Simultaneous hydrogen utilization and in situ biogas upgrading in an anaerobic reactor

The possibility of converting hydrogen to methane and simultaneous upgrading of biogas was investigated in both batch tests and fully mixed biogas reactor, simultaneously fed with manure and hydrogen. Batch experiments showed that hydrogen could be converted to methane by hydrogenotrophic methanogenesis with conversion of more than 90% of the consumed hydrogen to methane. The hydrogen consumption rates were affected by both (hydrogen partial pressure) and mixing intensity. Inhibition of propionate and butyrate degradation by hydrogen (1 atm) was only observed under high mixing intensity (shaking speed 300 rpm). Continuous addition of hydrogen (flow rate of 28.6 mL/(L/h)) to an anaerobic reactor fed with manure, showed that more than 80% of the hydrogen was utilized. The propionate and butyrate level in the reactor was not significantly affected by the hydrogen addition. The methane production rate of the reactor with H₂ addition was 22% higher, compared to the control reactor only fed with manure. The CO₂ content in the produced biogas was only 15%, while it was 38% in the control reactor. However, the addition of hydrogen resulted in increase of pH (from 8.0 to 8.3) due to the consumption of bicarbonate, which subsequently caused slight inhibition of methanogenesis. Biotechnol. Bioeng. 2012; 109:1088–1094. © 2011 Wiley Periodicals, Inc.     

Integrated biogas upgrading and hydrogen utilization in an anaerobic reactor containing enriched hydrogenotrophic methanogenic culture

Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO₂, biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic‐ and thermophilic anaerobic cultures were enriched to convert CO₂ to CH₄ by addition of H₂. Enrichment at thermophilic temperature (55°C) resulted in CO₂ and H₂ bioconversion rate of 320 mL CH₄/(gVSS h), which was more than 60% higher than that under mesophilic temperature (37°C). Different dominant species were found at mesophilic‐ and thermophilic‐enriched cultures, as revealed by PCR–DGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH₄ content, around 95% at steady‐state, at gas (mixture of biogas and hydrogen) injection rate of 6 L/(L day). The increase of gas injection rate to 12 L/(L day) resulted in the decrease of CH₄ content to around 90%. Further study showed that by decreasing the gas–liquid mass transfer by increasing the stirring speed of the mixture the CH₄ content was increased to around 95%. Finally, the CH₄ content around 90% was achieved in this study with the gas injection rate as high as 24 L/(L day). Biotechnol. Bioeng. 2012; 109: 2729–2736. © 2012 Wiley Periodicals, Inc.     

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.     

Hollow fiber membrane based H2 diffusion for efficient in situ biogas upgrading in an anaerobic reactor

Bubbleless gas transfer through a hollow fiber membrane (HFM) module was used to supply H₂ to an anaerobic reactor for in situ biogas upgrading, and it creates a novel system that could achieve a CH₄ content higher than 90 % in the biogas. The increase of CH₄ content and pH, and the decrease of bicarbonate concentration were related with the increase of the H₂ flow rate. The CH₄ content increased from 78.4 % to 90.2 % with the increase of the H₂ flow rate from 930 to 1,440 ml/(l?day), while the pH in the reactor remained below 8.0. An even higher CH₄ content (96.1 %) was achieved when the H₂ flow rate was increased to 1,760 ml/(l?day); however, the pH increased to around 8.3 due to bicarbonate consumption which hampered the anaerobic process. The biofilm formed on the HFM was found not to be beneficial for the process since it increased the resistance of H₂ diffusion to the liquid. The study also demonstrated that the biofilm formed on the membrane only contributed 22–36 % to the H₂ consumption, while most of the H₂ was consumed by the microorganisms in the liquid phase.     

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.     

Feeding approaches for biogas production from animal wastes and industrial effluents

Different feeding approaches were applied to a 5 l anaerobic digester in order to improve the biogas production. During operation, the reactor was fed with a mixture (9.7% w/v total solids (TS) and 7.6% w/v volatile solids (VS) in average) of pig manure with fish oil waste and waste from bentonite of edible oil filtration process, at different intervals of 24, 12 and 4 h at 15 days of hydraulic retention time. Production and quality of the biogas were practically constant at 183.7 ml (average) of biogas per gram of volatile solids available in the reactor per day, and the best biogas composition was 73.6% v/v CH₄ and 26.4% v/v CO₂.     

Assessing the potential for up-cycling recovered resources from anaerobic digestion through microbial protein production

Anaerobic digesters produce biogas, a mixture of predominantly CH₄ and CO₂, which is typically incinerated to recover electrical and/or thermal energy. In a context of circular economy, the CH₄ and CO₂ could be used as chemical feedstock in combination with ammonium from the digestate. Their combination into protein-rich bacterial, used as animal feed additive, could contribute to the ever growing global demand for nutritive protein sources and improve the overall nitrogen efficiency of the current agro- feed/food chain. In this concept, renewable CH₄ and H₂ can serve as carbon-neutral energy sources for the production of protein-rich cellular biomass, while assimilating and upgrading recovered ammonia from the digestate. This study evaluated the potential of producing sustainable high-quality protein additives in a decentralized way through coupling anaerobic digestion and microbial protein production using methanotrophic and hydrogenotrophic bacteria in an on-farm bioreactor. We show that a practical case digester handling liquid piggery manure, of which the energy content is supplemented for 30% with co-substrates, provides sufficient biogas to allow the subsequent microbial protein as feed production for about 37% of the number of pigs from which the manure was derived. Overall, producing microbial protein on the farm from available methane and ammonia liberated by anaerobic digesters treating manure appears economically and technically feasible within the current range of market prices existing for high-quality protein. The case of producing biomethane for grid injection and upgrading the CO₂ with electrolytic hydrogen to microbial protein by means of hydrogen-oxidizing bacteria was also examined but found less attractive at the current production prices of renewable hydrogen. Our calculations show that this route is only of commercial interest if the protein value equals the value of high-value protein additives like fishmeal and if the avoided costs for nutrient removal from the digestate are taken into consideration.     

Biogas upgrading using single-walled carbon nanotubes by molecular simulation

Abstract

Biogas from anaerobic digestion of biological wastes is a renewable energy resource that mainly contains CH₄, CO₂, trace amounts of H₂S and a fraction of H₂O vapour. In order to transfer biogas into biomethane to meet the standards for use as vehicle fuel or for injection in the natural gas grid, removing H₂S from biogas in advance is necessary. In addition, biogas is usually saturated with water vapour. It is significant to study the effect of the presence of H₂O on the biogas separation performance. Adsorption of H₂S/CO₂/CH₄ and H₂O/CO₂/CH₄ ternary mixtures using single-walled carbon nanotubes (SWCNT) were investigated via the Grand Canonical Monte Carlo (GCMC) method. We studied the effects of carbon nanotube diameter, –COOH modification, temperature and pressure on H₂S adsorption. The results indicate that the presence of hydrophilic –COOH groups does affect the separation of H₂S/CO₂/CH₄ mixtures. Temperature swing adsorption is more suitable than pressure swing adsorption for the separation of H₂S/CO₂/CH₄ mixtures. The effect of water vapour on the separation of CO₂/CH₄ was also investigated. The result shows that the presence of H₂O has little effect on the selectivity of CO₂/CH₄ in pristine CNT, but the selectivity of CO₂/CH₄ with the presence of H₂O is markedly enhanced after modification in –COOH modified SWCNT with specific modification degree. It is expected that this work could provide some useful information for biogas upgrading.     

Research on soybean protein wastewater treatment by the integrated two-phase anaerobic reactor

The start-up tests of treating soybean protein wastewater by the integrated two-phase anaerobic reactor were studied. The results showed that the soybean protein wastewater could be successfully processed around 30 days when running under the situation of dosing seed sludge with the influent of approximately 2000 mg/L and an HRT of 40 h. When the start-up was finished, the removal rate of COD by the reactor was about 80%. In the zone I, biogas mainly revealed carbon dioxide (CO₂) and hydrogen (H₂). Methane was the main component in the zone 2 which ranged from 53% to 59% with an average of 55%. The methane content in biogas increased from the zone I to II. It indicated that the methane-producing capacity of the anaerobic sludge increased. It was found that the uniquely designed two-phase integrated anaerobic reactor played a key role in treating soybean protein wastewater. The acidogenic fermentation bacteria dominated in the zone I, while methanogen became dominant in the zone II. It realized the relatively effective separation of hydrolysis acidification and methanogenesis process in the reactor, which was benefit to promote a more reasonable space distribution of the microbial communities in the reactor. There were some differences between the activities of the sludge in the two reaction zones of the integrated two-phase anaerobic reactor. The activity of protease was higher in the reaction zone I. And the coenzyme F₄₂₀ in the reaction zone II was twice than that in the reaction zone I, which indicated that the activity of the methanogens was stronger in the reaction zone II.     

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.     

Evaluation of Slow Release Substrates for Anaerobic Bioremediation

A variety of food-grade organic substrates were evaluated to identify materials that could be used to support long-term anaerobic bioremediation processes in the subsurface. In this work, the rate and extent of biogas production was used as an indicator of the potential for substrate fermentation to H₂ and acetate, the primary electron donors used in reductive dechlorination. The rate and extent of biogas (primarily CO₂+ CH₄) evolution varied widely between the different substrates. For many of the substrates, biogas generation declined to very low levels within 100 days of substrate addition. However, a few substrates including several vegetable oils and sucrose esters of fatty acid (SEFAs) did support biogas production for extended time periods. Column studies demonstrated that both soybean oil and a SEFA could support sulfate reduction, methanogenesis and reductive dechlorination of perchloroethene (PCE) to cis-dichloroethene (cis-DCE) for over 14 months. The slower degradation rate of the SEFAs could be used to control substrate degradation rate in the subsurface, increasing substrate lifetime and reducing the required reinjection frequency.     

Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation

The optimization of a two-phase thermophilic anaerobic process treating biowaste for hydrogen and methane production was carried out at pilot scale using two stirred reactors (CSTRs) and without any physical/chemical pre-treatment of inoculum. During the experiment the hydrogen production at low hydraulic retention time (3d) was tested, both with and without reject water recirculation and at two organic loading rate (16 and 21 kgTVS/m³d). The better yields were obtained with recirculation where the pH reached an optimal value (5.5) thanks to the buffering capacity of the recycle stream. The specific gas production of the first reactor was 51 l/kgVS_fed and H₂ content in biogas 37%. The mixture of gas obtained from the two reactors met the standards for the biohythane mix only when lower loading rate were applied to the first reactor, with a composition of 6.7% H₂, 40.1% CO₂ and 52.3% CH₄ the overall SGP being 0.78 m³/kgVS_fed.     

Mass spectrometric control of the thermophilic anaerobic digestion process based on levels of dissolved hydrogen

Summary The continuous and simultaneous monitoring of dissolved CH₄ and H₂ in samples from a laboratory scale thermophilic anaerobic digester contents by use of a silicone rubber-covered probe has enabled control of methanogenesis: regulation of the hydrogen signal in a closed feedback loop was by controlled addition of the carbon source. Dissolved hydrogen became apparent in this system at a lower loading rate than was obtained for a mesophilic anaerobic digestion system (Whitmoreet al., 1986). Controlling the supply of glucose (25 mM) at a dilution rate of 0.02 h^–1 and at progressively lower preset hydrogen levels allowed methanogenesis to be significantly prolonged before inhibition of the process occurred.     

Microbial Methanogenesis and Acetate Metabolism in a Meromictic Lake

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH₄ and CO₂ in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Temperature sensitivity of greenhouse gas production in wetland soils of different vegetation

Organic matter decomposition regulates rates of carbon loss (CO₂ and CH₄) in wetlands and has implications for carbon sequestration in the context of changing global temperature. Here we determined the influence of temperature and vegetation type on both aerobic and anaerobic decomposition of organic matter in subtropical wetland soils. As in many other studies, increased temperature resulted in higher rates of respiration and methanogenesis under both aerobic and anaerobic conditions, and the positive effect of temperature depended on vegetation (source of carbon substrate to soil). Under anaerobic incubations, the proportion of gaseous C (CO₂ and CH₄) lost as CH₄ increased with temperature indicating a greater sensitivity of methanogenesis to temperature. This was further supported by a wider range of Q₁₀ values (1.4–3.6) for methane production as compared with anaerobic CO₂ (1.3–2.5) or aerobic CO₂ (1.4–2.1) production. The increasing strength of positive linear correlation between CO₂:CH₄ ratio and the soil organic matter ligno-cellulose index at higher temperature indicated that the temperature sensitivity of methanogenesis was likely the result of increased C availability at higher temperature. This information adds to our basic understanding of decomposition in warmer subtropical and tropical wetland systems and has implications for C models in wetlands with different vegetation types.     

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.     

Methanogenesis in McLean Bog,an Acidic Peat Bog in Upstate New York: Stimulation by H2/CO2 in the Presence of Rifampicin,or by Low Concentrations of Acetate

Acidic peat bog soils produce CH₄ and although molecular biological studies have demonstrated the presence of diverse methano-genic populations in them, few studies have sustained methanogenesis by adding the CH₄ precursors H₂/CO₂ or acetate, and few indigenous methanogens have been cultured. McLean Bog is a small (ca. 70 m across), acidic (pH 3.4–4.3) Sphagnum -dominated bog in upstate New York. Although addition of H₂/CO₂ or 10 mM acetate stimulated methanogenesis in soils from a nearby circumneutral-pH fen, neither of these substrates led to sustained methanogenesis in McLean Bog soil slurries. After a brief period of stimulation by H₂/CO₂, methanogenesis in McLean Bog soil declined, which could be attributed to buildup of large amounts of acetic acid produced from the H₂/CO₂ by acetogens. Addition of the antibiotic rifampicin inhibited acetogenesis (carried out by Bacteria) and allowed methanogenesis (carried out by Archaea) to continue. Using rifampicin, we were able to study effects of temperature, pH, and salts on methanogenesis from H₂/CO₂ in McLean Bog soil samples. The enriched H₂/CO₂-utilizing methanogens showed an optimum for activity near pH 5, and a temperature optimum near 35°C. Methanogenesis was not stimulated by addition of 10 mM acetate, but it was stimulated by 1 mM acetate, and multiple additions were consumed at increasing rates and nearly stoichiometrically converted to CH₄. In conclusion, we have found that both hydrogentrophic and aceticlastic methanogens are present in McLean Bog soils, and that methanogenic activity can be stimulated using H₂/CO₂ in the presence of rifampicin, or using low concentrations of acetate.     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review

Among different conversion processes for biomass, biological anaerobic digestion is one of the most economic ways to produce biogas from various biomass substrates. In addition to hydrolysis of polymeric substances, the activity and performance of the methanogenic bacteria is of paramount importance during methanogenesis. The aim of this paper is primarily to review the recent literature about the occurrence of both acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of particulate biomass to methane (not wastewater treatment), while this review does not cover the activity of the acetate oxidizing bacteria. Both acetotrophic and hydrogenotrophic methanogens are essential for the last step of methanogenesis, but the reports about their roles during this phase of the process are very limited. Despite, some conclusions can still be drawn. At low concentrations of acetate, normally filamentous Methanosaeta species dominate, e.g., often observed in sewage sludge. Apparently, high concentrations of toxic ionic agents, like ammonia, hydrogen sulfide (H₂S) and volatile fatty acids (VFA), inhibit preferably Methanosaetaceae and especially allow the growth of Methanosarcina species consisting of irregular cell clumps, e.g., in cattle manure. Thermophilic conditions can favour rod like or coccoid hydrogenotrophic methanogens. Thermophilic Methanosarcina species were also observed, but not thermophilic Methanosaetae. Other environmental factors could favour hydrogentrophic bacteria, e.g., short or low retention times in a biomass reactor. However, no general rules regarding process parameters could be derivated at the moment, which favours hydrogenotrophic methanogens. Presumably, it depends only on the hydrogen concentration, which is generally not mentioned in the literature.     

Monochloro- and dichloroacetic acids as carbon and energy sources for a stable,methanogenic mixed culture

A stable methanogenic mixed culture was enriched from an industrial environment to utilize chloroacetate as sole carbon and energy source for growth. It immobilized spontaneously on activated charcoal and grew reproducibly on this carrier in a fluidized bed reactor when supplied with an anaerobic mineral salts medium. Substrate disappearance was complete. Methane, CO₂ and chloride ions were conclusively identified as the metabolic products and quantified. The growth yield from chloroacetate was about 1 g of protein/mol of carbon. The calculated degradation rate in the fluidized bed reactor was 0.2 to 0.8 mmol/l·h. The first metabolic intermediate from [2–¹³C]monochloroacetate in portions of biofilm-coated carrier was shown by ¹³C-NMR to be glycolate, from which ¹³CO₂ and ¹³CH₄ were formed. Glycolate was formed in an oxygen-insensitive hydrolysis, but its conversion to CO₂ and CH₄ was strictly anaerobic and sensitive to inhibition by bromoethanesulfonate. Degradation of [1-¹⁴C]-and [2-¹⁴C]-chloroacetate each yielded the same amount of [¹⁴C]-methane. We thus presume glycolate to be cleaved to CO₂ and H₂, which were the substrates for methanogenesis. Dehalogenation was limited to chlorobromo-, iodo- and dichloroacetate. These four compounds and glycolate were utilized as the sole carbon and energy sources by the methanogenic mixed culture.