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Organics, H 2

DISSIMILATORY IRON REDUCTION. Carbon Dioxide, H 2 O. Organics, H 2. e. Fe(II) Mn(II). Fe(III) Mn(IV). Geobacter metallireducens growing on Mn(IV) oxide. "If it were not for the bacterium GS-15 we would not have radio and television today." Gerrit L. Verschuur in Hidden Attraction:

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Organics, H 2

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  1. DISSIMILATORY IRON REDUCTION Carbon Dioxide, H2O Organics, H2 e Fe(II) Mn(II) Fe(III) Mn(IV)

  2. Geobacter metallireducens growing on Mn(IV) oxide

  3. "If it were not for the bacterium GS-15 we would not have radio and television today." Gerrit L. Verschuur in Hidden Attraction: The History and Mystery of Magnetism, Oxford University Press, 1993

  4. Importance of Dissimilatory Fe(III) Reduction Geological and Microbiological evidence suggests that Fe(III) reduction was the first form of microbial respiration on Earth Fe(III) reduction has an important influence on soil chemistry and water quality Microbial Fe(III) reduction plays an important role in the modern carbon cycle -in oxidizing organic matter and in controlling methane production Fe(III) reduction is an important natural process for the bioremediation of organic contaminants in the subsurface and this process can be stimulated Fe(III)-reducing microorganisms have the ability to reduce and immobilize uranium and other metal contaminants in the subsurface Fe(III)-reducing microorganisms have the ability to transfer electrons directly to Electrodes, providing a novel strategy for energy harvesting

  5. CH2O + SO4= CO2 + HS- 2 CH2O CH4 + CO2 Fe(III) Sediment-Water Interface CH2O + O2 CO2 + H2O Aerobic Respiration Nitrate Reduction CH2O + NO3- CO2 + N2 or NH4+ Fe(III) Fe(II) CH2O + Mn(IV) CO2 + Mn(II) Mn(IV) Reduction Fe(III) Reduction CH2O + Fe(III) CO2 + Fe(II) Fe(II) Fe(II) Sulfate Reduction Methanogenesis

  6. Oxidation of Acetate by Geobacter Acetate Carbon Dioxide e Fe(III) Fe(II)

  7. Oxidation of Hydrogen by Geobacter H2O H2 e Fe(III) Fe(II)

  8. Degradation of Aromatic Contaminants by Geobacter Aromatic Compounds Carbon Dioxide e Fe(III) Fe(II)

  9. Complex Organic Matter Plus Fe(III) Aromatic Compounds Hydrolysis Acetate Minor fermentation acids Sugars, Amino Acids Fe(II) CO2 Metabolism by Geobacter Hydrolysis Fermentative Microorganisms H2 Hydrolysis Long Chain Fatty Acids

  10. Degradation of Aromatic Contaminants by Geobacter Aromatic Compounds Carbon Dioxide e Fe(III) Fe(II)

  11. Zone of Intense Fe(III) Reduction

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

  13. AEROBIC U(VI) ANAEROBIC U(VI) U(VI) Organics U(VI) U(VI) U(VI) Groundwater Flow CO2 U(IV) Precipitation Subsurface Uranium Ore Deposition

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

  15. % of total clones recovered as Geobacteraceae

  16. 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

  17. 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-

  18. Mechanism for the Regeneration of Soluble U(VI) From Precipitated U(IV) in Subsurface Sediments NO3- U(VI) Fe(II) Fe(III) NH4+ U(IV)

  19. N2 Concentrated U(VI) extract Precipitate with U(VI) Reduction Pump or Gravity feed Nitrate Solution (Low conc.) DO < 1mg/L Injection Gallery } Zone of U(VI) Removal Groundwater Flow U(VI) U(VI) NO3- NO3- U(VI) U(VI) U(VI) SO42- NO3- U(VI) NO3- U(VI) NO3- NO3- U(VI) NO3- U(VI) U(IV) Fe(II) Fe(III) Geobacter NO3- NH4

  20. 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- C2H4O2 2 CO2

  21. Who is enriched on the “anaerobic” (anode) electrode? % of clone library Delta-proteobacteria enriched to over 75% of population

  22. Fe(III)-Reducing Bacteria Thermotoga maritima Thermus sp. SA-01 Thermoterrobacterium ferrireducens Deferribacter thermophilus Bacillus infernus Geothrix fermentens Geovibrio ferrireducens Pelobacter propionicus Geobacter chapellei Geobacter humireducens Geobacter arculus Geobacter sulfurreducens Thiobacillus ferroxidans Geobacter hydrogenophilus Geobactermetallireducens Desulfuromonas palmitatis Desulfuromonas chloroethenica Ferrimonas balearica Geobacteraceae Desulfuromonas acetexigens Pelobacter acetylinicus Pelobacter venetianus Aeromonas hydrophila Pelobacter carbinolicus Desulfuromonas acetoxidans Desulfuromusa bakii Shewenella putrefaciens Desulfuromusa kysingii Shewenella saccharophilia Desulfuromusa succinoxidans Shewenella alga Desulfobacter propionicus Ferribacterium limneticum Sulfospirrillum barnseii

  23. Fe(II) Fe(III) Oxide Fe(III) It was Initially Considered That Microorganisms Must Contact Insoluble Fe(III) Oxides in Order to Reduce Them )

  24. Fe(II) Model for Fe(III) Oxide Reduction by Geobacter Fe(III) Oxide Fe(III) C < Release of Shuttle Fe(II) Fe(III) Fe(II) Microbially Produced Reduced Shuttle >C ) Fe(III) Oxide Fe(III) Oxide Fe(III) Fe(III) Fe(III) Oxidized Shuttle C < C < Models for Fe(III) Oxide Reduction by Shewanella and Geothrix

  25. 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

  26. 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.

  27. 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

  28. ROLE CATEGORY % Occurrence % Occurrence in Geobacter in Database Amino acid biosynthesis 2.1 2.1 Purines, pyrimidines, nucleosides, and nucleotides 1.0 1.6 Fatty acid and phospholipid metabolism 0.7 1.4 Biosynth. of cofactors, prosthetic groups & carriers 2.2 2.3 Central intermediary metabolism 1.1 1.9 Energy metabolism 6.1 6.3 Transport and binding proteins 3.1 4.9 DNA metabolism 1.7 2.5 Transcription 0.8 1.1 Protein synthesis 2.8 4.3 Protein fate 2.3 2.6 Regulatory functions 4.6 2.9 Cell envelope 2.3 2.9 Cellular processes 4.0 3.3 Other categories 0.8 1.3 Unknown functions 1.7 2.8 Unclassified 7.4 7.3 Conserved hypothetical 13.0 20.6 Hypothetical 42.3 27.9

  29. r2= 0.70 p< 0.01 GS AF PA AA

  30. Fe(III)-NTA Fe(III)-Oxide Absorbance

  31. 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 0 0 20 40 60 80 100 120 Hours

  32. Orf1 Orf2 FerB Orf3 Orf4 FerA Genes Organization of the 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.

  33. 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

  34. Geobacter sulfurreducenscytochrome expression Fumarate Fe(III)

  35. Cytochrome Expression Patterns

  36. Soluble Fe(III) Mn(IV)OOHin Fe(III)OOHin Specific production of flagella by Geobacter when grown on insoluble substrates Bar = 1µm

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