Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium

Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified β-oxidation to H₂/CO₂ and acetate. These intermediates are converted to CH₄/CO₂ by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO₂/H₂ and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H₂-producing syntroph–methanogen partnership that may serve to improve community stability.     

Methanogenesis from Sucrose by Defined Immobilized Consortia

A bacterial consortium capable of sucrose degradation primarily to CH₄ and CO₂ was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H₂, and CO₂ (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH₄. Significant decreases in yields of CH₄ and rates of CH₄ production were observed if any component of the consortium was omitted. Approximately 70% of the CH₄ generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH₄ and rates of CH₄ production from sucrose similar to those of nonimmobilized cells. The rate of CH₄ production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4°C.     

Variation of carbon isotope fractionation in hydrogenotrophic methanogenic microbial cultures and environmental samples at different energy status

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H₂ plus CO₂ (), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C‐isotope fractionation was correlated to the Gibbs free energy change (ΔG) of CH₄ formation from H₂ plus CO₂ and that the relation can be described by a semi‐Gauss curve. The derived relationship was used to quantify the average ΔG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H₂ concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH₄, exhibited increasingly smaller , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the ¹³C flux from microbial sources of atmospheric CH₄.     

Response of Methanogens in Arctic Sediments to Temperature and Methanogenic Substrate Availability

Although cold environments are major contributors to global biogeochemical cycles, comparatively little is known about their microbial community function, structure, and limits of activity. In this study a microcosm based approach was used to investigate the effects of temperature, and methanogenic substrate amendment, (acetate, methanol and H₂/CO₂) on methanogen activity and methanogen community structure in high Arctic wetlands (Solvatnet and Stuphallet, Svalbard). Methane production was not detected in Stuphallet sediment microcosms (over a 150 day period) and occurred within Solvatnet sediments microcosms (within 24 hours) at temperatures from 5 to 40°C, the maximum temperature being at far higher than in situ maximum temperatures (which range from air temperatures of -1.4 to 14.1°C during summer months). Distinct responses were observed in the Solvatnet methanogen community under different short term incubation conditions. Specifically, different communities were selected at higher and lower temperatures. At lower temperatures (5°C) addition of exogenous substrates (acetate, methanol or H₂/CO₂) had no stimulatory effect on the rate of methanogenesis or on methanogen community structure. The community in these incubations was dominated by members of the Methanoregulaceae/WCHA2-08 family-level group, which were most similar to the psychrotolerant hydrogenotrophic methanogen Methanosphaerula palustris strain E1-9c. In contrast, at higher temperatures, substrate amendment enhanced methane production in H₂/CO₂ amended microcosms, and played a clear role in structuring methanogen communities. Specifically, at 30°C members of the Methanoregulaceae/WCHA2-08 predominated following incubation with H₂/CO₂, and Methanosarcinaceaeand Methanosaetaceae were enriched in response to acetate addition. These results may indicate that in transiently cold environments, methanogen communities can rapidly respond to moderate short term increases in temperature, but not necessarily to the seasonal release of previously frozen organic carbon from thawing permafrost soils. However, as temperatures increase such inputs of carbon will likely have a greater influence on methane production and methanogen community structure. Understanding the action and limitations of anaerobic microorganisms within cold environments may provide information which can be used in defining region-specific differences in the microbial processes; which ultimately control methane flux to the atmosphere.     

Effect of pre-aeration and inoculum on the start-up of batch thermophilic anaerobic digestion of municipal solid waste

In this study, a short pre-aeration step was investigated as pre-treatment for thermophilic anaerobic digestion of the organic fraction of municipal solid waste (OFMSW). It was found that pre-aeration of 48 h generated enough biological heat to increase the temperature of bulk OFMSW to 60 °C. This was sufficient self-heating of the bulk OFMSW for the start-up of thermophilic anaerobic digestion without the need for an external heat source. Pre-aeration also reduced excess easily degradable organic compounds in OFMSW, which were the common cause of acidification during the start-up of the batch system. Careful consideration however must be taken to avoid over aeration as this consumes substrate, which would otherwise be available to methanogens to produce biogas. To accelerate methane production and volatile solids destruction, the anaerobic digestion in this study was operated as a wet process with the anaerobic liquid recycled through the OFMSW. Appropriate anaerobic liquid inoculum was found to be particularly beneficial. It provided high buffer capacity as well as suitable microbial inoculum. As a result, acidification during start-up was kept to a minimum. With volatile fatty acids (VFAs-acetate in particular) and H₂ accumulation typical of hydrolysis and fermentation of the easily degradable substrates during start-up, inoculum with high numbers of hydrogenotrophic methanogens was critical to not only maximise CH₄ production but also reduce H₂ partial pressure in the system to allow VFAs degradation. In a lab-scale bioreactor, the combined pre-aeration and wet thermophilic anaerobic digestion was able to stabilise the OFMSW within a period of only 12 days. The stabilised inert residual material can be used as a soil amendment product.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor

The effect of different electron acceptors on substrate degradation was studied in pure and mixed cultures of various hydrogenotrophic homoacetogenic, methanogenic, sulfate-reducing, fumarate-reducing and nitrate-ammonifying bacteria. Two different species of these bacteria which during organic substrate degradation produce and consume hydrogen, were cocultured on a substrate which was utilized only by one of them. Hydrogen, which was excreted as intermediate by the first strain (and reoxidized in pure culture), could, depending on the hydrogen acceptor present, also be used by the second organism, resulting in interspecies hydrogen transfer. The efficiency of H₂ transfer was similar when methanol, lactate or fructose were used as organic substrate, although the free energy changes of fermentative H₂ formation of these substrates are considerably different. In coculture experiments nitrate or fumarate>sulfate> CO₂/CH₄>sulfur or CO₂/acetate were the preferred electron acceptors, and an increasing percentage of H₂ was transferred to that bacterium which was able to utilize the preferred electron acceptor. In pure culture the threshold values for hydrogen oxidation decreased in the same order from 1,100 ppm for homoacetogenic bacteria to about 0.03 ppm for nitrate or fumarate reducing bacteria. The determined H₂-threshold values as well as the percentage of H₂ transfer in cocultures were related to the Gibbs free energy change of the respective hydrogen oxidizing reaction.Parts of this work (grant to R C-R) was supported by the European Community (ST2A-0022)     

Functional-Structural Analysis of Nitrogen-Cycle Bacteria in a Hypersaline Mat from the Omani Desert

Anaerobic microbial activity in northern peat soils most often results in more carbon dioxide (CO ₂ ) production than methane (CH₄) production. This study examined why methanogenic conditions (i.e., equal molar amounts of CH₄ production and CO₂ production) prevail so infrequently. We used peat soils from two ombrotrophic bogs and from two rheotrophic fens. The former two represented a relatively dry bog hummock and a wet bog hollow, and the latter two represented a forested fen and a sedge-dominated fen. We quantified gas production rates in soil samples incubated in vitro with and without added metabolic substrates (glucose, ethanol, H₂/CO₂). None of the peat soils exhibited methanogenic conditions when incubated in vitro for a short time (< 5 days) and without added substrates. Incubating some samples > 50 days without added substrates led to methanogenic conditions in only one of four experiments. The anaerobic CO₂:CH₄ production ratio ranged from 5:1 to 40:1 in peat soil without additions and was larger in samples from the dry bog hummock and forested fen than the wet bog hollow and sedge fen. Adding ethanol or glucose separately to peat soils led to methanogenic conditions within 5 days after the addition by stimulating rates of CH₄ production, suggesting CH₄ production from both hydrogenotrophic and acetoclastic methanogenesis. Our results suggest that methanogenic conditions in peat soils rely on a constant supply of easily decomposable metabolic substrates. Sample handling and incubation procedures might obscure methanogenic conditions in peat soil incubated in vitro.     

Direct conversion of cellulose to methane by anaerobic fungus Neocallimastix frontalis and defined methanogens

Co-cultures of N. frontalis with a formate-utilizing methanogen, Methanobacterium formicicum and/or an aceticlastic methanogen, Methanosaeta concilii, were performed for methane production from cellulose. In the co-culture with M. formicicum, ca. 16 mM CH₄ was produced after 7 days without accumulation of H₂ and formate. In the co-culture with M. concilii, 12 mM CH₄ was produced after 17 days with decreasing acetate production. In the tri-culture of N. frontalis with M. formicicum and M. concilii, 24 mM CH₄ was produced after 17 days where acetate still remained at 23 mM, but production of lactate and ethanol decreased. When a 4-times concentrated culture broth of M. concilii was inoculated in this tri-culture system in a bioreactor, 150 mM CH₄ was produced after 24 days by feeding of cellulose, although 57 mM acetate still accumulated.     

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Hydrogen and methane production from desugared molasses by a two‐stage thermophilic anaerobic process was investigated in a series of two up‐flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen‐producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H₂/day/L, corresponding to a yield of 132 mL H₂/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH₄/day/L, corresponding to a yield of 239 mL CH₄/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H_2, 38.7% CO₂, and 44.8% CH₄, containing approximately 15% energy by hydrogen is viable to be bio‐hythane.     

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

Salinity Constraints on Subsurface Archaeal Diversity and Methanogenesis in Sedimentary Rock Rich in Organic Matter

The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl⁻ in a subsurface shale rich in CH₄ derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH₄-producing zone lacked electron acceptors such as O₂, NO₃⁻, Fe³⁺, or SO₄²⁻. Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl⁻ concentrations above ~1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H₂/CO₂) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic Methanohalophilus species and a hydrogenotrophic Methanoplanus species at high salinity and a single phylotype closely related to Methanocorpusculum bavaricum at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH₄-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.     

Isolation and Characterization of Acetate-Utilizing Anaerobes from a Freshwater Sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5–1 μm × 3–4 μm long). Doubling times with acetate at 30–35°C were 5.6–8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related toMethanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5–0.7 μm × 1.5–3.5 μm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30°C the doubling times with acetate were 3.9–5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H₂, CO₂, and sulfate. The closest relative of strain ASRB-Zg isDesulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5–0.7 μm × 0.7–1 μm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20–25°C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H₂, CO₂, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg isVariovorax paradoxus.     

Evaluation of Three Electron-Donor Permeable Reactive Barrier Materials for Enhanced Reductive Dechlorination of Trichloroethene

Understanding the fate of complex electron-donor materials is important for developing efficient biostimulation strategies to treat ground water contamination by chlorinated ethenes (CEs). The fermentation product distributions and H₂ production of common permeable reactive barrier (PRB) carbon substrates (dairy whey, sodium lactate syrup, and Hydrogen Release Compound [HRC]) were monitored as measures of substrate efficiency in aquifer microcosms spiked with trichloroethene (TCE). In long-term experiments, the fermentation of PRB substrates to slow-degrading organic acids maintained low H₂ partial pressures (≤ 10^?3.5) that, as previous studies suggest, may give competitive advantage to dechlorinators over hydrogenotrophic methanogens. Whey-amended and lactate-amended microcosms exhibited faster complete dechlorination and, according to organic acid carbon flow, higher rates of fermentation to acetate. In HRC-amended microcosms, propionate appeared to serve as a carbon sink that prolonged dechlorination. Upon complete dechlorination, whey microcosms contained the highest percentage of organic acid carbon. Native Dehalococcoides populations increased by 3 orders of magnitude (per g sediment) in whey-amended microcosms. Whey's efficiency improved in microcosms prepared with aquifer sediment and water from within a downgradient whey PRB. Results suggested whey loading values of 0.2 kg/m³ may be appropriate under sufficiently reducing conditions to efficiently stimulate hydrogenotrophic and potentially actetotrophic dechlorinating populations. Renewal of whey PRBs may, however, be required. Implications for further long-term study of cost-efficiencies are discussed.     

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C₃–C₆), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Bioconversion of organic carbon to CH4 and CO2

The activities of populations in complex anaerobic microbial communities that perform complete bioconversion of organic matter to CH₄ and CO₂ are reviewed. Species of eubacteria produce acetate, H₂, and CO₂ from organic substrates, and methanogenic species of archaebacteria transform the acetate, H₂, and CO₂ to CH₄. The characteristics and activities of the methanogenic bacteria are described. The impact of the use of H₂ by methanogens on the fermentations that produce acetate, H₂, and CO₂ and the importance of syntrophy in complete bioconversion are discussed.     

Evidence for iron‐mediated anaerobic methane oxidation in a crude oil‐contaminated aquifer

In a methanogenic crude oil contaminated aquifer near Bemidji, Minnesota, the decrease in dissolved CH₄ concentrations along the groundwater flow path, along with the positive shift in δ¹³C_CH4 and negative shift in δ¹³C_DIC, is indicative of microbially mediated CH₄ oxidation. Calculations of electron acceptor transport across the water table, through diffusion, recharge, and the entrapment and release of gas bubbles, suggest that these processes can account for at most 15% of the observed total reduced carbon oxidation, including CH₄. In the anaerobic plume, the characteristic Fe(III)‐reducing genus Geobacter was the most abundant of the microbial groups tested, and depletion of labile sediment iron is observed over time, confirming that reduced carbon oxidation coupled to iron reduction is an important process. Electron mass balance calculations suggest that organic carbon sources in the aquifer, BTEX and non‐volatile dissolved organic carbon, are insufficient to account for the loss in sediment Fe(III), implying that CH₄ oxidation may also be related to Fe(III) reduction. The results support a hypothesis of Fe(III)‐mediated CH₄ oxidation in the contaminated aquifer.     

Determination of isotope fractionation factors and quantification of carbon flow by stable carbon isotope signatures in a methanogenic rice root model system

Methanogenic processes can be quantified by stable carbon isotopes, if necessary modeling parameters, especially fractionation factors, are known. Anoxically incubated rice roots are a model system with a dynamic microbial community and thus suitable to investigate principal geochemical processes in anoxic natural systems. Here we applied an inhibitor of acetoclastic methanogenesis (methyl fluoride), calculated the thermodynamics of the involved processes, and analyzed the carbon stable isotope signatures of CO₂, CH₄, propionate, acetate and the methyl carbon of acetate to characterize the carbon flow during anaerobic degradation of rice roots to the final products CO₂ and CH₄. Methyl fluoride inhibited acetoclastic methanogenesis and thus allowed to quantify the fractionation factor of CH₄ production from H₂/CO₂. Since our model system was not affected by H₂ gradients, the fractionation factor could alternatively be determined from the Gibbs free energies of hydrogenotrophic methanogenesis. The fractionation factor of acetoclastic methanogenesis was also experimentally determined. The data were used for successfully modeling the carbon flow. The model results were in agreement with the measured process data, but were sensitive to even small changes in the fractionation factor of hydrogenotrophic methanogenesis. Our study demonstrates that stable carbon isotope signatures are a proper tool to quantify carbon flow, if fractionation factors are determined precisely.