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Modeling Subsurface Bioremediation by Geobacter

Modeling Subsurface Bioremediation by Geobacter. Acetate. Carbon Dioxide. e. Geo bacter. U(VI). U(IV). Uranium Contamination Removal Documented: Groundwaters from DOE Hanford Site Surface water from DOI site Washings from DOD contaminated soil.

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Modeling Subsurface Bioremediation by Geobacter

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  1. Modeling Subsurface Bioremediation by Geobacter

  2. Acetate Carbon Dioxide e Geo bacter U(VI) U(IV) Uranium Contamination Removal Documented: Groundwaters from DOE Hanford Site Surface water from DOI site Washings from DOD contaminated soil

  3. Acetate-Dependent Metal Reduction at Site 1103 % Fe (II) U(VI) (µM) and NO3- (mM)

  4. 107 80 8 105 7 70 106 104 6 60 105 5 50 103 104 Nanograms of Geobacter 16S rDNA per gram of sediment Number of Geobacter sequences per gram of sediment Percent Fe(II) micromolar U(VI) 4 40 103 100 3 30 100 20 2 10 10 1 10 0 10 5 15 20 0 40 25 5 30 35 10 15 25 35 30 20 40 Time (days) Time (days) MPN and TaqMan results from site 1103 U(VI) and Fe(II) concentrations over time at site 1103

  5. % of total clones recovered as Geobacteraceae

  6. CO2 CH3COO- Fe(II) Fe(III) U(IV) U(VI) Acetate Solution (Low Conc.) DO < 1mg/L N2 Pump or Gravity feed Acetate Injection Gallery Zone of U(VI) Removal } Groundwater Flow U(VI) U(VI) CH3COO- U(VI) U(VI) U(VI) CH3COO- CH3COO- U(VI) CH3COO- U(VI) U(VI) U(VI) U(VI) U(VI) CH3COO-

  7. Subsurface Environments in Which Geobacteraceae Predominate 1. Fe(III) reduction zone of petroleum-contaminated aquifers 2. Uranium-contaminated subsurface sediments in which metal reduction was artificially stimulated 3. Field studies in which subsurface metal reduction was artificially stimulated 4. Fe(III) reduction zone of landfill-leachate contaminated aquifers 5. Diversity of Fe(III)-reducing aquatic sediments 6. On energy-harvesting electrodes in sediments Geographic Range:Minnesota, Mississippi, Wisconsin, Massachusetts, New Mexico, Canada, Switzerland, Netherlands Note: No Shewanella detected even with Shewanella-specific PCR primers

  8. Fe(III)-Reducing Microorganisms Can Use Electrodes as an Electron Acceptor Harvesting Power From Aquatic Sediments and Other Sources of Waste Organics Cathode Reaction: 2O2 +8H+ + 8e- 4H2O Sediment Battery 4H2O 2O2 cathode water 8e- Rload sediment 8H+ 1 - 10 cm anode Anode Reaction: C2H4O2 + 2H2O  2 CO2 +8H+ + 8e- e C2H4O2 2 CO2 Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485

  9. Who is enriched on the “anaerobic” (anode) electrode? % of clone library Over 75% of Anode population Delta-proteobacteria, primarily Geobacteraceae

  10. Environmental Genomics with Geobacter Geobacter provides a rare instance in Environmental Microbiology in which it is possible to study organisms in pure culture that are closely related to the microorganisms that are known to be responsible for an process of interest in the environment. The study of Geobacter physiology is likely not only to indicate how these organisms reduce metals and electrodes in the subsurface, but also to elucidate other physiological factors which make microorganisms effective competitors in subsurface environments.

  11. Elucidation of the Mechanisms for Electron Transfer in G. sulfurreducens • Closely related to Geobacters that predominate in various environments • Genome of G. sulfurreducens available • Genetic system has been developed • Methods for mass culturing available • Techniques available for anaerobic biochemistry • Can readily be grown in chemostats to provide physiologically consistent cells

  12. Data Provided by Barbara Methe, TIGR r2= 0.70 p< 0.01 GS AF PA AA

  13. Geobacter sulfurreducenscytochrome expression Fumarate Fe(III)

  14. Cytochrome Expression Patterns

  15. Orf1 Orf2 FerB Orf3 Orf4 FerA Genes Organization of a known duplication region of G. sulfurreducens genome • The ferA gene, encoded an outer membrane 89kD c-type cytochrome shares 79% identical sequences with the ferB gene • Gene duplication: Two open reading frames-Orf1 and Orf2 preceded the ferB gene have 99.95% same sequences as those preceded ferA respectively.

  16. Fe(III) Reduction by Mutants in the 89 kDa Cytochrome (ferA) and Its Homologue (ferB) 60 ferA:: kan 50 40 Fe(II) mM 30 20 Wild type 10 ferB::cam 0 0 20 40 60 80 100 120 Hours

  17. Soluble Fe(III) Mn(IV)OOHin Fe(III)OOHin Specific production of flagella by Geobacter when grown on insoluble substrates Bar = 1µm Childers, Ciufo, and Lovley. 2002. Nature (in press)

  18. 1 µm Pili production inGeobacter

  19. Fe(III) oxide reduction of pilA mutant

  20. Reduction of soluble Fe(III) by pilA 60 wild type 50 pilA mutant 40 Fe(II) mM 30 20 10 0 0 5 10 15 20 25 30 35 40 45 Hours

  21. Figure 4 Chemotaxis assays using motile G. metallireducens. Plugs contained a, chemotaxis buffer; b,10 mM MnCl; c, Mn(IV) oxides; d, 10 mM FeSO4; e, 50 mM Fe(III) oxides; f,10 mM Fe(III) oxides + 0.2 mM AQDS. Arrows emphasize bright ring of cells around agarose plugs.

  22. NO3 NO3 Motility is Not Needed to Access Soluble Electron Acceptors NO3 NO3 NO3 Motility is Needed to Find and Access Insoluble Electron Acceptors Fe(III) Oxide

  23. Construction of a BAC library from sediment Collect sediment BAC Cloning Vector Extract DNA Clone fragments Screen Library ATCGATCAGCTCAGC GCATCAGCAGCTACG TAGCATCAGCATAAT GCATCGACGATCAGC GCATACGTAGCATCG Excise DNA in agarose plugs Partial SgrA1 digest (Yields 40-100 kb Fragments) Electrophorese on low melting point agarose Sequence

  24. Desulfuromonas BAC clone from Uranium Bioremediation of Shiprock UMTRA Site 1 10,000 20,000 TCA cycle TCA cycle 16s FtsZ TCA cycle 20,001 30,000 40,000 tRNA synthase TCA cycle glucogenesis 40,001 50,000 60,000 Nucleotide Hypothetical Carbon metabolism Cell division Unknown rRNA Membrane Translation

  25. Future Directions • More biochemical and genetic evaluation of potential electron carriers involved in electron transfer to metals and electrodes • Begin evaluation of intermediary metabolism, stress response, and growth under nutrient-limiting conditions found in subsurface • Begin evaluation of regulatory mechanisms • Functional Genomics a. Proteomics (Carol Giometti, Argonne National Laboratory) b. Expression Analysis with DNA-Microarrays (Barbara Methe, TIGR) c. Genetic Studies

  26. Future Directions (continued) • In Silico Biology (Bernhard Palsson) Development of a computer model of Geobacter functioning • Comparative Environmental Genomics Genomic comparison of three genomes of pure cultures (G. sulfurreducens, G. metallireducens, D. acetoxidans) with Geobacter genomes recovered from subsurface environments • Application of results from studies outlined above to measuring and predicting the activity of Geobacter in the subsurface

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