that natural attenuation of heavy metals is occurring in Mn-oxide rich soil horizons that range in pH

from 5-7. Microorganisms are known to oxidize Mn(II) and precipitate Mn-oxides at pH ~7 under

oxic conditions, however little is known about Mn(II)-oxidizing bacteria (MOB) at pH ~5 and in the

presence of heavy metals. The objectives of this work were (1) to isolate MOB from two sites in the

contaminated Ronneburg area at pH 5.5 and 7 and (2) to evaluate the biological formation of Mn-

oxides. Five and 4 strains were isolated from the Gessen Creek and Gessenwiese test field,

respectively, which were confirmed to be MOB using the Leucoberbelin blue spot test. These MOB

were found to be members of the - and -Proteobacteria and Bacteroidetes, including the genera

Pseudomonas, Janthinobacterium, Duganella, and Flavobacterium. Mn-oxides formed were

investigated with FTIR and preliminary data suggests that all isolates may be producing Birnessite.

The ability of the isolates to oxidize Mn was evaluated in liquid cultures. The data revealed that the

ability to oxidize Mn(II) varied among the different strains and in the presence of a mixture of

heavy metals (Cd, Co, Cu, Zn, Ni). Pseudomonas- and Janthinobacterium-relates strains oxidized

the most Mn(II). In addition, localization of Mn-oxides relative to bacterial cells is being evaluated

with scanning electron microscopy (SEM). This work has shown that the isolates obtained from

regions of natural attenuation at Ronneburg are capable of forming Mn-oxides. Future work is

needed to fully evaluate the minerals formed and their interactions with contaminating heavy metals

and radionuclides.


16

Heavy metals influence on the chemical composition of Acidiphilium cryptum JF-5

Valerian Ciobotă1, Eva-Maria Burkhardt

2, Petra Rösch

1, Kirsten Küsel

2, Jürgen Popp

1, 3

1Friedrich Schiller University, Institute of Physical Chemistry, Jena, Germany;

2Friedrich Schiller

University, Institute of Ecology, Jena, Germany; 3Institute of Photonic Technology, Jena, Germany

Investigations focused on the diversity of microbial community in different heavy metals

contaminated sites reveal the presence of various Acidiphilium species. To gain a better

understanding of the role of these organisms in the remediation process and their resistance

mechanisms towards different toxic metals, a study dedicated to the changes induced by various

heavy metals in microorganisms is required. An elegant method for chemical composition analysis

of single bacterial cells is micro-Raman spectroscopy. Various studies demonstrate that Raman

spectroscopy can be successfully applied in bacterial identification field. A possible problem for the

bacteria identification by means of Raman spectroscopy represents the accumulation of storage

material within the bacterial cells. Many microorganisms produce polyhydroxybutyrate (PHB) as

carbon and energy storage material. Since PHB can be accumulated in high amounts within the

bacterial cells, it is expected that the Raman signals from the above mentioned substance partially

or totally overlap the bands from others cell components.

For our study, A. cryptum JF-5 was investigated with respect to the influence of Cd, Cu, Cr and Ni

on the chemical composition of the cells. A. cryptum JF-5 is a dissimilatory Fe- and Cr- reducing

bacterium which is able to use Fe3+

or oxygen as electron acceptor and to produce high amounts of

PHB. The obtained results confirmed that the effect of heavy metals on the chemical composition of

the cells depends on the heavy metal type and concentration. Different resistance strategies

appeared to be used by A. cryptum JF-5 to adapt to the new environmental conditions.


17

UMBRELLA: Using microbes and plants for the remediation of heavy metal contaminated

sites

Francesca Langella, Goetz Haferburg, Erika Kothe

Institute of Microbial Phytopathology, Neugasse 25, Friedrich Schiller University, Jena

The project UMBRELLA, that is supported by the European Community, is aiming to develop

innovative techniques for soil remediation. This includes milestones in biotechnology including

microbial consortia and specific plants to find ways for improving phytoextraction or

phytostabilization, in either case reducing metal contaminations resulting from mining activities to

enter ground and surface waters. This will be achieved by creating a tool-box of microorganisms

specific for each soil to develop cost-efficient and sustainable measures for soil remediation at sites

spread throughout Europe. The contaminated areas presented in this presentation include

Ronneburg (Germany), Zlatna (Romania), and Kopparberg (Sweden), thus covering already a wide

array of environmental conditions with cold, humid and dry climates.

Microbes isolated from each site for the tool-box will be inoculated in soils collected from the

contaminated sites, in the presence of plants which have been selected for their potential of growing

at the sites in order to allow decontamination via accumulation in harvestable biomass

(phytoextraction). Consequently, the isolated microorganisms were screened for their ability to

enhance plant growth, mobilize inorganic phosphate and, fix nitrogen often limited in contaminated

low fertility soils, produce plant growth hormones, produce chelators for heavy metals and, at the

same time, to be resistant against the prevalent heavy-metals. In addition to isolation, specifically

from soil samples collected from the rhizosphere of plants growing naturally in these harsh

environments, the microbial communities at these sites are characterized physiologically and

identified using 16S rDNA sequencing.

In a second step, the selected strains with plant growth promoting effects will be studied in

greenhouse experiments in inoculated pots in the presence of plants able to promote metal

extraction, such as sunflower (Helianthus annuus), Triticale and Solidago. The selection of these

plants was based on good performance at the three investigated sites, to test for optimal microbe-

plant interactions for metal uptake into plant biomass, accompanied by localization of metals in the

plant tissues and in-situ metal speciation in plant biomass. The tool-box will later be provided to

end-users for remediation actions in order to guarantee future applicability of this technology across

Europe.


18

Using soil bioaugmentation with selected bacterial strains, native to the abandoned mine

dumps of Ingurtosu (Sardinia) to assess heavy metal bioavailability by adapting the

fumigation-extraction method

Laura Emili1, S. Marinari

1, C. Alisi

2, F. Tasso

2, A. R. Sprocati

2

1Dept.of Agrobiology and Agrochemistry, Univ. of Tuscia, Viterbo, Italy;

2ENEA, Technical Unit

for Environmental Characterization, Prevention and Remediation, CR-Casaccia, Italy

The traditional technologies for soil remediation of abandoned mining areas are inappropriate

essentially due to the environmental impact and to the costs. The bioremediation could represent an

unconventional solution to restore a good level of soil quality, but it requires an effort to evaluate its

efficiency and sustainability. To this purpose, a better comprehension of the contribution of

microorganisms to metals mobility in soil and a better accuracy of methods used to evaluate the fate

of heavy metals into soil microbial biomass are required, mostly considered in relation to plants,

which could provide the removal of the metals from soil. In this frame the present study aims to

three objectives: i) selection of microbial strains indigenous of the mining site of Ingurtosu

(Cagliari-Italy) able to resist heavy metals and to be plant-growth-promoters; ii) evaluation of

microbial bioaugmentation efficiency in relation to plants growth; iii) to investigate the reliability of

the Fumigation-Extraction method applied to the evaluation of heavy metal bioavailability and

bioaccumulation into soil microbial pool. Results concerning microbial community

characterization, selection of a consortium suitable to the UMBRELLA tool-box for

phytoremediation microbial assisted and some preliminary results about the assessment of the

Fumigation-Extraction method are presented. The study regarding the effect of bioaugmentation on

the heavy metal mobility is under investigation.

Session 3 - Fungal impact on biogeochemical cycles Talks

19

Interplay between the heavy metal and oxidative stress tolerances of fungi – a biotechnology-

oriented approach

Thoughts coming from the transcriptome-wide and proteome-wide oxidative stress responses

of the filamentous fungus Aspergillus nidulans

István Pócsi

Department of Microbial Biotechnology and Cell Biology, University of Debrecen, Hungary

In terms of biotechnology, there are numerous promising molecular targets to engineer toxic

metal/metalloid tolerant fungi for several bioremediation purposes. Among them, the

overexpression of certain elements of the antioxidative defense system represents an exceptionally

effective tool to reach the goals. Considering the wealth of literature available in this field, co-

expression of Cu/Zn-superoxide dismutase and Cu-chaperones, overexpression of glutathione

synthase as well as regulators of the oxidative stress response may result in stress-tolerant

phenotypes in a wide array of fungi. Owing to the wide-spread application of different and robust „-

omics‟ technologies, data on the molecular background of the heavy metal and oxidative stress

tolerances of the aspergilli are accumulating fast. The genetic engineering of this important fungal

genus is remarkably sophisticated and, hence, we can assume that engineered Aspergillus strains

will appear in versatile toxic metal removal technologies in the near future. The filamentous fungus

model organism Aspergillus nidulans possesses a complex and robust stress defense system with

large gene groups regulated by changes in the intracellular glutathione/glutathione disulphide

concentration ratios and in the superoxide and peroxide concentrations. In our experiments, DNA-

microarray expression data supported proteome changes with 34-40 % coincidence, and among the

most interesting oxidative stress induced proteins are thioredoxin reductase, nitroreductase,

flavohemoglobin, and elements of protein degradation – possible future targets to increase the

heavy metal tolerance as well.


20

Interaction of U(VI) with Schizophyllum commune

Alix Günther, A. Roßberg, J. Raff, G. Bernhard

Forschungszentrum Dresden-Rossendorf e.V, Institute of Radiochemistry, 01314 Dresden

Biosorption processes of heavy metals and actinides like uranium by fungal cells play an important

role in the mobilisation or immobilisation of these elements in nature. Different sorption

experiments of U(VI) with Schizophyllum commune at different initial uranium concentrations and

therewith connected varying initial uranium speciation showed high uranium sorption capacities in

the pH range from 4 to 7. Formed uranium complexes were investigated by means of time-resolved

laser-induced fluorescence spectroscopic measurements (TRLFS). The obtained spectra show

clearly differences between the emissions properties of the uranium species on fungi and the

dissolved uranium species in the initial mineral medium. While the oxidation state of uranium

remained unchanged during the sorption process, the uranium speciation changes significantly.

Extra- and/or intracellular phosphate groups are the predominant binding places of uranium due to

our first spectroscopic results. X-ray absorption fine structure spectroscopic measurements

(EXAFS) were performed for a structural characterisation of formed uranium-fungi species and a

verification of the TRLFS results.


21

Characterization of genes involved in the biodegradation in Schizophyllum commune

Soumya Madhavan, Katrin Krause, Erika Kothe

Friedrich Schiller University, Institute of Microbiology, Microbial Phytopathology, D-07743 Jena,

Germany

Schizophyllum commune, a saprophytic white rot fungus is able to degrade complex organic

molecules including lignin. Previous study reveals that it could also able to release carbon from the

surface of black slate by releasing enzymes capable of efficient degradation.

In this study, S. commune is used to investigate the genes involved in the degradation of organic

material by creating a knock-out strain with increased gene targeting efficiencies. In molecular

biology, gene manipulation is often achieved by integration of extra chromosomal DNA into a

target site through homologous recombination.To favour homologous recombination, a strain with

impaired repair is constructed by knocking out ku70 gene.This is achieved by constructing a gene

targeting vector by cloning the amplified genomic flanking regions of ku70 with a selectable marker

gene ura1. The genome of S. commune reveals 16 FOLymes (Fungal Oxidative Lignin enzymes).

These enzymes are potentially involved in lignin degradation which comprise of lignin oxidases

(LO) and lignin degrading auxiliary (LDA) enzymes that generate H2O2 for peroxidases. Genes

with higher impact for degradation were selected based on microarray and Real Time PCR data

comparing the expression level in presence and absence of black slate. The knock-out strain with

higher gene targeting efficiencies will be used for gene deletion approaches for the functional

analyses.


22

Microbial influence on the alteration of organic coatings on sand grains

Steffi Rothhardt1, Gerd Gleixner², Erika Kothe³, Reinhard Gaupp

1

1 Institute of Geosciences, Friedrich Schiller University Jena;

2 Institute of Microbiology –

Microbial Phytopathology, Friedrich Schiller University Jena; 3 Max Planck Institute of

Biogeochemistry, Jena

Organically coated sand particles are found below brown coal seams. These sands retain dissolved

metals by adsorption from bypassing groundwaters. They consequently might be used as adsorbent

to purify low heavy metal contaminated water. However, exposure of these organically coated

surfaces to oxygenated environment might induce microbial degradation. We investigate the

microbial effect on the morphology and chemistry of these organic coatings.

To study the microbial influence on the alteration of organic coatings on sand we analysed the sand

particles before and after the incubation with the fungus Schizophyllum commune using agar

medium. Organic coatings are partial dissolved after the penetration of fungal hyphae because the

basidiomycete is able to degrade organic matter. The interaction of the fungi and the organic

coatings bear different characteristic structures: etch pits, etched channels and secondary mineral

formation. Quartz surfaces appear to remain unchanged. These degradation structures are a sign of

the fungal ability to dissolve complex organic structures beside lignin.

Organic coatings on sand are removable by microbial activity. Because microbial biomass can also

retain metals we will investigate the prevailing mechanism in further experiments. To understand

the effect of substrate quality on the degradation mechanisms we will compare the proteomic

fingerprints of the fungus Schizophyllum commune grown on different substrates.


23

Function of microorganisms in black slate degradation

Anne-Gret Seiferta, Susan Trumbore

a,b, Xiaomei Xu

b, Dachung Zhang

b, Erika Kothe

c, Reinhard

Gauppd, Gerd Gleixner

a

aMax Planck Institute for Biogeochemistry, Jena;

bDepartment of Earth System Science, University

of California, Irvine, CA; cMicrobial Phytopathology, Institute of Microbiology, Friedrich Schiller

University, Jena; dInstitute for Geosciences, Friedrich Schiller University, Jena

Coupling between microbial metabolisms, reactions at the mineral surfaces and element transport at

the mineral-microbe interface are essential processes for the biogeochemical functioning of the

environment. Hereby, the principle role of microorganisms in black slate degradation is well

recognized. However, information is lacking on the contribution of individual groups of

microorganisms and on the effect of labile carbon sources to the degradation process.

Therefore, we investigated the contribution of fungi, Gram-positive and Gram-negative bacteria in

the degradation process using a column experiment and on natural waste piles. Investigations were

performed using low metamorphic black slates. Phospholipid fatty acids (PLFA) were extracted to

investigate microbial communities. We used both the compound specific 14

C and 13

C signal of the

PLFA to quantify carbon uptake from black slates and modern carbon sources.

Both, in a laboratory study and at natural sites, we measured uptake of black slate carbon into

microbial biomass. Radiocarbon measurements on individual PLFA indicated that fungi and Gram-

positive bacteria were best adapted to black slate degradation. PLFA composition could clearly

discriminate between microbial communities in different black slate treatments. Gram-positive

bacteria were most abundant in control columns and on bare sites. Moderate priming with glucose

and plant material resulted in increased degradation of black slate carbon. The effect of priming is

PLFA specific. Results of compound specific radiocarbon and stable isotope analysis also show that

a higher glucose uptake resulted in PLFA representative for Gram-negative bacteria in almost no

black carbon uptake, whereas the availability of glucose positively affected black carbon uptake

into Gram-positive bacteria.

Oxidation of organic carbon from black slates may supply energy for growth of heterotrophs, which

if growing on surfaces, could inhibit pyrite oxidation by out-competing more slowly growing

chemolithoautotrophic organisms for oxygen, nutrients or attachment to the mineral surface. The

presence of specific inoculums like Streptomyces and Schizophyllum might suppress pyrite

oxidation and thus lower the amounts of heavy metals in the aqueous phase. The results suggest,

that encouraging the growth of heterotrophic, especially filamentous microbes can lower the effects

of pyrite oxidation and that microorganisms are able to retain substantial amounts of heavy metals.

Session 4 – Microbial communities Talks

24

Microbial in situ degradation of contaminants in soils and sediments – chances and limitations

in complex contaminated sites

Matthias Kästner1, Petra Bombach

1, Anko Fischer

2, H. H. Richnow

2, Marcus Hirsch

3, Holger Weiß

3

1Department of Environmental Biotechnology (formerly Bioremediation),

2Department of Isotope

Biogeochemistry, 3Department of Groundwater Remediation, Helmholtz-Centre for Environmental

Research, 04318 Leipzig, Germany

([email protected] / Fax: ++49/341/235-1471 / Phone: ++49/341/235-1235)

For successful application of Natural Attenuation processes, a reliable assessment of the in situ

turnover of a contaminant in a contaminated aquifer is essential. Here, we review and present

details of two developed approaches for the assessment of in situ biodegradation:

a) determination of biodegradation caused by microbial metabolism in a contamination plume by

compound specific isotope analysis (CSIA) and

b) determination of the actual degradation under the respective environmental conditions in the

aquifer by using in situ microcosms (BACTRAP®) amended with 13

C-labeled substrates as tracer

compounds. The methods were applied in site assessment studies at the European reference sites

Zeitz (BTEX contamination) and Bitterfeld (chlorobenzene contamination) in Germany.

Based on stable isotope fractionation analysis, the degradation occurring under anoxic

biogeochemical conditions at a respective site could be calculated for the contamination plume.

This has been shown for benzene and toluene at the Zeitz site and partly for chlorobenzene at the

Bitterfeld site. Using the in situ microcosm approach with 13

C-labeled compounds, the microbial in

situ degradation under strictly anaerobic conditions was proven for benzene, toluene, and

chlorobenzene at the sites. The transformation of 13

C-carbon of the labelled substrate into microbial

fatty acids indicates the assimilation of the carbon from the pollutant. The results led to the first full

size remediation measure approved by local authorities in Germany for Monitored Natural

Attenuation at the Zeitz site. However, additional site remediation approaches (air sparging, steam

injection, radiowave heating) clearly indicated the presence of high amounts of non-aqueous phase

liquids in the source of the contamination which need to be excavated before a real decrease of the

contamination by Natural Attenuation can be expected.


25

Microbial communities involved in iron oxidation at an acid mine drainage-affected creek

Felix Beulig, Denise Akob, Maria Fabisch, Kirsten Küsel

Friedrich Schiller University Jena, Institute of Ecology, 07743 Jena, Germany

Former mining activities in the area of Ronneburg, Germany have resulted in contamination of the

Gessen Creek with acid mine drainage. Sediments of the Gessen Creek now have a wide pH range,

ranging across 3 sampling sites from acidic (pH 2.8) over moderately acidic (pH 4.4) to near neutral

(pH 6.4). Previous work at the creek has shown that microbial iron cycling is actively occurring.

The objectives of this study were to i) characterize present and metabolically active bacterial

communities in Gessen Creek sediments and ii) isolate iron-oxidizing bacteria (FeOB) at near

neutral pH. Members of the Betaproteobacteria dominated microbial communities in all 3 sediment

sites, as shown by 16S rRNA gene cloning and sequencing. The majority of Betaproteobacteria-

related clones were related to known FeOB and the distribution of phylogenetic groups of FeOB

was observed to shift with changes in pH in the creek. In acidic sediments, clones were related to

FeOB, e.g. Ferrovum sp. and also surprisingly Sideroxydans sp., as well as iron-reducing bacteria

(FeRB), e.g. Acidiphilium sp. Whereas, in moderately acidic sediments clones were dominated by

FeOB within the genera Gallionella, Ferritrophicum and Sideroxydans. At the near neutral pH site,

a comparison of DNA- and RNA-based clone libraries indicated that both FeRB, e.g., Geobacter

sp., and FeOB, e.g., Rhodobacter sp., Acidovorax sp. and Thiomonas sp., are present and

metabolically active. FeOB were isolated from the near neutral creek sediment site under

microaerophilic, lithoautotrophic conditions at pH 5.5. A total of 4 novel isolates were obtained that

are new species with respect to the closest characterized strains. The strains showed affiliation to

members of the genera Thiomonas and Bordetella, which have not been reported previously to

oxidize iron. The results show that the distribution of the present and active community of FeOB in

the Gessen Creek is strongly dependent on geochemistry, e.g., pH. The isolation of metabolically

active and dominant FeOB strains provides the possibility for future studies on mechanisms of

metal attenuation by FeOB.


26

Interaction of microorganisms with clay minerals

Wafaa Zidan

1, Rachid Ayach

2, Götz Haferburg

2, Urlich Bläß

1, Dirk Merten

1, Erika Kothe

2,

Reinhard Gaupp1

1 Institute of Geosciences, Burgweg 11, Friedrich Schiller University, 07749 Jena, Germany

[email protected] 2 Institute of Microbiology, Neugasse 25, Friedrich Schiller University, 07743 Jena, Germany

Clays and clay minerals are common components in soils, sediments, and sedimentary rocks, and

they play an important role in many environmental processes such as nutrient cycling, plant growth,

contaminant migration, organic matter maturation, and petroleum production. On one side, Fe(III)

reduction in crystalline clay minerals causes fundamental changes to the physicochemical properties

of soil clays such as swellability, exchange capacity, and flocculation properties, affecting their

characteristics in agricultural and contaminant dynamics contexts, on the other side, numerous

studies have shown that interactions of microorganisms with solid adsorbents leads to an increase in

biomass, growth rate and the production of enzymes and metabolites. Changes of size, shape and

porosity of fungal pellets as well as of morphology of microbial hyphae and their interaction with

clay species will influence toxic metal sorption properties on such biosorbants.

Fungi have been found to excrete higher concentrations of more effective chelating agents than, for

example, bacteria. However, bacteria are more abundant and exist in extreme environments that

fungi cannot tolerate.

To characterize microbial influence on clay mineral dissolution and surface alteration, the fungi

Schizophyllum commune and the bacteria Streptomyces acidscabies are incubated with two rich iron

clay minerals (nontronite and chlorite). Different analytical techniques (ICP(OES/MES), SEM,

TEM, XPS, AFM, VSI, STXM, etc., are used to analyse different components (liquid cultures, clay

minerals, and microbial surfaces)


27

Comparison of surface morphology and cell adhesion mechanisms of pyrrhotite in the

presence of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

Juliane Hopf, Kilian Pollok, Falko Langenhorst

Bayerisches Geoinstitut, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany

Pyrrhotite (Fe1-xS) is an abundant sulfide mineral in natural rocks and mine wastes. Microbes can

significantly accelerate its rate of oxidation and thereby enhances the production of sulfuric acid,

causing environmental problems known as Acid Mine Drainage (AMD).

Polished pyrrhotite surfaces were chemically and morphologically analyzed after abiotic and biotic

driven oxidation experiments under acidic conditions. The experiments revealed that the Fe- and S-

oxidizing bacterium Acidithiobacillus ferrooxidans (A. f.) distintly augments the oxidation of

pyrrhotite and particularly favors the dissolution of troilite (FeS) lamellae which are embedded in

the Fe-deficient pyrrhotite matrix. The S-oxidizing bacteria Acidithiobacillus thiooxidans (A. t.) was

tested to examine possible differences in bacterial dissolution behavior and cell adhesion

mechanisms on the mineral surface. The surface morphology of two abiotic/biotic altered pyrrhotite

surfaces were measured with confocal surface microscopy (µSurf) and scanning electron

microscopy (SEM) at various reaction times (7, 14 and 28 days).

A. f. develops a whitish biofilm on the pyrrhotite surfaces, whereas A. t. does not form a visible

biofilm. After washing (biofilm removal), both strains show cell residues on the surface. A. t. shows

a stronger cell adhesion on the surface than A. f. Contrary to observations of bacterial pyrite

oxidation, both bacterial strains show no preferred orientation for cell attachment on the pyrrhotite

surface. Troilite lamellae dissolve to a similar extent in both biotic experiments, but not in the

control. A. f. increases surface roughness slightly faster than A. t. (hardly any change in control). To

investigate the impact of biofilm formation on mineral dissolution, experiments were performed

with cells separated from the mineral using a dialysis capsule. These experiments revealed a slower

increase in surface roughness compared to free cells, indicating that bacterial substances below

20.000 Da are actively involved in the dissolution process of pyrrhotite.


28

Seven years test‐site “Gessenwiese” – an intermediate result

Frank Schindler, Martin Reinicke, Erika Kothe

Institute of Microbiology – Microbial Phytopathology, Friedrich Schiller University Jena

A site in Eastern Thuringia near Ronneburg was investigated with regard to effects of acid mine

drainage (AMD). Low‐grade black shale uranium ores were leached at the heap “Gessenhalde” in

1970–1989. Sulphuric acid (10 g/l) and AMD were used for leaching. As a result of the barrier the

leachate with a pH below 3 and high metal load infiltrated the glacial sediments below the heap. A

high heavy metal load is typical for the heap. In 2004 the field site “Gessenwiese” was created in

the north‐western part of the former “Gessenhalde”.

The inhomogeneous, organically depleted substrate was mixed with compost and topsoil. After that,

the soil was inoculated with the fungus Glomus intraradices Sy 167 and two Streptomyces strains.

This different treatments lead to an increased mycorrhizal rate after inoculation, positive effects on

microbial cell number and for better plant growth and diversity because of an improved nutrient

supply.

Phytoextraction, biostabilisation and bioimmobilization strategies are under estimation in order to

investigate the effects on soil and plants as strategies for bioremediation. Also population dynamics

and their seasonal changes are a point of interest.

Session 5 – Plant interactions Talks

29

Metal uptake into the roots is efficiently controlled by the endophytic fungus Piriformospora

indica

Ralf Oelmüller

Plant Physiology, Friedrich Schiller University Jena, Dornburger Str. 159, D-07743 Jena

The endophytic fungus Piriformsopora indica interacts with the roots of all plant species tested and

promotes their growth, seed and biomass production as well as their resistance to biotic and abiotic

stress. We use a forward genetic approach with Arabidopsis to identify genes and components,

which are targets of the fungus in the plants and which can be used for biotechnological

applications.

Here, I focus on two ions, which are central regulators of many plant pathways, calcium and iron.

Mutants impaired in calcium and iron uptake and metabolism uncovers a so far unexpected

complexity in the regulation of plant fitness and in their ability to shape microbial communities in

the rhizophere.


30

Biogas Potential of Plant Species Accumulating Heavy Metals

Thi Thu Ha Chu, Bernd Bilitewski, Christina Dornack, Veit Grundmann, Ulrike Lange

Institute of Waste Management and Contaminated Site Treatment, Dresden University of

Technology, Pratzschwitzer Str. 15, 01796 Pirna, Germany

Increasing amounts of CO2 which occur due to emissions from fossil fuel combustion are the major

impact causing climate change and most concerned of the anthropogenic factors. Production and

consumption of biogas which are considered as the processes with the net effect of CO2 emission

equals to zero become an increasing demand of many countries. The utilization of plant residues,

which have been grown to remediate heavy metal contaminated soil, as a source of sustainable

energy brings economic, societal and environmental benefits. This research work is aimed at

evaluating the use of mustard (Brassica juncea L.), triticale (x. Triticosecale Wittmack) and

sunflower (Helianthus annuus L.) in a combination approach – phytoremediation and anaerobic

digestion.

Different parts of plant materials were used, including mustard (pod and stem), triticale (spica, stem

and root) and sunflower (blossom, leaf and stem). The roots of triticale accumulated the highest

concentrations of all 12 elements, especially Al, Fe and Mn. The potential of biogas produced from

the contaminated and loaded plant residues was investigated through batch fermentation under

mesophilic conditions. The results of the investigation indicated that heavy metal concentrations in

the majority of the input materials did not inhibit the anaerobic process, however some samples

should be further determined before the final conclusion. After 42 days of fermentation, spica of

triticale produced the highest biogas yield and methane yield with 806 and 440 lN/kg VS,

respectively. Mustard samples produced lower yields of biogas and methane. Sunflower samples,

without washing with water after being harvested, were digested 60 days. Their biogas and methane

yields were lowest. Depending on different input materials of the reactors, digestates from mustard,

triticale and sunflower experiments can be used as fertilizer for agriculture soil with the ranges 8.3-

12.3, 10.2-12.1, and 11.7-14.6 t FM/ha/ in three years, respectively. These amounts can supply the

required nutrients such as N, P and S for some crops, on the other hand the amount of heavy metals

are under the safe limitation.


31

Testing different phytoremediation methods for the Zlatna tailing dams, Romania

Paula Stancu1, A. Nicoara

2, A. Neagoe

2, V. Iordache

2, G. Udubasa

1

1Faculty of Geology, Doctoral School – Minerology Department,University of Bucharest, Romania,

[email protected]; 2Faculty of Biology, Research Center for Ecological Services,

University of Bucharest, Romania

Engineering remediation methods such as covering contaminated areas with cleaner soil give good

results in short term, but they are costly and problematic in terms of logistics and may fail in the

long run. Customized methods adapted to a specific type of site should give better long term results

with less of an initial impact on the area.

The aim of own study was to test different phytoremediation methods, determining which is best

one for vegetating mine tailings to stabilize heavy metals and prevent erosion. We performed two

small scale pot experiments using mine tailing substrate from the Mica Valley, Zlatna, cultivated

with different plant species. In the first experiment we used a mixture of Festuca rubra L. (an

excluder species) and Melilotus albus L. (an acidophilic species). Experimental variants were

unpolluted soil as positive reference, contaminated soil from the small tailing dam, contaminated

soil from the small tailing dam inoculated with mychorrizal fungi; contaminated soil from the big

tailing dam amended with top soil; contaminated soil from the big tailing dam, amended with green

manure (Rhizobium).

In the second pot experiment we used Agrostis capillaris L. (an acidophilic species), and the

amendments for this one were reference soil, calcium carbonate and zeolitic material applied or not

in a mixture. As an inoculum it was used a mixture of three fungi (Glomus intraradices, G.

etunicatum and G. claroideum) and two streptomycetes (S. tendae and S. acidiscabies). Because the

heterogeneity of these substrates is huge, pH values of the substrate ranging between 2.43 and 7.66

and Pb content between 28.79 and 2689 μg/g d.w, we used a strongly homogenized composite

sample.

The inoculations had positive effects on plant parameters, especially on biomass but also on

oxidative stress. Regarding metal concentrations in plants the particular effect of the inoculation is

metal specific. In some cases we found a decrease in the concentration of metal in plants which

could be considered another positive effect and seems to be due to reducing of the metals

availability as a result of amendments. The more pronounced effects of green manure and top soil

were a decrease of the oxidative stress and an increase of biomass production.

Key words: tailing dam, heavy metals, fungi and streptomycetes inoculum, ammendments


32

Mycorrhiza - succession in petri dish

Steffi Formann, Sophia Wirth, Katrin Krause, Matthias Gube, Erika Kothe

Friedrich Schiller University of Jena, Institute of Microbiology, Microbial Phytopathology,

Neugasse 25, D-07743 Jena, Germany

Mycorrhiza is known to help plants to colonize and to survive in low nutrient soil contaminated

with heavy metals. Through different strategies the mycorrhizal fungus is able to bind heavy metals

and to reduce the contact of heavy metal and plant. Within this, fungi well support the process of

remediation.

Sterile co-cultures with tree seedlings of pine and spruce demonstrated a primary succession in petri

dish. We started the experiment with inoculation of the arbuscular mycorrhizal fungus Glomus

intraradices followed by the “early stage” ectomycorrhizae with Pisolithus tinctorius and Paxillus

involutus. The “late stage” ectomycorrhiza using Tricholoma vaccinum completed the succession

experiment. Different techniques like the preparation of thin sections of mycorrhizal roots and the

staining of the fungal tissue showed vesicles of arbuscular mycorrhiza and the fungal sheath with

the Hartig`net of ectomycorrhiza. Antagonistic growth experiments using different conditions like

competition with and without heavy metal content in the growing media gave insights in the

behaviour of the fungi.

By the help of these results we will focus now on microcosm experiments in a bigger scale to

simulate this succession in a heap like the uranium mining heap of Ronneburg, Thuringia,

Germany.


33

Arbuscular mycorrhizal fungi mediating differential distribution of heavy metals in plants

Katarzyna Turnau1, P. Ryszka

1, E. Orlowska

3, J. Mesjasz-Przybylowicz

2, W. Przybylowicz

2

1

Institute of Environmental Sciences of the Jagiellonian University, ul. Gronostajowa 7, 30-387

Kraków, Poland, E-mail: [email protected]; 2

Materials Research Department, iThemba LABS,

PO Box 722, Somerset West 7129, South Africa; 3Department of Molecular Biology, University of

Aarhus, Gustav Wieds 10 C, 8000 Aarhus C, Denmark

Mutualism in terrestrial, multicellular and parenchymatous plants seems to be ancestral, although,

the mechanisms of interactions between symbionts and the role of partners in element distribution

still remains unknown. In the present study the distribution of elements within mycothallic and

nonmycothallic gametophytes and early stages of sporophyte development of fern Pellaea viridis

(member of Pteridaceae) was investigated using micro-PIXE. Gametophytes collected from soil

samples of the Agnes Mine ultramafic area near Barberton (South Africa) were grown on both the

original soil and on a plant growth substratum obtained from the local botanical garden. Soil was

inoculated with Glomus tenue. Mycothallic ferns had significantly more Mg, Ca, V, Al, Si than

nonmycothallic ones. Within young gametophyte the cells that were colonized by the fungi and also

forming rhizoids accumulated more Zn, Mn, Fe, Si, Cu, P, S, Ca and Cl than the rest of the

gametophyte. In older gametophytes the content of Ni, Si, Ti, Mn, Fe Rb, Br, Cu and Sr was even

higher than in other parts of the ferns. The results strongly support the role of mycorrhizal plants in

detoxification of potentially toxic elements and selective transfer of nutrients from the soil into the

shoots. For the first time this was visualized in case of gametophytes and young sporophytes of the

ferns but probably is similar in case of other mycorrhizal plants.

Session 6 – Soil-water system and nutrition Talks

34

First come first grow? Development of ectomycorrhiza communities

Jens Wöllecke

Brandenburg University of Technology Cottbus, Chair of Soil Protection and Recultivation

Post mining landscapes are most suitable for investigations of the colonization process, the

development of initial fungi communities, and the following primary succession in virgin

substrates. Within the Lusatian lignite mining district (East Germany) ectomycorrhizal fungi (ECM)

as root symbionts of woody plants are of great importance for the development of young trees in

reclaimation areas. The parent material is characterized by the lack of nutrients, organic material

and any organisms. All species, including ECM, have to re-enter those sites to establish a new

biocoenosis. Quercus rubra and Pinus sylvestris seedlings free of mycorrhizae were grown in the

greenhouse and planted together with cuttings of a Populus-clone on two different mining sites,

representing different soil conditions. These plants developed mycorrhizae with the fungi already

established an active mycelium in the substrate. The following primary succession of ECM

communities was studied in a false time series of red oak (Quercus rubra L) stands from 5 to 40

years. The temporal change of the composition of the exploration types within the ECM

communities, the mycorrhiza frequency and species diversity as well as other factors like predation

influencing fungal distribution will be discussed.


35

The influence of microorganism amendments on plant growth and heavy metal faith in the

rhizosphere

Andrei Nicoara1, Virgil Iordache

1, Aurora Neagoe

1, Erika Kothe

2

1Research Center for Ecological Services, Faculty of Biology, University of Bucharest, Romania;

2Institute of Microbiology - Microbial Phytopathology, Friedrich Schiller University Jena, Germany

[email protected]

Mycorrhizal fungi and bacteria are vital for plant growth and a rich vegetal cover will sustain rich

microbial communities, preventing soil erosion and immobilizing toxic elements.

To test the influence of plants and microorganisms on heavy metal movements in contaminated soil

we have set up a mid-scale lysimeter experiment using soil monoliths prelevated from a former

industrial area near Bucharest, Romania. The soil was contaminated with lead, copper and zinc by

atmospheric deposition. Metal concentrations are in the range of 2000 mg/Kg for lead, 250 mg/Kg

for copper and 500 mg/Kg zinc, with the highest concentrations at a depth of 0-10 cm.

The plants chosen for the experiment were sunflower for the first season and rye for the second

season. Experimental variants were: unamended soil as a negative control, soil amended with

Glomus intraradices mycorrhizal fungus and soil amended with Glomus intraradices and and a

mixture of Streptomyces tendae and Streptomyces acidiscabies. Plant growth was assessed by

measuring biomass and size of individuals, while the health of plants was measured through

assimilating pigment and oxidative stress enzyme assays. Metal faith was investigated by measuring

metal concentrations in the soil, percolation water and in different parts of the plants.

Microorganism amendments had a dramatic effect on plant growth and heavy metal assimilation.

Amended plants were significantly larger in size than the negative control. Protein concentrations

were much higher in amended plants. The leaves of amended plants had lower concentrations of

oxidative stress related enzymes.

Mycorhyzation was also analyzed. No arbuscules or vesicles were observed in the roots of negative

control plants from the first season, while amended plants showed extensive mycorrhization. The

experiment will have a total of four cultures, two with sunflower and two with rye.

Key words: lysimeter, heavy metals, Helianthus annuus L., Secale cereale L., bioremediation


36

Community-scale analysis of the ability of ectomycorrhizal fungi to mobilize mineral

nutrients from soil organic matter in forest ecosystems

1Felicia Gherghel,

1J. Garbaye,

2A. Franc,

3M. A. Ali,

1P. E. Courty,

4C. Damon,

3M. Duchemin,

5S.

Egli, 7J. Ernst,

4L. Fraissinet-Tachet,

5S. Hutter,

4R. Marmeisse,

5M. Peter,

3C. Plassard,

6K. Pritsch,

7F. Richard,

1F. Rineau,

2M. Roy,

6M. Schloter,

7M. A. Selosse

1INRA Nancy (France),

2INRA Bordeaux (France),

3INRA Montpellier (France),

4CNRS Lyon

(France), 5WSL Zurich (Switzerland),

6TUM Munich (Germany),

7CNRS Montpellier (France)

The FUNDIV project aimed at exploring in situ the functional diversity of ectomycorrhizal

communities, the key to understanding their contribution to ecological processes of interest for

sylviculture, soil conservation and landscape management. An experimental approach has been

chosen based on ecosystem manipulation and response modelling: relevant variables concerning the

functionality of ectomycorrhizas (mobilization of mineral elements from soil organic matter) have

been measured in three field experiments with various environmental and sylvicultural controlled

factors. FUNDIV data collecting was terminated in 2009 and we present here the results of

preliminary data analyses.

Eight enzyme activities secreted by ectomycorrhizas and involved in organic matter decomposition

have been measured in spring and autumn for two years in control and treated plots of a network of

ten forest ecosystem manipulation experiments (water regime, soil chemistry, soil compaction, leaf

damage) in a range of site types in west-central Europe. The taxonomic status of the fungal

symbionts has also been determined by ribotyping.

The analysis of variance (site, sampling date, treatment, taxa) of the functional structure of the

ectomycorrhizal communities thus described reveals that the enzymatic activity pattern is mainly

driven by the taxa, then by the site and the sampling date, while the treatments contribute to the

total variance for a few percents only. All communities share the same structure pattern with a few

dominant species and many less abundant ones. The species differ from site to site but are

moderately affected by the treatments. Dominant species display higher enzymatic activities than

rare species, what is consistent with competitive exclusion on functions.

These results show that the overall ability of the ectomycorrhizal community in a forest ecosystem

is very dependant on the assemblage of symbiotic fungal species and on the site characteristics, but

that this community is very resilient towards short-term environmental disturbance.

Keywords: ectomycorrhiza, community, enzymatic activities, diversity


37

Heavy metal and radionuclide balances in a phytoremediation field study

Daniel Mirgorodsky, Delphine Ollivier, Dirk Merten, Georg Büchel

FSU Jena, Institute of Geosciences, Burgweg 11, D-07749 Jena, Germany

Large sites with a slight contamination by heavy metals and metall(oid)s were in the past a problem

for remediation procedures since the “traditional” processes are to expensive for an application on

such large sites.Phytoremediation can be an alternative for such areas since it is cost effective.

Phytoremediation was applied to a former U mining area near Ronneburg, East Germany. On this

area, a low grade uranium ore leaching dump was situated, that was later removed during

remediation actions. Today, a slightly to intermediately contaminated underground with restrictions

of land use is remaining.

To enhance the efficiency of phytoremediation procedures quantification of HM/R transport in the

soil-water-plant system is of utmost importance.

Lowering of metal leaching could be an important advantage of in situ immobilization treatments,

and may play a role in groundwater protection and reduction of metal dispersion. In the present

study a lysimeter approach was used to evaluate and compare the effect of different soil amendment

strategies (increasing pH and organic matter content) and additives (nitrogen-phosphor-potassium

fertilizer (NPK), mycorrhiza + Streptomyces (MS)) on the percolation of metals and on HM/R

uptake in plants. One lysimeter contains an undisturbed soil monolith containing a black layer

where accumulation of Pb, Ni, Co and Zn occurred. Two lysimeters were filled with homogenized test

field substrate (TF control) in addition of calcareous topsoil (MIX) and mycorrhiza and

Streptomyces (TF+MS). All lysimeters were planted with identical plant mixture of Triticale,

Helianthus annuus and Brassica juncea.

In general, HM/R concentration of seepage water increased after homogenization of the soil due to

the increase of the mobile fraction. The addition of the soil amendments calcareous topsoil

(MIX+NPK) and mycorrhiza and Streptomyces (TF+MS+NPK) decreased significantly HM/R

concentration of seepage water in comparison to undisturbed soil (monolith). Here, the seepage

water loads for Al, Ni, Zn, Co, U and La are orders of magnitude higher for the monolith as with

soil amendments MIX+NPK and TF+MS+NPK.

With information of in-situ HM/R content in a soil-water-plant system and the knowledge about

(in)- and outputs by plants and/or seepage water, quantification of HM/R cycling in the system soil-

water-plant can be carried out.


38

Sustainable remediation of heavy metal contaminated sites

Kersten Roselt

JENA-GEOS®-Ingenieurbüro GmbH, Saalbahnhofstraße 25c, 07743 Jena

Soil contamination caused by immission through various paths, generates the forming of different

types of contamination, which vary in spreading and intensity. Therefore, genesis of soil

contamination crucially determines risk analysis as well as solutions to related conflicts at current

or future utilization of burdened areas.

The problem will be illustrated by evaluation and remediation of a previously used phosphor

factory in Thuringia, the world‟s largest Cd-pollution which is known so far.

Termination of production in 1988 resulted in regression of brownfield immission. This is proven

by sudden regression of concentrations within series of measurements to dust precipitate, to surface

water and its suspension and also to annual rings of trees, however, not to soil and groundwater.

Within an observation period about 15 years temporal and spatial variances of soil contamination

state can be detected for heavy metals (Cd, Zn, As, …), too. Thus, the actuality of data has to be

scrutinized in the space of spatial analysis about distribution of soil contamination. Critical analysis

about source and quality of data, its factually logical weighting and a modern geo-statistical

evaluation create basis for distinction of use-based areas, which are marked by limit exceedance of

contamination, preferably illustrated in a GIS (site atlas). Thereby, results about contamination

types essentially are important regarding to prevention of distorted outcomes.

Integration of specific re-use projects within the analysis leads to the effect that questions about

active defence of environmental danger don‟t have to be solved by specially fitted remediation

methods. In this way, decontamination or immobilization can be integrated in the concept of

scheduled structural projects according to principle of commensurability. Thereby, enormous

optimization potentials can be occurred.

In this context the project optirisk shall be referenced. optirisk comprises the optimization of site

development concepts for polluted, unused properties. The project deals with the improvement of

chances for reactivation ecological burdened brownfields with the objective of reducing land

consumption at Greenfield. Employing a so-called Integrated Site Development Concept an

optimization of investment needs associated with the reduction in costs for revitalization will be

achieved on the basis of a risk prognosis model. Thereby, the utilizations of site-specific renewable

energy and heat storage potentials also are regarded. You can find the “Guideline to optimization of

site development concepts for polluted, unused properties", further information and other useful

tools, in English too, at www.optirisk.de.

Posters

39

Abstracts of the posters

(in alphabetical order)

Posters

40

Copper – load of Austrian vineyard soils

Andreas Baumgarten

AGES Institute for Soil Health and Plant Nutrition, Vienna, Austria

In the course of the evaluation of the Austrian programme for environmentally sound agriculture

(ÖPUL) the load of vineyard soils with copper has been assessed. Routinely, only the EDTA –

extractable amount is measured, but threshold values are related to aqua regia extractable amounts.

Based on a representative sample, the relation between EDTA- and aqua regia extractable amounts

was calculated, showing a coefficient of determination R = 0.879.

According to this relation, 48% of the samples taken in the north eastern production area were

above the Austrian threshold of 60mg/kg Cu. In the southeastern production area, only 18% of the

samples were above this limit. Similar to the regulations concerning the application of sewage

sludge, the use of plant protection compounds containing copper should not be used on these plots.

Posters

41

The effect of Sb(V) on the transformation of ferrihydrite to goethite

Ralph M. Bolanz1, J. Maizlan

1, S. Ackermann

2, T. Y. Reich

3, M. Kersten

3

1Inst. of Geosciences, Friedrich Schiller University, Jena, Germany ([email protected]);

2SGS Institute Fresenius GmbH, Kölliken, Switzerland;

3Inst. of Geoscience, Johannes-Gutenberg-

University, Mainz, Germany

Many aspects of the environmental chemistry of antimony, from its global cycling to its local

speciation, are yet relatively poorly understood [1]. Antimony can become a pollutant of soils and

sediments, especially in acid mine drainage systems and shooting ranges, where it can become

adsorbed by iron oxides. The most common Fe oxide in these settings is the poorly crystalline 2-

line ferrihydrite, which can be transformed upon ageing to goethite or hematite [2]. The rate and

products of the transformation depend on temperature, pH, and the presence of adsorbing ions or

molecules [3]. The focus of this study was the formation rate of goethite from synthetic ferrihydrite

in the presence of Sb(V).

To verify the association of Sb(V) with ferrihydrite, EXAFS spectra were collected at the Super-

XAS beamline at the Swiss Light Source (SLS). In addition to the first coordination sphere (6 O

atoms at 1.98 Å), two Fe atoms at the distance ~3.11 Å and four Fe atoms at the distance ~3.57 Å

were detected. This result can be interpreted as a strong inner-sphere edge- and corner-sharing

complexation on the ferrihydrite surface and agrees well with the conclusions of related studies of

antimony adsorption onto Fe oxides [4, 5].

Fig. 1: Full width at half maximum (FWHM) of the (101) peak of goethite (Pnma setting) as a function of ageing time.

Series 0 shows the formation of goethite without Sb(V), whereby the Sb(V) concentration increases from series 1 to

series 5.

2-line ferrihydrite was doped with different concentrations of Sb(V) by KSb(OH)6. During aging of

the doped ferrihydrite at 60 °C, twelve samples were taken, washed, dried and measured by X-ray

diffraction (XRD). An increase of the FWHM with increasing Sb(V) indicates retardation of the

transformation of ferrihydrite to well-crystalline goethite, similarly as already known for silicate

[3]. [1] Filella, M., Williams, P.A., Belzile, N. (2009) Environ. Chem., 6, 95–105. [2] Cudennec, Y., Lecerf, A. (2006) J.

Solid State Chem., 179, 716–722. [3] Cornell, R.M. (1987) Z. Planzenern. Bodenk., 150, 304-307. [4] Mitsunobu, S.,

Harada, T., Takashi, Y. (2006) Environ. Sci. Tech. 40, 7270 - 7276. [5] Ackerman, S., Gieré, R., Newville, M.,

Majzlan, J. (2009) J. Sci. total Environ., 407, 1669 – 1682.

Posters

42

Developing and banch-scale testing of soft decontamination-remediation methods to be used

in highland of Georgia

V. Gvakharia, Archil Chirakadze, M. Mirtskhulava, L. Sakhvadze, R. Gigauri, I. Chelidze, T.

Adamia, Z. Buachidze

St. Andrew the First Called Georgian University of Georgian Patriarchate, Scientific-Research Firm

“Gamma”, National Center of Disease Control and Public Health, Tbilisi Georgia

([email protected])

Vast amount of industrial, toxic and potentially toxic waste (about 200 mil tons of As, Mn, Cu, Zn,

Fe, coal wastes and slag, containing heavy and noble metals) is accumulated in Georgia. This

causes morbidity of the residents, impacts the quality of environment and hinders tourism in the

state. Development and related growing load on the natural environment affects quality of air,

drinking and surface water, cause soil contamination and hinders production of ecologically clean

agricultural products causing deterioration of the human health. The situation is aggravated by the

fact that amount of the toxic waste is growing by 4-5 mil tons annually. The hot spots need

immediate and effective remediation.

As and some of its compounds are ranked as toxic to wildlife and humans, persisting in the

environment, accumulating in living organisms and capable to cause cancer and genetic damage.

Today the level of As contamination of soils in two highland regions of Georgia – Racha and Lower

Svaneti – is the highest in the world and causes the anomalously high level of extension of heart

ischemia and hypertensive disease in this region, stimulates the process of depopulation (the

number of children per family sharply decreased from 2.8 to 1).

Last year the systematical study of the environment contaminated with As (wastes and soils) was

carried out which showed that the main “secondary” sources of pollution are the destroyed

storehouses and poorly managed landfills and tailings with arsenic containing compounds. At the

same time a number of 'soft' methods were developed and banch-scale tests were carried out.

Experiments showed that developed methods can be successfully combined and can serve as a basis

for the low-cost technologies for combined physical, chemical and biological utilization of arsenic-

containing wastes and bioremediation of contaminated soils in the areas mentioned above. The 5-

years plan of urgent decontamination and remediation of Racha and Lower Svaneti was developed.

The appropriate costs must be very low due to the usage of the developed novel technologies and

must not exceed 600,000 USD during all five years instead of more than 10,000,000 USD required

for methods usually applied for decontamination and remediation of arsenic-contaminated areas.

Posters

43

Identification of a gene product important for caesium accumulation in Arabidopsis thaliana –

the role of SEC22

Stephan Dräxl┼, Ulrike Kanter

○, Sabine Heuck

┼, Anton R. Schäffner

○

┼Institute of Radiation Protection, Helmholtz Zentrum München,

○Institute of Biochemical Plant

Pathology, Helmholtz Zentrum München

Analysis of Caesium (Cs+) uptake and transport mechanisms in plants is carried out not only for the

purpose of reducing radio-caesium contamination after nuclear fallout, but can also give deeper

insight into the regulation of ion homeostasis in general or Cs+ accumulation in particular. Cs

+ has

physicochemical features similar to that of potassium (K+) and thus is transported mainly via

potassium channels and pumps, but several findings point towards a discrimination of the two ions.

Concentration measurements in a whole genome yeast mutant library identified a number of genes

influencing specifically the Cs+ content of the cells. Amino acid homologies to the according yeast

proteins as well as further bioinformatical approaches identified possible candidates in the model

plant A. thaliana. Knock-out lines for those candidates were screened for significantly altered Cs+

accumulation behaviour via the root uptake way using radioactive tracer. The aim was to identify

genetic factors that influence Cs+ concentration in the plant but that are at the same time not

essential for the basic ion homeostasis of the cell. This kind of factors would establish a working

platform for manipulation of Cs+ accumulation which could finally be assigned to crop plants, and

might reveal new facts about the regulation of ion uptake in eukaryotic cells.

We identified a strong and specific phenotype showing a reduced concentration of Cs+ in the knock-

out of the gene SEC22, which is in accordance with the behaviour of the according yeast-mutant. A

set of diverse studies is applied to achieve a more detailed characterization of the mutant and to

assign SEC22‟s role in Cs+ accumulation. Recent results in yeast also imply a possible use in

phytoremediation.

Posters

44

Role of soil supplements in the plant microbial cycle of trace metals

Gerhard Gramss, Hans Bergmann†

Friedrich-Schiller-University, Institute of Earth Sciences, Burgweg 11, D-07749 Jena, Germany

Microbial solubilization and transformation of metals bound to rocks, minerals, or organic matter

and their subsequent plant availability depend on the nature of soil nutrient resources. Most

bacteria, mitosporic fungi, and ascomycetes expect the supply of external nutrients in dormancy,

while others degrade lignocelluloses and humic substances (HS) upon consume of NO3- and other N

sources. Basidiomycetes are more efficient in the degradation of soil organics, use Norg to

accumulate NH4+ in soil (up to 500 %) and do predominantly not compete for the plant‟s nitrate

resources.

All bacteria and fungi diminish/elevate soil pH within the first 30 d of carbohydrate/organic N

supplementation. The resulting one-basic bacterial acetic, butyric, formic, lactic, and propionic

acids acidify soils pH < 5, whereas the common malic/fumaric, and, in part, malonic and isocitric

acids also act as metal phytochelants. Fungi form, beneath the one-basic acetic, propionic, and

pyruvic acids higher concentrations of oxalic and malic/fumaric acids. The generation of ammonia

from organic N by all microorganisms is cause of pH increases.

At pH 4 to 5, cation exchange by, and chemical reactions of minerals with, H+, elevate the

concentration of dissolved metal cations dramatically over those of their otherwise dominant HS

ligand. This gives room for the formation of low-stability (non-HS) complexes of high plant

availability. Whereas bacteria and several mitosporers do little to degrade and solubilize additional

HS ligands, basidiomycetes dissolve high concentrations of HS by peroxidases, oxalic, and malic

acids, forcing metal cations to form the less plant available HS complexes even at low pH.

In the ageing carbohydrate-amended soil, microbial degradation of carboxylic acids which is linked

with consume of H+ and the liberation of OH

- results in alkalination and thus in the formation of

stable and less plant available metal-HS complexes. In contrast, the initially alkalinized, organic-N-

amended soil acidifies by nitrification of the resulting ammonium.

Uptake of the (enzyme-) proteid-associated trace metals, (Cd), Co, Cu, Mn, Ni, and Zn depends

more on the plant‟s ability to form high concentrations of the respective proteids than on metal

speciation in the soil solution. Cutting the plant‟s N supply by bacteria and lower fungi or

generating soil mineral N species by basidiomycetes has thus more influence on the plant‟s metal

homeostasis than differences in the nature of metal complexation and plant availability. It is shown

that external ammonium supply is optimum to support plant protein formation and uptake of trace

metals made plant available in a soil which acidifies by nitrification. Acidifying soil with

carbohydrate supplements results in the depletion of N, in plants low in protein and trace metals,

and in the formation of carboxylic acids which are phytotoxic in their undissociated state.

Posters

45

Heavy metal uptake of mushrooms from a former uranium mining site in Eastern Thuringia

Matthias Gube, Erika Kothe

Friedrich-Schiller-University Jena, Institut of Microbiology – Microbial Phytopathology, Neugasse

25, D-07743 Jena

The uptake of heavy metals by fungi has great impact on public health and the environment.

Consumption of edible mushrooms collected on contaminated sites may pose a health thread. On

the other hand, knowledge on uptake capacities and mechanisms can potentially facilitate

ecotoxicological risk assessment or bioremediation applications.

The former uranium mining district in Eastern Thuringia and Saxony is well suited to study the

impact of heavy metals. Mining of the sulfidic ore was undertaken between 1949 and 1990, with an

outcome of more than 220,000 tons of uranium. Today, the remnants of mining include various

heaps in different stages of restoration.

Element contents of fruiting bodies collected at the northwestern part of the former Lichtenberg pit

near Ronneburg/ Thuringia were analysed by ICP-MS to evaluate their individual uptake capacities

in relation to bioavailable and total soil content. Accumulation of certain heavy metals varies

greatly among species. In some cases, this reflects phylogenetic relationships, as shown in the

considerable cadmium accumulation capacity of Agaricaceae and Lycoperdaceae. Certain species,

such as Thelephora terrestris, are hyperaccumulators of multiple elements, often widely surpassing

total soil content. Element uptake is in many cases two to three magnitudes higher than soil element

contents considered as bioavailable, suggesting increased availability of mineral bound elements for

fungi.

Posters

46

Phytoremediation of slightly HM/R contaminated soils and subsequent utilization of the

plants

Lukasz Jablonski, S. Willscher, P. Werner

Institute of Waste Management and Contaminated Site Treatment, Technical University. Dresden,

Pratzschwitzer Str. 15, 01796 Pirna

In the last 20 years, great progress has been made in the field of remediation of heavy metal

contaminated sites. However, there are very large areas with slight contaminations of heavy metals;

their remediation is still a problem due to the clean-up costs. The phytoremediation represents an

emerging and sustainable technology for such slightly contaminated sites.

A research project on this subject is carried out on a former uranium mining site where in the past a

leaching of U and radionuclides was carried out. Main topics of this research project shall be the

investigation of the phytoextraction of heavy metals and radionuclides (HM/R) out of the soil, a

balance of their mass transport into the underground, as well as the final utilization of the plants

with a mass balance of their waste streams.

In this part of investigations, possibilities of the maximization of heavy metal uptake by selected

plants were investigated by means of pot, as well as field experiments. Variants of soil

improvements (compost, lime, sand) are scrutinized in combination with fertilization (NPK) and

chelating agents (EDTA, NTA). So far, field and pot experiments with potatoes (Solanum

tuberosum) and topinambur (Jerusalem artichoke) confirmed the ability of those two species to

grow well on HM/R contaminated test field substrate and their resistance to unfavorable conditions

for plant growth (low pH 4.4, low content of available phosphorus and nitrogen, compact structure

of soil). First germination and small scale pot experiments with sugarbeet (Beta vulgaris) show

great importance of further investigation of these plants on a field, or by means of large scale pot

experiments.

A final utilization of the plant biomass as CO2-neutral energy source was investigated in further

experiments. Fermentation experiments with triticale grain from test field gave 89% and 91.5%

yield of ethanol compared to an uncontaminated triticale, and 96.8-97% utilization of reduced

substances. The fertilizer value of the resulting distillery grains does not differ from the distillery

grains of conventional materials.

Another fermentation experiment with Solanum tuberosum and different heavy metal additions to

the fermentation pulp gave as well satisfactory results (the lowest heavy metal addition results in

100.44% of uncontaminated alcohol yield) and proved the enrichment of heavy metals mainly in the

solid phase of distillery pulps.

Linking the phytoremediation with a subsequent winning of an energy carrier (ethanol) seems to be

a sustainable remediation method, and costs of remediation and restoration of soil function can be

reduced by the benefit from the derived ethanol.

Posters

47

Distribution of trace elements in leaves of Helianthus annuus

Anika Kötschau1, G. Büchel

1, J. W. Einax

2, W. von Tümpling

3, D. Merten

1

1Friedrich Schiller University Jena, Institute of Geosciences, Applied Geology, Burgweg 11, 07749

Jena, Germany; 2Friedrich Schiller University Jena, Institute of Inorganic and Analytical Chemistry,

Environmental Analysis, Lessingstraße 8, 07743 Jena, Germany; 3Helmholtz-Centre for

Environmental Research – UFZ, Water Analytics and Chemometrics, Brückstraße 3a, 39114

Magdeburg, Germany

Trace elements like Cu, Ni, Mn and Zn are essential needed for healthy growth and life of plants,

since they have several important functions like being part of enzymes in the organism. However,

an excess of trace elements in the surrounding soil could lead to disease and death, especially if

they are bioavailable, due to low pH. Some plants are able to resist high contents of these elements,

since they either exclude them or accumulate them without being destroyed. A plant which is

known to grow under increased trace element concentrations in soil and low pH is Helianthus

annuus (Sunflower). It accumulates trace elements in roots, shoots and leaves. To test if and how

the elemental distribution in leaves changes throughout the time and under different growth

conditions several pot experiments were carried out. H. annuus was planted in pots filled with

contaminated soil substrate from the Gessenwiese (pH ranges around 4-5) and grown in the

greenhouse during summer/autumn 2010. The Gessenwiese is situated in a former mining area near

Ronneburg (Thuringia, Germany), which is polluted with heavy metals like Cu, Ni, Mn, Zn, U and

rare earth elements. Over a time period of 12 weeks H. annuus has been grown and harvested every

2 weeks. The leaves were separated according their growth state. Ca, Cd, Ce, Co, Cr, Cu, Eu, Fe, K,

La, Mn, P, Pb, U and Zn were analyzed in the leaves by Laser Ablation-Inductively Coupled

Plasma-Mass Spectrometry (LA-ICP-MS). In addition the binding sites of the elements in the plant

tissue are of interest. Therefore a sequential plant extraction was performed. Fine grounded plant

material was extracted in three steps: 1st step - shaking in ethanol for 24 h, 2nd step - shaking in de-

ionized water for 24 h, 3rd step -microwave-assisted digestion with concentrated HNO3. During the

first and second step trace elements bound to small organic molecules like amino acids and those

which are kept in vacuoles or appear free in the cell plasma are extracted. During the last step the

elements bound to the cell wall are extracted. To find elements with similar behavior a cluster

analysis was done for every step and compared with each other. Cluster analysis is an unsupervised

chemometrical method for pattern recognition.

Posters

48

Impact of environment(tal stress) on mycorrhiza

Katrin Krause, Christine Steinbach, Felicia Gherghel, Erika Kothe

Friedrich Schiller University Jena, Institute of Microbiology, Neugasse 25, D-07743 Jena,

Germany, [email protected]

Mycorrhizal fungi have deep impact on nutrition and health of plants, especially under stress

conditions as heavy metals.

Field experiments showed that mycorrhizal profile depends on the bioavailability of heavy metals.

Investigations of gene expression indicated specific expression in lab experiments with microarray

assay too. Different basidiomycetes Tricholoma vaccinum, Paxillus involutus, and for control the

wood rotting fungus Schizophyllum commune were used for the comparison of expression pattern

after the growth on heavy metal and fungicide containing media.

Using an in vitro model system, differential gene expression in Tricholoma vaccinum spruce

ectomycorrhiza was investigated and genes expressed mycorrhiza specific but also in adding toxic

substances in pure cultures of the fungus were identified.

Posters

49

Improvement of phytoremediation of metal-contaminated substrates and gainful utilisation of

contaminated plant material

Daniel Mirgorodsky1, D. Ollivier

1, D. Merten

1, G. Büchel

1, S. Willscher

2, J. Wittig

2, L. Jablonski

2,

P. Werner2

1Institute of Geosciences, Friedrich Schiller University, Burgweg 11, 07749 Jena, Germany

2Institute of Waste Management and Contaminated Site Treatment, Technical University Dresden,

Pratzschwitzer Str. 15, 01796 Pirna

The Ronneburg mining district provided more than half of the East German uranium production

(about 220 kt of U3O8). 1990, the newly founded Wismut GmbH began a major effort to remediate

the area. However, elevated concentrations of heavy metals including radionuclides (HM/R) are

still found in groundwater and in soils, especially in cemented layers.

Phytoremediation is a technique that uses plants to extract HM/R from the soil and/or to immobilise

them so that they are no longer a risk for groundwater and food chain. This technique is well

adapted to large surfaces and low to medium contamination. It is an innovative, in situ, cost-

effective strategy which contributes to the improvement of the physical, chemical and ecological

quality of the soil.

In a joint project remediation is optimized by applying phytostabilization and phytoextraction

methods combining microbial, soil and plant techniques both at the field- and the lab scale. The

addition of biological and biochemical activators increased stress tolerance of plants. Fertilization

significantly increased biomass of triticale and Brassica napus thus improving bioremediation

success. Furthermore, availability of HM/R to seepage water and biota could be significantly

reduced by increasing pH.

Concentrations of heavy metals are typically higher in roots and shoots than in blossoms and seeds.

However, also within the plant compartments the binding strength differs from element to element

demanding different strategies for the utilisation of the material. Alcoholic fermentation was shown

to be very well suited since the fermentation of Solanum tuberosom, Helianthus tuberosus and

triticale does not interfere with high heavy metal concentrations allowing both efficient production

of bio-ethanol and high accumulation of heavy metals in the residue.

Posters

50

Development and application of low-carbon technologies for utilization of manganese-

containing waste and producing of high-grade manganese ferroalloys

Merab Mirtskhulava, V. Gvakharia, A. Chirakadze

National Center of Disease Control and Public Health, Scientific-Research Firm “Gamma”,

St. Andrew the First Called Georgian University of Georgian Patriarchate, Tbilisi Georgia

([email protected])

Manganese is the main mineral wealth of Georgia. Total amount of Mn ore in Chiatura district is

estimated to be around 115 mil tons. It is used in steel, ferroalloys, aluminum cans, fungicides,

fertilizers and electronics production. However, development of this resource generated significant

amount of waste. Today, total amount of Mn waste (ferroalloy slag, solid tailings) already disposed

in Zestaphoni-Chiatura district is about 80 mil tons, while total amount to be disposed in the area

during the next 50-60 years is expected to be another 60-80 mil tons. Results of population survey

have shown that prevalence of respiratory and nervous system diseases are twice higher in the town

of Chiatura where the basic Mn mines are located than in the adjacent villages. Also food poisoning

rate is rather high. Taking into account the level of the Mn pollution in Zestaphoni-Chiatura regions

the full-scale remediation of the sites is rather complex and requires significant financial input

(several hundreds of mil of dollars), which is unaffordable for Georgia. The challenges of global

climate change can only be solved with rapid development and diffusion of low carbon

technologies, both for energy supply and energy efficiency. This is recognized in statements by

governments, business and in international agreements and declarations such as those adopted by

the G8 and in the Bali Action Plan. Global warming is mainly caused by greenhouse gases. The

emissions of CO2 have been dramatically increased within the last 50 years and are still increasing

by almost 3% each year. At the date CO2 emissions worldwide by year are estimated to be about

32-40 billion tons. Considerable part of CO2 direct emissions (2.0-2.5%) is related with Mn

ferroalloys industry: metallic manganese, low- and middle carbon ferromanganese,

silicomanganese, high-carbon ferromanganese, etc. Therefore, the development of novel

technologies reducing the CO2 emissions, caused by Mn ferroalloys, is significantly important for

mitigation of greenhouse effect and global warming. Moreover, producing and use of new

ferroalloys with increased de-oxidation efficiency can also contribute to carbon abatement in steel

industry. The rapid growth of prices of metallic manganese and high grade manganese ferroalloys

provides the economical effectiveness and high profitability of producing these alloys from

microwave pretreated and enriched wastes and low grade ores. Both methods proposed to develop

and to test in this work became prospective only recently due to of this sharp rise of prices and

serious improvements of both methods. They can provide in Georgia and Ukraine the low-cost

profitable industrial annual utilization of about 3 mil tons of wastes reducing the greenhouse gas

emissions for about 2 mil tons per year. The global effectiveness of the proposed project could be

the reducing of CO2 emissions for about 15 mil tons per year.

Posters

51

Changes in microbial populations over time in an AMD affected field site

Martin Reinicke, Frank Schindler, Erika Kothe

Friedrich Schiller University Jena, Institute of Microbiology-Microbial phytopathology, Neugasse

25, 07743 Jena, Germany

Microbial communities play an essential role in the global metabolic cycles of carbon, nitrogen,

sulfur and other elements. Especially in contaminated regions, microbes are important parts of the

ecosystem influencing element mobility. The microbial communities have to cope with these

contaminants, which are providing an additional selective pressure.

At our study and sampling site near Ronneburg (Thuringia, Germany), heavy metals were

mobilized via leaching processes driven by acidic mine drainage (AMD) and microbial leaching

with Acidithiobacillus ferrooxidans. During the leaching process, the leachate percolated through

the isolation layer and contaminated the underground with mobilized heavy metals and salts.

After removal of the heap material, the salt and heavy metal rich sediment led to a pH in the range

of 3 to 4.5, while the content of organic matter is very limited. As a result of these parameters an

obvious decrease in numbers of cfu per gram soil of 100 to 1000fold were observed in comparison

to an uncontaminated soil. The test field Gessenwiese was installed 2001. Three large plots were

prepared with different treatments: 5cm with topsoil or compost and no amendment as control.

To understand the interdependencies between affecting conditions and to investigate the influence

of heavy metals, the population dynamics and growth characteristics of single isolates were

investigated including both cultivation-dependent and DNA-based fingerprinting methods. Plating

strain isolation, direct cell counts and respiration measurements were used to establish surface and

vertical profiles at the heavy metal contaminated field site to follow microbial diversity over time.

Long time observation of the living cells showed that the cell number decreases slowly after

addition of compost. The positive effect of the compost and topsoil addition to fertilize substrates

and promote plant growth can still be visualized of plant growth. Plating experiments showed a

majority of Gram-positive bacteria with the ability to form spores. With molecular methods, it was

able to detect also Gram-negative microbes, which usually are predominant and widespread in soil.

Cloning of 16S rRNA genes helps to describe the entire microbial community. A 16S rRNA gene

bank is set up to identify the active microbial consortia.

Posters

52

OPTIRISK®

Optimization of the site development for polluted, unused properties based on identification and

monetary valuation of liability and investment

Old sites – New energy

Energetic re-use of fallow, ecologically burdened sites in the rural space of Thuringia

Soil classification and land use specification of the State of Qatar

Kersten Roselt

JENA-GEOS® -Ingenieurbüro GmbH, Saalbahnhofstraße 25c, 07743 Jena

Posters

53

Raman spectroscopic contribution to the field of bioremediation

Angela Walter1, P. Rösch

1, T. Bocklitz

1, W. Schumacher

1, S. Erdmann

3, M. Reinicke

3, E. Kothe

3, J.

Popp 1,2

1Institut für Physikalische Chemie der Friedrich-Schiller-Universität Jena, Helmholtzweg 4, 07743

Jena, Germany; 2Institut für Photonische Technologien, Albert-Einstein-Str. 9, 07745 Jena,

Germany; 3Institut für Mikrobiologie der Friedrich-Schiller-Universität Jena, Neugasse 25, 07743

Jena, Germany

Raman spectroscopy reveals molecular information and can be applied in a non-invasive manner. It

covers already many fields concerning microbial identification on species and strain level

processing micro-Raman spectra of single cells. The application of sophisticated chemometrical

methods has been established and is under ongoing development.1 Therefore, Raman spectroscopy

can be applied to investigate microbes on single cell level and e.g. to study interactions of biotic and

abiotic soil elements.

We demonstrate the classification of Streptomyces exposed to different Ni2+

concentration on single

cell level by micro-Raman spectroscopy. Unknown concentrations are predicted successfully within

defined concentration ranges. Resistances are, among other genes, often encoded on plasmids and

thus we question what is spectroscopically and statistically required to detect differences induced by

plasmid pDrive transformed into an Escherichia coli strain. The classification and identification

potential of Raman spectroscopy for differentiating bacterial cultures due their plasmid content is

investigated. To further elucidate the potential of Raman spectroscopy, the influence of the

antibiotic ampicillin is discussed.

To extend the studies from prokaryotic to eukaryotic organisms, the fungi Schizophyllum commune

is studied spatially resolved. Here, non-invasive and labelfree mitochondria localization was

achieved. The capability of in vivo application with additional chemical information via resonance

Raman spectroscopy and recording times near video rates by means of Coherent anti-Stokes Raman

scattering (CARS) as well as cytochrome quantification is demonstrated.2

References:

[1] M. Harz, P. Rösch, K.-D. Peschke, O. Ronneberger, H. Burkhardt and J. Popp, Analyst, 2005,

130, 1543–1550.

[2] A. Walter, S. Erdmann, T. Bocklitz, E. M. Jung, N.Vogler, D. Akimov, B. Dietzek, P. Rösch, E.

Kothe and J. Popp, Analyst, 2010, 135, 908-917

Posters

54

Molecular and microscopical characterization of the algae mat from the „Gessenhalde“

(Ronneburg/Eastern Thuringia) and the proof of the retention of uranium

Isabel Zirnstein1, Evelyn Krawczyk-Bärsch

1, Thuro Arnold

1, Isolde Röske

2

1 Forschungszentrum Dresden-Rossendorf e.V., Institute of Radiochemistry, P.O. Box 510119, D –

01314 Dresden, Germany; 2 Dresden University of Technology, Institute of Microbiology, D-01062

Dresden, Germany

Next to the test-field site of the former uranium leaching dump “Gessenhalde” (Ronneburg/Eastern

Thuringia), which is part of a research program of the Friedrich-Schiller University, biofilms were

sampled from a creek and investigated by PCR and cloning, light microscopy, CLSM and

TEM/EDX. The molecular and microscopical characterization of the eukaryote diversity showed a

wide spectrum of free living eukaryote microorganism, like the filamentary green algae Microspora

and Klebsormidium as the dominant eukaryote. Flagellates, ciliates and rotaroria are present as

representatives of the protozoa in minor amount. The water of the creek is characterized by a low

pH of 3.3 – 3.9 and a uranium concentration, which ranges seasonally between 1x10-6

M/L and

6x10-6

M/L. Following up the question if the algae mat may have immobilized uranium by

biosorption or bioaccumulation the filamentary algae were selected and investigated

microscopically and spectroscopically by CLSM and TEM/EDX. Since the results did not provide

any microscopic and spectroscopic evidence for the presence of uranium immobilization, the

filamentary algae were cultured in rain water with a pH adjusted to 6.0. After the addition of

uranium as UO2(ClO4)2 in the ecologically relevant concentration of 1x10-5

M/L the algae showed

characteristic fluorescence spectra in the wavelength range of 480-560 nm, which is typical for

uranium (VI). The results demonstrated that the actual in-situ geochemical conditions of the creek

from the "Gessenhalde" inhibit the retention of uranium by the algae mat. The removal of uranium

from the aqueous phase requires the cultivation of algae in a neutral media.

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