Topic 7: BioGeoChemistry – Anaerobic Respirations

1. Topic 7: BioGeoChemistry – Anaerobic Respirations Overview Anaerobic respirations of inorganic electron acceptors Aerobic oxidation of the endproducts of anaerobic respirations Cycles (C, N, S, Fe) Industrially and environmentally relevant reactions

2. Topic 7: BioGeoChemistry – Anaerobic Respirations Examples of Examinable Material (will need own complementation of knowledge gaps from internet) The processes: • Sulfate reduction, oxidation of sulfur/sulfides • Nitrate reduction (denitrification), nitrification • CO2 reduction (methanogenesis), methane oxidation • Iron reduction (Geobacter), Iron oxidation • Any others? • Ecological role of the processes • Economic, commercial, applied role of the processes • Reaction, Organism,

3. CO2 + H2O <--> H2CO3<--> HCO3- <--> CO32- H+ H+ Simple carbon cycle R1: Photosynthesis • CO2 + H2O  CH2O + O2 • Electron donor: • H2O (the oxygen atom) • Oxidation state from -II  0 • Electron acceptor: • CO2 (the C atom) • Oxidation state from +4  0 • Reaction is endergonic. How does it work? • Light energy to drive reaction “uphill” • Terrestrial plants and marine microalgae

4. Simple Carbon cycle R2: Respiration • CH2O + O2  CO2 +H2O+ new biomass • Electron donor: organic carbon • Electron acceptor: O2 • Exergonic, releasing energy (ATP) for growth • Reactions stoichiometrically reverts photosynthesis. • For mature ecosystems (e.g. rain forest) respiration balances exactly photosynthetic activity • Sustained Net O2 production (or CO2 consumption) needs deposition of organics

5. Role of Bacteria in Nature CO2 CH2O O2 Electron Acceptor Electron Donor H2O Energy Oxygen cycle and simplified carbon cycle

6. More complex carbon cycle also involves: • Methane cycle • Anaerobic respirations

7. How Can Life without Oxygen Work? • O2 = principal electron acceptor of aerobic life. • Without O2 a different e- acceptor needs to be found. • Fermentations (e.g. lactic, ethanolic) have used internally created e- acceptors for no gain in ATP (no respiration). • Now we will deal with e-acceptors that allow ATP generation via respiration (ETC, proton gradient, ATP-synthase) • Bacteria are the only life forms capable of using electron acceptors other than O2 (anaerobic respiration). • The use of alternative electron acceptors dramatically changes the chemistry of the environment

8. What are the Typical Electron Accepting Reactions? O2 +  H2O (aerobic respiration) 4e- SO42- +  H2S (sulfate reducing bacteria) 8e- Fe3+ +  Fe2+ (iron reducing bacteria) 1e-

9. What are the Typical Electron Accepting Reactions? • S +  H2S (sulfur reducing bacteria) • 2e- • NO3- +  N2 (denitrifying bacteria) • 5e- • NO3- +  NH3 (nitrate ammonification) • 8e- • CO2 + 8e-  CH4 (methane producing bacteria)

10. What are the Typical Electron Accepting Reactions? • O2 + 4e- H2O (aerobic respiration) • SO42- + 8e-  H2S (sulfate reducing bacteria) • Fe3+ + 1e-  Fe2+ (iron reducing bacteria) • S + 2e-  H2S (sulfur reducing bacteria) • NO3- + 5e-  N2 (denitrifying bacteria) • NO3- + 8e-  NH3 (nitrate ammonification) • CO2 + 8e-  CH4 (methane producing bacteria)

11. What Happens with the Electron Acceptors after Accepting Electrons? By accepting electrons, the acceptors they turn into reduced species. Reduced species are reducing agents that dramatically change the chemistry of the environment If in contact with O2 , reduced species can become electron donors for specialised lithotrophic bacteria The continued cycle of electron acceptors to reduced species and back to electron acceptors is a typical part of biogeochemical cycling. The S, N, Fe cycle are typical examples.

12. Classification of Microbial Metabolic Types • Examples: • Algae: Photo-Litho-Autotroph, • Bacteria, Fish: Chemo-organo-heterotroph • Thiobacillus: Chemo-litho-autotroph • Photosynthetic bacteria: Photo-Organo-heterotroph • Our focus : Anaerobic Heterotophs and Aerobic lithotrophs

13. Life without O2: Alternative Electron Acceptors 1 Electron Acceptors Measure for Energy Released with H2 as Electron Donor (log K) O2 H2O 21 NO3- N2 21 NO3-  NH4- 15 Fe3+ Fe2+ 8 SO42- H2S 5 So  H2S 3 CO2 CH4 2 CO2 Acetate ? HumOx HumRed ? adapted from Stumm & Morgan Observation: There is a sequence of use of electron acceptors which is according to the energetic usefulness (redox potential) O2 , nitrate, humic acids, ferric iron, sulfate, CO2

15. Electron Acceptors are Reduced and can become e- Donors Electron Donors Electron Acceptors organic O2 H2O H2S  So NO3- N2 So SO42- NO3- NH4+ Fe2+ Fe3+ Fe3+ Fe2+ NO2- NO3- SO42- H2S H2 H+ So H2S CO  CO2 CO2 Acetate NH4 NO2- CO2 CH4 HumR HumOx HumOx  HumR

16. Interconnection Between Different Electron Donors and Acceptors Electron Donors H2 H+ CH4 CO2 H2S  So So SO42- Fe2+ Fe3+ NO2- NO3- Electron Acceptors CO2 CH4 So H2S SO42- H2S Fe3+ Fe2+ NO3- NH4 NO3- N2 O2 H2O

17. Simple Sulfur cycle

18. CO2 CH4 H2 SO4 2- H2S NO3- NH4+ Competition for Electron Donor by Different Acceptor Systems • General observations with anaerobic respirations: • Threshold level for minimum degradable substrate concentrations decreases with the redox potential of the electron acceptor • Using H2 as a model substrate • (accounting for about 30 % of energy flow in anoxic environments): • Organisms with more “powerful” electron acceptor out-compete others by keeping the H2 concentration below “detection limit” of competitors. • This explains the apparent preference for using best electron acceptors (most positive redox potential) first. Time

19. S v = vmax ------- kM S + How does the threshold for electron donor (e.g. H2) affect the kinetics of uptake rate (not growth rate)? What is the relationship between substrate concentration (S) and its uptake rate (v) ? vmax (h-1) v (h-1) substrate limitation Effect of threshold (e.g. H2) because of back-reaction) S (g/L) kM Growth- Michaelis Menten model

20. Effect of Maintenance Coefficient (mS) on growth Rate The negative specific growth rate (µ) observed in the absence of substrate (when S = 0) (cells are starving, causing loss of biomass over time) is the decay rate mS*Ymax µ (h-1) 0 S(g/L) - mS*Ymax

22. Sulfate Reduction (SRB) Sulfate is a suitable and abundant alternative electron acceptor Typical reactions: 4 H2 + SO42- + H+ HS- + 4 H2O CH3-COO- + SO42- HS- + 2 HCO3- Organisms: Sulfate Reducing Bacteria (SRB), strictly anaerobes, Desulfovibrio, Desulfobacter, etc. Electron donors: small molecules (breakdown products from fermentations, or geologically formed, e.g. H2, acetate, organic acids, alcohols) Reduce also elemental sulfur, sulfite and thiosulfate to H2S Ubiquitous

23. Bacterial Sulfate Reduction When Does it Occur ? • In the presence of organic substances , after depletion of oxygen nitrate and ferric iron  sulfate reduction is next • Initially in sediments, Rates from 0.01 to 10 mM/day • Typically in sediments but also on surfaces (ships) underneath biofilms • Within flocs or intestines of marine animals • Sulfide reacts chemically as a reducing agent (e.g. with O2 or Fe3+)  elemental sulfur formation • Formation of FeS and FeS2 black color of sediment • In typical reduced sediments (e.g. mangroves, estuaries SR may be higher than O2 use) • Thiosulfate disproportionation (SO32-) into sulfate and sulfide

24. Dissimilatory Sulfate Reduction by SRB • Organisms: Sulfate Reducing Bacteria (SRB), strictly anaerobe • Desulfovibrio, Desulfobacter, etc. • Use of small compounds (H2, acetate, other organic acids alcohols but • not polymers, proteins, carbohydrates, fats) • Cooperation with fermentative bacteria needed to degrade detritus • End product sulfide (H2S  HS- + H+) is toxic, reactive, explosive • Typical reactions: • 4 H2 + SO42- + H+ HS- + 4 H2O • CH3-COO- + SO42- HS- + 2 HCO3- • Reduce also elemental sulfur, sulfite and thiosulfate to H2S

25. SRB Significance in Marine Environments Ecologically: playing a major role in sulfur cycle and sediment activities sulfide = O2 scavenger  “negative oxygen concentration” responsible for sulfur deposits (H2S + O2 S + H2O) P-release from sediments Economically: End product H2S: poisonous, explosive, corrosive, malodorous Corrosion of submersed steel structures (e.g. platforms, bridges) Corrosion of oil pipelines (inside and outside) Lethal gas emissions on offshore platforms Petroleum degradation (burning sour gas: H2S + O2 SO2)

26. S-ox: Volcanic Sulfur Springs E.g. New Zealand Yelllowstone National Park

27. SRB Morphology Typical shape of sulfate reducing bacteria (SRB) of the type Desulfovibrio.

28. SO42- HS- SRB Fe2+ H2 H+ FeS e- Steel Corrosion current SRB Role in Corrosion of Steel • Electrons on the steel surface produce stabilising H2 layer • bacteria use H2 as the electron donor for sulfate reduction • this removes electrons and leaves the iron positively charged • The positive charge favours the release of Fe2+ into solution • Ongoing process causes corroding electron flow and weight loss • Bacteria feed on electricity • Cathodic protection

29. SRB Damage to Pipeline Microbially influenced corrosion of marine oil pipeline showing typical pitting corrosion.

30. “SRB in Petroleum Industry” Research at Murdoch Q: Where do SRB in oil pipelines come from? A: Mostly as a biofilm attached inside the pipes. Method SRB monitoring during pig runs. Q: Are SRB supported by corrosion ? A: SRB can grow on corroding iron. Cathodic protection enhances their growth. Q: Are treatments effective against SRB? A: SRB can degrade organic biocides.

31. Dissimilatory Nitrate Reducing Bacteria • Dentrification (nitrate to N2) typically involves the aerobic bacteria • Organic electron donor + NO3- N2 • Bacteria use complex substrates • Further details in lecture on N-cycle • In sediments nitrate ammonification can play important role • Organic electron donor + NO3- NH3 • Nitrate ammonification is due to anaerobic bacteria (e.g. SRB)

32. Dissimilatory Iron Reducing Bacteria • Organisms: Various anaerobic bacteria, no specific group • e.g. Geobacter • e- donors: mainly small compounds • Typical reaction: • H2 + 2 Fe3+ 2 Fe2+ + 2 H+ • Reaction results in lowering of redox potential • Reduce also Manganese, elemental sulfur and other metals (e.g. uranium) • End product is magnetite (Fe3O4) and other compounds (black precipitates) • Significance of iron reduction is still being underestimated • Recent research: electricity production using ferric iron reducing bacteria

33. CO2 or HCO3- Reduction (Methane Producing Bacteria) • CO2 is even more abundant than sulfate but difficult to use • By Methane Producing Bacteria (Archeae) • Strictly anaerobic requiring a redox potential of less than -350mV • Highly oxygen sensitive: • 4 H2 + HCO3- + H+  CH4 + 3 H2O • Very limited substrate spectrum (H2, acetate, methanol) • Syntrophic associations are formed with fermenting bacteria • Because of poor solubility (bubble formation) some methane from sediments escapes into atmosphere (greenhouse gas)  True removal of BOD from water.

34. Aerobic Re-oxidation Processes 1 - Sulfide and Fe2+ • Contact of reduced (black sediments) with O2 : • bacterial oxidation of sulfide and Fe2+ occurs. • Beggiatoa: 2 H2S + O2  2 S0 + H2O (white algae) • Thiobacillus: H2S + 2 O2 H2SO4 (sulfuric acid) •  very low local pH values of <1. •  Further microbial pipeline corrosion. • Also insoluble species are re-oxidized e.g. pyrite (FeS2) bio-leaching of minerals). • Elemental sulfur is often produced as intermediate (white precipitate (“white smoker”, “white algae”)

35. Aerobic Re-oxidation Processes 2 - Ammonia NH4+ oxidation is energetically difficult and slow and requires oxygen as electron acceptor. Organisms: Nitrosomonas, Nitrobacter, two step process. NH4 uptake also possible by assimilation of phytoplankton.

36. Fate of Sulfide in the Presence of Oxygen • Contact of reduced sulfide (H2S or FeS) with air  • spontaneous oxidation (H2S) to insoluble S • Microbial Oxidation: • (a) Beggiatoa: 2 H2S + O2  2 So + H2O (“white algae”) • (b) Thiobacillus: H2S + 2 O2 H2SO4 (sulfuric acid) •  very low local pH values of <1. •  Further microbial pipeline corrosion. • Also insoluble species are re-oxidized e.g. pyrite (FeS2) • (bio-leaching of minerals). • Elemental sulfur is often produced as intermediate • (white precipitate (“white smoker”, “white algae”) • Together, sulfate reduction and sulfide oxidation can close the sulfur cycle.

37. Depth Profile of Aerobic Anaerobic Interface Brown high Eh NO3- O2 Fe3+ Microbial S conversion Sulfate NH4+ Fe 2+ HS- Sulfide Black Low Eh H2 , CH4 Depth Highest chemical and biological activity at the interface (presence of electron donors and acceptors) Concentration

38. Scheme of Ocean Hydrothermal Ventfrom Ocean Ridges • Extreme Life Conditions: • Anaerobic, hydrogen driven • Strong temperature gradients • High pressure • Origin of life is thought to have been thermophilic, with H2 and So from volcanic sources as e-donor and acceptor. www.jamstec.go.jp/jamstec-e/ bio/subext/thergane.html

39. Sulfur Cycle at Hydrothermal Vents H2S + O2  S, SO42- SO42- Biological oxidation H2S Geochemical Reduction Similar principle in sewer pipes

40. “Black Smokers” releasing reduced sufur and iron (e.g. FeS) as potential electron donors for bacteria.

41. S oxidising Bacteria as primary producers White “snowblower” producing suspended sulfur bacteria in snow flake type aggregates during a volcanic eruption Woods Hole Oceanographic Institute East Pacific Rise.

42. Tubeworms (Riftia) living in association with sulfur oxidising bacteria

43. Tubeworms (Riftia) living in association with sulfur oxidising bacteria • Dark food-chain Independent of Sunlight ? Micheal Degruy

44. Deep-sea mussel Bathymodiolus thermophilus using symbiotic sulfur bacteria. Photo by Richard A. Lutz

45. Galatheid crabs lining a fissure at a hot vent on the East Pacific Rise feeding on sulfur bacteria. Courtesy Woods Hole Oceanographic Institution

46. Tubeworms (Riftia) living in association with sulfur oxidising bacteria

47. Anaerobic Oxidation of Sulfide There are principally two conditions allowing sulfur cycle in the absence of oxygen: 1. Presence of light and phototrophic bacteria: Very colorful, play a role in microbial mats Can use light that is not suitable for algae Green sulfur bacteria (S outside, Chlorobium) Purple sulfur bacteria (S inside, Chromatium) 2. Presence of other “powerful e-acceptors (e.g. nitrate, Fe3+) are available Thiomargareta a recent discovery

48. Thiomargareta namibiensis

49. Nitrate storage

50. Thiomargareta namibiensis