Hydrogen metabolism in a mildly acidic lake sediment (Knaack Lake)

Hydrogen metabolism was studied in anoxic Knaack Lake sediments by measuring the in situ concentrations of dissolved H₂, as well as the V_max, turnover rate constant, and K_m for H₂. The results show that the relatively low rate of H₂/CO₂-dependent methanogenesis is paralleled by a low turnover of the dissolved H₂ pool. H₂-dependent acetate formation did not appear to be of importance based on the discrepancy of the K_m for H₂ consumption between the sediment and the prevalent homoacetogenic microbial population. In this mildly acidic lake sediment, H₂ turnover apparently was limited by H₂ production from organic matter. During incubation of sediment under a gaseous headspace, H₂ escaped from the aqueous phase, and steady state concentrations of dissolved H₂ were significantly lower than under in situ conditions. H₂ concentrations increased upon addition of various organic substrates. H₂ turnover within the sediment appeared unrelated to the concentration of H₂ detected in the water column, especially in the epilimnetic water layers.     

Gas Metabolism Evidence in Support of the Juxtaposition of Hydrogen-Producing and Methanogenic Bacteria in Sewage Sludge and Lake Sediments

We developed new techniques to measure dissolved H₂ and H₂ consumption kinetics in anoxic ecosystems that were not dependent on headspace measurements or gas transfer-limited experimentation. These H₂ metabolism parameters were then compared with measured methane production rates, and estimates of H₂ production and interspecies H₂ transfer were made. The H₂ pool sizes were 205 and 31 nM in sewage sludge from an anaerobic digestor and in sediments (24 m) from Lake Mendota, respectively. The H₂ turnover rate constants, as determined by using in situ pool sizes and temperatures, were 103 and 31 h⁻¹ for sludge and sediment, respectively. The observed H₂ turnover rate accounted for only 5 to 6% of the expected H₂-CO₂-dependent methanogenesis in these ecosystems. Our results are in general agreement with the results reported previously and are used to support the conclusion that most of the H₂-dependent methanogenesis in these ecosystems occurs as a consequence of direct interspecies H₂ transfer between juxtapositioned microbial associations within flocs or consortia.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H₂ or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H₂ was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H₂ sinks when external H₂ was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H¹⁴CO₃⁻. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H₂ was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H₂ was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H₂ or by a cross-epithelial H₂ transfer from the anterior gut regions, which may create microniches favorable for H₂-dependent acetogenesis.     

Methanogenic Pathway and Archaeal Community Structure in the Sediment of Eutrophic Lake Dagow: Effect of Temperature

Methanogenic degradation of organic matter is an important microbial process in lake sediments. Temperature may affect not only the rate but also the pathway of CH₄ production by changing the activity and the abundance of individual microorganisms. Therefore, we studied the function and structure of a methanogenic community in anoxic sediment of Lake Dagow, a eutrophic lake in north-eastern Germany. Incubation of sediment samples (in situ 7.5°C) at increasing temperatures (4, 10, 15, 25, 30°C) resulted in increasing production rates of CH₄ and CO₂ and in increasing steady-state concentrations of H₂. Thermodynamic conditions for H₂/CO₂ -dependent methanogenesis were only exergonic at 25 and 30°C. Inhibition of methanogenesis with chloroform resulted in the accumulation of methanogenic precursors, i.e., acetate, propionate, and isobutyrate. Mass balance calculations indicated that less CH₄ was formed via H₂ at 4°C than at 30°C. Conversion of ¹⁴CO₂ to ¹⁴CH₄ also showed that H₂/CO₂ -dependent methanogenesis contributed less to total CH₄ production at 4°C than at 30°C. [2–¹⁴ C]Acetate turnover rates at 4°C accounted for a higher percentage of total CH₄ production than at 30°C. Collectively, these results showed a higher contribution of H₂-dependent methanogenesis and a lower contribution of acetate-dependent methanogenesis at high versus low temperature. The archaeal community was characterized by cloning, sequencing, and phylogenetic analysis of the 16S rRNA genes retrieved from the sediment. Sequences were affiliated with Methanosaetaceae, Methanomicrobiaceae, and three deeply branching euryarchaeotal clusters, i.e., group III, Rice cluster V, and a novel euryarchaeotal cluster, the LDS cluster. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that 16S rRNA genes affiliated to Methanosaetaceae (20–30%), Methanomicrobiaceae (35–55%), and group III (10–25%) contributed most to the archaeal community. Incubation of the sediment at different temperatures (4–30°C) did not result in a systematic change of the archaeal community composition, indicating that change of temperature primarily affected the activity rather than the structure of the methanogenic community.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.     

Typical methanogenic inhibitors can considerably alter bacterial populations and affect the interaction between fatty acid degraders and homoacetogens

The effects of two typical methanogenic inhibitors [2-bromoethanesulfonate (BES) and chloroform (CHCl₃)] on the bacterial populations were investigated using molecular ecological techniques. Terminal restriction fragment length polymorphism analyses (T-RFLP) in combination with clone library showed that both the toxicants not only inhibited methanogenic activity but also considerably altered the bacterial community structure. Species of low % G + C Gram-positive bacteria (Clostridiales), high % G + C Actinomycetes, and uncultured Chloroflexi showed relatively greater tolerance of CHCl₃, whereas the BES T-RFLP patterns were characterized by prevalence of Geobacter hydrogenophilus and homoacetogenic Moorella sp. In addition, due to indirect thermodynamic inhibition caused by high hydrogen partial pressures, the growth of obligately syntrophic acetogenic Syntrophomonas and Syntrophobacter was also affected by selective inhibition of methanogenesis. Interestingly, by comparing the fermentative intermediates detected in BES- and CHCl₃-treated experiments, it was furthermore found that when methanogenesis is specifically inhibited, the syntrophic interaction between hydrogen-producing fatty acid degraders and hydrogen-utilizating homoacetogens seemed to be strengthened.     

Methane Production from Formate by Syntrophic Association of Methanobacterium bryantii and Desulfovibrio vulgaris JJ

Coculture of a sulfate-reducing bacterium, when grown in the absence of added sulfate, with Methanobacterium bryantii, which uses only H₂ and CO₂ for methanogenesis, degraded formate to CH₄. A pure culture of Desulfovibrio vulgaris JJ was able to produce small amounts of H₂. Such a syntrophic relationship might provide an additional way to avoid formate accumulation in anaerobic environments.     

Carbon and Electron Flow in Mud and Sandflat Intertidal Sediments at Delaware Inlet, Nelson, New Zealand

An investigation of carbon and electron flow in mud and sandflat intertidal sediments showed that the terminal electron acceptor was principally sulfate and that the carbon flow was mainly to CO₂. Studies with thin layers of sediment exposed to H₂ showed that methane production accounted for virtually none of the H₂ utilized, whereas sulfate reduction accounted for a major proportion of the gas uptake. At all sampling sites except one (site B7), rates of methanogenesis were low but sulfate concentrations in the interstitial water were high (>18 mM). At site B7, the sulfate concentrations declined with depth from 32 mM at 2 cm to <1 mM at 10 cm or below, and active methanogenesis occurred in the low-sulfate zone. Sulfate-reducing activity at this site initially decreased and then increased with depth so that elevated rates occurred in both the active and nonactive methanogenic zones. The respiratory index (RI) [RI = ¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism at site B7 ranged from 0.98 to 0.2 in the depth range of 2 to 14 cm. Addition of sulfate to sediment from the low-sulfate zone resulted in an increase in RI and a decrease in methanogenesis. At all other sites examined, RI ranged from 0.97 to 0.99 and was constant with depth. The results suggested that although methanogenesis was inhibited by sulfate (presumably through the activity of sulfate-reducing bacteria), it was not always limited by sulfate reduction.     

Effect of Fall Turnover on Terminal Carbon Metabolism in Lake Mendota Sediments

The carbon and electron flow pathways and the bacterial populations responsible for the transformation of H₂-CO₂, formate, methanol, methylamine, acetate, ethanol, and lactate were examined in eutrophic sediments collected during summer stratification and fall turnover. The rate of methane formation averaged 1,130 μmol of CH₄ per liter of sediment per day during late-summer stratification versus 433 μmol of CH₄ per liter of sediment per day during the early portion of fall turnover, whereas the rate of sulfate reduction was 280 μmol of sulfate per liter of sediment per day versus 1,840 μmol of sulfate per liter of sediment per day during the same time periods, respectively. The sulfate-reducing population remained constant while the methanogenic population decreased by one to two orders of magnitude during turnover. The acetate concentration increased from 32 to 81 μmol per liter of sediment while the acetate transformation rate constant decreased from 3.22 to 0.70 per h, respectively, during stratification versus turnover. Acetate accounted for nearly 100% of total sedimentary methanogenesis during turnover versus 70% during stratification. The fraction of ¹⁴CO₂ produced from all ¹⁴C-labeled substrates examined was 10 to 40% higher during fall turnover than during stratification. The addition of sulfate, thiosulfate, or sulfur to stratified sediments mimicked fall turnover in that more CO₂ and CH₄ were produced. The addition of Desulfovibrio vulgaris to sulfate-amended sediments greatly enhanced the amount of CO₂ produced from either [¹⁴C]methanol or [2-¹⁴C]acetate, suggesting that H₂ consumption by sulfate reducers can alter methanol or acetate transformation by sedimentary methanogens. These data imply that turnover dynamically altered carbon transformation in eutrophic sediments such that sulfate reduction dominated over methanogenesis principally as a consequence of altering hydrogen metabolism.     

Methane production in littoral sediment of Lake Constance

Maximum rates of CH₄ production in the littoral sediment were observed in 2–5 cm depth. The CH₄ production rates increased during the year from about 5 mmol m⁻²d⁻¹ in December to a maximum of about 95 mmol m⁻²d⁻¹ in September. CH₄ production rates showed a temperature optimum at 30°C and an apparent activation energy of 76 kJ mol⁻¹. A large part of the seasonality of CH₄ production could be ascribed to the change of the sediment temperature. Most of the produced CH₄ was lost by ebullition. Gas bubbles contained about 60–70% CH₄ with an average δ¹³C of −56.2% and δD of −354%, and 2% CO₂ with an average δ¹³C of −14.1% indicating that CH₄ was produced from methyl carbon, i.e. mainly using acetate as methanogenic substrate. This result was confirmed by inhibition of methanogenesis with chloroform which resulted in an accumulation rate of acetate equivalent to 81% of the rate of CH₄ production. Most probable numbers of methanogenic bacteria were in the order of 10⁴ bacteria g⁻¹d.w. sediment for acetate-, methanol- or formate-utilizing, and of 10⁵ for H₂-utilizing methanogens. The turnover times of acetate were in the order of 2.3–4.8 h which, with in situ acetate concentrations of about 25–50 μM, resulted in rates of acetate turnover which were comparable to the rates of CH₄ production. The respiratory index (RI) showed that [2−¹⁴C]acetate was mainly used by methanogenesis rather than by respiratory processes, although the zone of CH₄ production in the sediment overlapped with the zone of sulfate reduction.     

Effects of Sulfuroxy Anions on Degradation of Pentachlorophenol by a Methanogenic Enrichment Culture

We studied the degradation of pentachlorophenol (PCP) under methanogenic and sulfate-reducing conditions with an anaerobic mixed culture derived from sewage sludge. The consortium degraded PCP via 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol and eventually accumulated 3-chlorophenol. Dechlorination of PCP and metabolites was inhibited in the presence of sulfate, thiosulfate, and sulfite. A decrease in the rate of PCP transformation was noted when the endogenous dissolved H₂ was depleted below 0.11 μM in sulfate-reducing cultures. The effect on dechlorination observed with sulfate could be relieved by addition of molybdate, a competitive inhibitor of sulfate reduction. Addition of H₂ reduced the inhibition observed with sulfuroxy anions. The inhibitory effect of sulfuroxy anions may be due to a competition for H₂ between sulfate reduction and dechlorination. When cultured under methanogenic conditions, the consortium degraded several chlorinated and brominated phenols.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Short-term effect of acetate and ethanol on methane formation in biogas sludge

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N₂/CO₂. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H₂+CO₂ as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H₂ did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.