Myristic acid supports the immediate inhibitory effect of lauric acid on ruminal methanogens and methane release

Two in vitro experiments were carried out with the Hohenheim gas test (HGT) apparatus in order to investigate dose-dependent effects and interactions of non-esterified lauric acid (C(12)) and myristic acid (C(14)) given either individually or in mixture on ruminal methanogens and methanogenesis. Special emphasis was also put on the relationship between effects on methane formation and methanogenic counts. The in vitro incubations were conducted in 10mL ruminal fluid and 20mL buffer solution and lasted for 24h. In the first experiment, 14 levels of C(12), C(14) and stearic acid (C(18); control) were supplied each in increasing steps of 2.5mg covering the range from 0 to 32.5mg. In the second experiment, dosages ranging from 2.5 to 30mg C(12) were supplemented in steps of 2.5mg either without or with 10, 20 or 30mg of C(14). Counts of total Archaea and individual methanogenic orders were determined by the fluorescence in situ hybridization technique using 16S rRNA oligonucleotide probes. In experiment 1, a methane-suppressing effect of more than 80% was achieved with a supply of 30mg C(12), whereas C(14) and C(18) had no effect. Incubation liquid counts of total Archaea and individual methanogenic orders (Methanococcales, Methanosarcinales, Methanomicrobiales and Methanobacteriales) exponentially decreased as a response to C(12) and C(14) to about the same degree (up to 90%) and, to a lesser extent, by C(18). The proportions of the orders of total methanogenic population were not altered by any of the fatty acids. In experiment 2, an additional supply of 10 or 20mg of C(14) supported the suppression of methanogenesis and methanogens by C(12) synergistically. Supplementing 30mg instead of 20mg of C(14) did not further increase the efficacy of C(12) in suppressing methane formation and methanogens. The study illustrated the advantage of using mixtures of C(12) and C(14) in ruminant nutrition to suppress methane emission since mixtures will reduce the amounts of the less palatable C(12) required in feed.     

Ruminal methanogenesis as influenced by individual fatty acids supplemented to complete ruminant diets

The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g x kg(-1) dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane-suppressing effect of medium-chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.     

The effect of vinyl acetate in acetoclastic methanogenesis

The influence of vinyl acetate (VA) in the methanogenesis was evaluated, by using an upflow anaerobic sludge blanket reactor of 1.5L. The reactor was operated at 33.5 g/L volatile suspended solids to 30±2 °C, a hydraulic residence time of 1 day, an organic loading rate of 1 kgCOD/m3/d of two different mixtures of VA and glucose. The VA was methanized to 81% when its proportion was of 10% into reactor loading rate, when VA proportion increased to 25%, the methane production rate decreased to 62% and the acetate production rate increased almost 8 times. These results indicated that VA was only hydrolyzed and glucose was not used as a co-substrate. The effect of glucose on VA methanogenic degradation was evaluated through batch reactors of 60 mL, concluding that the glucose supported the methanogenesis without favoring the VA elimination. On the other hand, the results of the sludge from the reactor in the presence of VA demonstrated that VA caused an irreversibly inhibition of acetoclastic methanogenesis when the anaerobic sludge was exposed to this compound.     

Pattern of non-methanogenic and methanogenic degradation of cellulose in anoxic rice field soil

Rice field soils turn anoxic upon flooding. The complete mineralization of organic matter, e.g. cellulose, to gaseous products is then accomplished by the sequential reduction of nitrate, ferric iron, sulfate and finally by methanogenesis. Therefore, the anaerobic turnover of [U-(14)C]cellulose was investigated in fresh, non-methanogenic and in preincubated, methanogenic slurries of Italian rice field soil. In anoxic soil slurries freshly prepared from air-dried soil [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in a ratio of 3:1. In methanogenic soil slurries, on the other hand, which had been preincubated for 45 days under anaerobic conditions, [U-(14)C]cellulose was converted to (14)CO(2) and (14)CH(4) in the ratio of 1:1. The turnover times (7-14 days) of cellulose degradation were not significantly different (P0.05) in fresh and methanogenic soil. Chloroform addition abolished CH(4) production, but only slightly (30%) inhibited cellulose degradation in both fresh and methanogenic soil. Under both soil conditions, [(14)C]acetate was the only labeled intermediate detected. A maximum of 24% of the applied radioactivity was transiently accumulated as [(14)C]acetate in both fresh and methanogenic soil slurries. However, when methanogenesis was inhibited by chloroform, 46% and 66% of the applied radioactivity were recovered as [(14)C]acetate in fresh and methanogenic soil, respectively. Only non-radioactive propionate accumulated during the incubation with [U-(14)C]cellulose, especially in the presence of chloroform, indicating that propionate was produced from substrates other than cellulose.     

Fate and transformation of thiocyanate and cyanate under methanogenic conditions

The fate of thiocyanate (SCN⁻) and cyanate (OCN⁻) under methanogenic conditions was investigated at 35 °C. Thiocyanate and cyanate were added to mixed methanogenic cultures along with an organic mixture. Thiocyanate was stable under these conditions, and had no adverse effect on methanogenesis at a concentration as high as 2.5 mM. In contrast, cyanate at a concentration as low as 0.3 mM initially inhibited methanogenesis but, after the complete removal of cyanate, methanogenesis gradually recovered. The inhibitory effect of cyanate on methanogenesis became more profound with repeated additions of cyanate. The transformation of cyanate followed the hydrolytic route to ammonia and bicarbonate under anaerobic conditions and its hydrolysis rate was enhanced by microbial activity. Cyanide was not detected as a cyanate transformation product under the methanogenic conditions of this study. Received: 13 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

Methylmercury Oxidative Degradation Potentials in Contaminated and Pristine Sediments of the Carson River, Nevada

Sediments from mercury-contaminated and uncontaminated reaches of the Carson River, Nevada, were assayed for sulfate reduction, methanogenesis, denitrification, and monomethylmercury (MeHg) degradation. Demethylation of [(sup14)C]MeHg was detected at all sites as indicated by the formation of (sup14)CO(inf2) and (sup14)CH(inf4). Oxidative demethylation was indicated by the formation of (sup14)CO(inf2) and was present at significant levels in all samples. Oxidized/reduced demethylation product ratios (i.e., (sup14)CO(inf2)/(sup14)CH(inf4) ratios) generally ranged from 4.0 in surface layers to as low as 0.5 at depth. Production of (sup14)CO(inf2) was most pronounced at sediment surfaces which were zones of active denitrification and sulfate reduction but was also significant within zones of methanogenesis. In a core taken from an uncontaminated site having a high proportion of oxidized, coarse-grain sediments, sulfate reduction and methanogenic activity levels were very low and (sup14)CO(inf2) accounted for 98% of the product formed from [(sup14)C]MeHg. There was no apparent relationship between the degree of mercury contamination of the sediments and the occurrence of oxidative demethylation. However, sediments from Fort Churchill, the most contaminated site, were most active in terms of demethylation potentials. Inhibition of sulfate reduction with molybdate resulted in significantly depressed oxidized/reduced demethylation product ratios, but overall demethylation rates of inhibited and uninhibited samples were comparable. Addition of sulfate to sediment slurries stimulated production of (sup14)CO(inf2) from [(sup14)C]MeHg, while 2-bromoethanesulfonic acid blocked production of (sup14)CH(inf4). These results reveal the importance of sulfate-reducing and methanogenic bacteria in oxidative demethylation of MeHg in anoxic environments.     

Temperature limitation of methanogenesis in aquatic sediments.

Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.     

Interactions between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion

The degradation of amino acids in anaerobic digestion was examined in terms of the interactions between amino-acid-degrading bacteria and methanogenic bacteria. Certain amino acids were degraded oxidatively by dehydrogenation, with methanogenic bacteria acting as H(2) acceptors. The inhibition of methanogenesis by chloroform also inhibited the degradation of these amino acids and/or caused variations in the composition of volatile acids produced from them. The presence of glycine reduced the inhibitory effect caused by chloroform, probably because glycine acted as an H(2) acceptor in place of methanogenic bacteria. This fact suggested that the coupled oxidation-reduction reactions between two amino acids-one acting as the H(2) donor and the other acting as the H(2) acceptor-may occur in the anaerobic digestion of proteins or amino-acid mixtures. The conversion of some proteins to volatile acids was not affected when methanogenesis was inhibited by chloroform. This suggested that the component amino acids of proteins may be degraded by the coupled oxidation-reduction reactions and that the degradation of proteins may not be dependent on the activity of methanogenic bacteria as H(2) acceptors.     

Inhibitory effects of nitrogen oxides on a mixed methanogenic culture

The effect of nitrate, nitrite, nitric oxide (NO), and nitrous oxide on a mixed, mesophilic (35 degrees C) methanogenic culture was investigated. Short-term inhibition assays were conducted at a concentration range of 10-350 mg N/L nitrate, 17-500 mg N/L nitrite, 0.02-0.8 mg N/L aqueous NO, and 19-191 mg N/L aqueous nitrous oxide. Simultaneous methane production and N-oxide reduction was observed in 10 and 30 mg N/L nitrate and 0.02 mg N/L aqueous NO-amended cultures. However, addition of N-oxide resulted in immediate cessation of methanogenesis in all other cultures. Methanogenesis completely recovered subsequent to the complete reduction of N-oxides to nitrogen gas in all N-oxide-amended cultures, with the exception of the 500 mg N/L nitrite- and 0.8 mg N/L aqueous NO-amended cultures. Partial recovery of methanogenesis was observed in the 500 mg N/L nitrite-amended culture in contrast to complete inhibition of methanogenesis in the 0.8 mg N/L aqueous NO-amended culture. Accumulation of volatile fatty acids was observed in both cultures at the end of the incubation period. Among all N-oxides, NO exerted the most and nitrate exerted the least inhibitory effect on the fermentative/methanogenic consortia. The effect of multiple additions of nitrate (300 mg N/L) on the same methanogenic culture was also investigated. Long-term exposure of the methanogenic culture to nitrate resulted in an increase of N-oxide reduction rates and decrease of methane production rates, which was attributed to changes in the microbial community structure due to nitrate addition.     

A comprehensive model of simultaneous denitrification and methanogenic fermentation processes

The denitrification process was incorporated into the IWA Anaerobic Digestion Model No. 1 (ADM1) in order to account for the effect of denitrification on the methanogenic fermentation process. The model was calibrated and optimized using previously published experimental data and kinetic parameter values obtained with a mixed, mesophilic (35°C) methanogenic culture. Model simulations were used to predict the effect of nitrate reduction on the methanogenic fermentation process in batch, semi‐continuous, and continuous flow reactors experiencing operational changes and/or system disturbances. The extended model clearly revealed the importance of substrate competition between denitrifiers and non‐denitrifiers as well as the impact of N‐oxide inhibition on process interactions between fermentation, methanogenesis, and denitrification. Biotechnol. Bioeng. 2010;105: 98–108. © 2009 Wiley Periodicals, Inc.     

Effect of sulfide on nitrate reduction in mixed methanogenic cultures

The effect of sulfide on nitrate reduction and methanogenesis was investigated in two mixed, mesophilic (35 degrees C) methanogenic cultures: sulfide-free and sulfide-acclimated (67 mg S/L total sulfide). A mixture of dextrin/peptone served as the carbon/electron donor source for the two stock cultures, as well as in all assays reported here. The sulfide-free enriched culture was amended with both nitrate (75-350 mg N/L) and sulfide (10-100 mg S/L). Denitrification was the predominant pathway at all sulfide levels tested and methanogenesis did not recover in any of the sulfide- and nitrate-amended cultures, except in the 10 mg S/L culture. Accumulation of denitrification intermediates such as NO and N(2)O took place, which irreversibly inhibited the methanogens and resulted in the complete cessation of methane production. In contrast, conversion of nitrate to nitrite and then to ammonia via dissimilatory nitrate reduction to ammonia (DNRA) prevented the accumulation of denitrification intermediates and led to the recovery of methanogenesis in the nitrate-amended, sulfide-acclimated, mixed methanogenic culture. The effect of the COD/N value on nitrate reduction was assessed with the sulfide-acclimated, methanogenic culture at COD/N values of 10, 20, and 60. As the COD/N value increased, the fraction of nitrate reduced through DNRA also increased. The results of this study have significant implications relative to the combined anaerobic treatment of carbon-, nitrogen-, and/or sulfur-bearing wastes.     

Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Thermophilic methanogenesis in a hot-spring algal-bacterial mat (71 to 30 degrees C).

Algal-bacterial mats which grow in the effluent channels of alkaline hot springs provided an environment suitable for studying natural thermophilic methane producing bacteria. Methane was rapidly produced in cores taken from the meat and appeared to be an end product of decomposition of the algal-bacterial organic matter. Formaldehyde prevented production of methane. Initial methanogenic rate was lower and methanogenesis became exponential when samples were permitted to cool before laboratory incubation. Methanogenesis occurred and methanogenic bacteria were present over a range of 68 to 30 degrees C, with optimum methanogenesis near 45 degrees C. The temperature distribution of methanogenesis in the mat is discussed relative to published results on standing crop, primary production, and decomposition in the thermal gradient. The depth distribution of methanogenesis was similar to that of freshwater sediments, with a zone of intense methanogenesis near the mat surface. Methanogenesis in deeper mat layers was very low or undetectable despite large numbers of viable methanogenic bacteria and could not be stimulated by addition of anoxic source water, sulfide, or a macronutrient solution.     

Thermophilic methanogenesis in a hot-spring algal-bacterial mat (71 to 30 degrees C).

Algal-bacterial mats which grow in the effluent channels of alkaline hot springs provided an environment suitable for studying natural thermophilic methane producing bacteria. Methane was rapidly produced in cores taken from the meat and appeared to be an end product of decomposition of the algal-bacterial organic matter. Formaldehyde prevented production of methane. Initial methanogenic rate was lower and methanogenesis became exponential when samples were permitted to cool before laboratory incubation. Methanogenesis occurred and methanogenic bacteria were present over a range of 68 to 30 degrees C, with optimum methanogenesis near 45 degrees C. The temperature distribution of methanogenesis in the mat is discussed relative to published results on standing crop, primary production, and decomposition in the thermal gradient. The depth distribution of methanogenesis was similar to that of freshwater sediments, with a zone of intense methanogenesis near the mat surface. Methanogenesis in deeper mat layers was very low or undetectable despite large numbers of viable methanogenic bacteria and could not be stimulated by addition of anoxic source water, sulfide, or a macronutrient solution.     

The bioenergetics of methanogenesis

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1

High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50–300 nmol min^–1 (mg protein)^–1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H₂ in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H₂ on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH₄/ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate. Received: 18 March 1996 / Accepted: 14 June 1996     

Biodegradation of dichloromethane and its utilization as a growth substrate under methanogenic conditions.

Biodegradation of dichloromethane (DCM) to environmentally acceptable products was demonstrated under methanogenic conditions (35 degrees C). When DCM was supplied to enrichment cultures as the sole organic compound at a low enough concentration to avoid inhibition of methanogenesis, the molar ratio of CH4 formed to DCM consumed (0.473) was very close to the amount predicted by stoichiometric conservation of electrons. DCM degradation was also demonstrated when methanogenesis was partially inhibited (with 0.5 to 1.5 mM 2-bromoethanesulfonate or approximately 2 mM DCM) or completely stopped (with 50 to 55.5 mM 2-bromoethanesulfonate). Addition of a eubacterial inhibitor (vancomycin, 100 mg/liter) greatly reduced the rate of DCM degradation. 14CO2 was the principal product of [14C]DCM degradation, followed by 14CH4 (when methanogenesis was uninhibited) or 14CH3COOH (when methanogenesis was partially or completely inhibited). Hydrogen accumulated during DCM degradation and then returned to background levels when DCM was consumed. These results suggested that nonmethanogenic organisms mediated DCM degradation, oxidizing a portion to CO2 and fermenting the remainder to acetate; acetate formation suggested involvement of an acetogen. Methanogens in the enrichment culture then converted the products of DCM degradation to CH4. Aceticlastic methanogens were more easily inhibited by 2-bromoethanesulfonate and DCM than were CO2-reducing methanogens. When DCM was the sole organic-carbon and electron donor source supplied, its use as a growth substrate was demonstrated. The highest observed yield was 0.085 g of suspended organic carbon formed per g of DCM carbon consumed. Approximately 85% of the biomass formed was attributable to the growth of nonmethanogens, and 15% was attributable to methanogens.     

Methanogenesis and methanogenic pathways in a peat from subarctic permafrost

Few studies have dealt so far with methanogenic pathways and populations in subarctic and arctic soils. We studied the effects of temperature on rates and pathways of CH4 production and on the relative abundance and structure of the archaeal community in a mildly acidic peat from a permafrost region in Siberia (67 degrees N). We monitored the production of CH4 and CO2 over time and measured the consumption of Fe(II), ethanol and volatile fatty 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 between 26 degrees C and 28 degrees C [4.3 micromol CH4 (g dry weight)(-1) day(-1)], but the activity was high even at 4 degrees C [0.75 micromol CH4 (g dry weight)(-1) day(-1)], constituting 17% of that at 27 degrees C. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Acetoclastic methanogenesis accounted for about 70% of the total methanogenesis. Most 16S rRNA gene sequences clustered with Methanosarcinales, correlating with the prevalence of acetoclastic methanogenesis. In addition, sequences clustering with Methanobacteriales were recovered. Fe reduction occurred in parallel to methanogenesis. At lower and higher temperatures Fe reduction was not affected by BES. Because butyrate was consumed during methanogenesis and accumulated when methanogenesis was inhibited (BES and CH3F), it is proposed to serve as methanogenic precursor, providing acetate and H2 by syntrophic oxidation. In addition, ethanol and caproate occurred as intermediates. Because of thermodynamic constraints, homoacetogenesis could not compete with hydrogenotrophic methanogenesis.     

Biodegradation of dichloromethane and its utilization as a growth substrate under methanogenic conditions.

Biodegradation of dichloromethane (DCM) to environmentally acceptable products was demonstrated under methanogenic conditions (35 degrees C). When DCM was supplied to enrichment cultures as the sole organic compound at a low enough concentration to avoid inhibition of methanogenesis, the molar ratio of CH4 formed to DCM consumed (0.473) was very close to the amount predicted by stoichiometric conservation of electrons. DCM degradation was also demonstrated when methanogenesis was partially inhibited (with 0.5 to 1.5 mM 2-bromoethanesulfonate or approximately 2 mM DCM) or completely stopped (with 50 to 55.5 mM 2-bromoethanesulfonate). Addition of a eubacterial inhibitor (vancomycin, 100 mg/liter) greatly reduced the rate of DCM degradation. 14CO2 was the principal product of [14C]DCM degradation, followed by 14CH4 (when methanogenesis was uninhibited) or 14CH3COOH (when methanogenesis was partially or completely inhibited). Hydrogen accumulated during DCM degradation and then returned to background levels when DCM was consumed. These results suggested that nonmethanogenic organisms mediated DCM degradation, oxidizing a portion to CO2 and fermenting the remainder to acetate; acetate formation suggested involvement of an acetogen. Methanogens in the enrichment culture then converted the products of DCM degradation to CH4. Aceticlastic methanogens were more easily inhibited by 2-bromoethanesulfonate and DCM than were CO2-reducing methanogens. When DCM was the sole organic-carbon and electron donor source supplied, its use as a growth substrate was demonstrated. The highest observed yield was 0.085 g of suspended organic carbon formed per g of DCM carbon consumed. Approximately 85% of the biomass formed was attributable to the growth of nonmethanogens, and 15% was attributable to methanogens.