Biogeochemistry of hydrogen and methane in anoxic environments: Thermodynamic and isotopic studies

Hydrogen (H₂) is a competitive substrate for several groups of bacteria, and is central to anaerobic metabolism. Hydrogen is maintained at low levels by competition between terminal electron accepting processes, which creates an extreme thermodynamic condition where the bacterially-mediated oxidation of many organic compounds is thermodynamically driven by H ₂ production. This process is referred to as interspecies H₂ transfer (IHT) and is generally studied by use of cocultures, consisting of H₂ producers and consumers.; In order to study H₂-producing organisms involved in IHT, a culture system was developed to externally control H₂ concentration. The vessel was used to: (i) decouple a syntrophic association in an ethanol consuming, methanogenic enrichment culture, (ii) grow pure cultures and study the bioenergetics of the ethanol-oxidizing, proton-reducing, Pelobacter acetylenicus, (iii) test the hypothesis that anaerobic methane oxidation results from a simple reversal of H ₂/CO₂ methanogenesis, and (iv) study the influence of H₂ concentration on carbon isotope fractionation during H₂/CO₂ methanogenesis by Methanobacterium strain Marburg,; Studies of ethanol oxidation show that syntrophic: associations can be decoupled using this method (i), and that pure cultures can be grown (ii). Results also show that P. acetylenicus (WoAcy1) grows with a thermodynamic yield near −25 kJ/mol, and confirms that this organism only conserves ∼1/3 of a mole of ATP per mole of ethanol oxidized.; Studies of anaerobic methane oxidation (iii) demonstrate that neither Methanobacterium strain Marburg, nor Methanosaeta thermophila (CALS-1) are capable of oxidizing methane by simply lowering the partial pressure of H₂. An alternative hypothesis based on thermodynamic calculations is presented which posits that both acetate and H₂ are intermediates in anaerobic methane oxidation.; Stable carbon isotope studies (iv) with Methanobacterium strain Marburg show that the carbon isotope fractionation factor (α-factor) varies from 1.023 with unlimited H₂, to 1.065 when H₂ is limiting. Additional studies with the aceticlastic M. thermophila demonstrate an α-factor of only 1.007 from the methyl position of acetate to methane.