Formate-Dependent Heterodisulfide Reduction in a Methanomicrobiales Archaeon.

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2 In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus copurified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr-associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2 We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order MethanomicrobialesIMPORTANCE Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments, such as in wastewater treatment facilities, anaerobic digesters, and the rumen, where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales.

Shifts of methanogenic communities in response to permafrost thaw results in rising methane emissions and soil property changes.

Permafrost thaw can bring negative consequences in terms of ecosystems, resulting in permafrost collapse, waterlogging, thermokarst lake development, and species composition changes. Little is known about how permafrost thaw influences microbial community shifts and their activities. Here, we show that the dominant archaeal community shifts from Methanomicrobiales to Methanosarcinales in response to the permafrost thaw, and the increase in methane emission is found to be associated with the methanogenic archaea, which rapidly bloom with nearly tenfold increase in total number. The mcrA gene clone libraries analyses indicate that Methanocellales/Rice Cluster I was predominant both in the original permafrost and in the thawed permafrost. However, only species belonging to Methanosarcinales showed higher transcriptional activities in the thawed permafrost, indicating a shift of methanogens from hydrogenotrophic to partly acetoclastic methane-generating metabolic processes. In addition, data also show the soil texture and features change as a result of microbial reproduction and activity induced by this permafrost thaw. Those data indicate that microbial ecology under warming permafrost has potential impacts on ecosystem and methane emissions.

The role of methanogens in acetic acid production under different salinity conditions.

In this study, a fed-batch acidogenic reactor was operated at a 3 d hydraulic retention time (HRT) and fed with alkaline pre-treated sludge to investigate salinity effects on methanogens' abundance, activities and their consumption of produced acetic acid (HAc) and total volatile fatty acids (VFAs). The salinity concentration was increased step-wise by adding sodium chloride. At 3‰ (parts per thousand) salinity, the average produced volatile fatty acids (VFAs) concentration was 2410.16 ± 637.62 mg COD L(-1) and 2.70 ± 0.36 L methane was produced daily in the acidogenic reactor. Further batch tests indicated methanogens showed a HAc degradation rate of 3.81 mg COD g(-1) VSS h(-1) at initial HAc concentration of 1150 mg COD L(-1), and showed tolerance up to 16‰ salinity (3.76 g Na(+) L(-1)) as indicated by a constant HAc degradation rate. The microbiological study indicated this can be related to the predominance of acetate-utilizing Methanosarcinaceae and Methanomicrobiales in the reactor. However, with salinity increased to 20‰ and 40‰, increases in VFAs and HAc production and decreases in methane production, methanogens population, acidogenic bacteria population and acidification extent were observed. This study demonstrated presence of acetate-utilizing methanogens in an acidogenic reactor and their high tolerance to salinity, as well as their negative impacts on net VFAs production. The results would suggest the presence of methanogens in the acidogenic reactor should not be ignored and the recovery of methane from the acidogenic reactor needs to be considered to avoid carbon loss.

Functional responses and adaptation of mesophilic microbial communities to psychrophilic anaerobic digestion.

Psychrophilic (<20°C) anaerobic digestion (AD) represents an attractive alternative to mesophilic wastewater treatment. In order to investigate the AD microbiome response to temperature change, with particular emphasis on methanogenic archaea, duplicate laboratory-scale AD bioreactors were operated at 37°C followed by a temperature drop to 15°C. A volatile fatty acid-based wastewater (composed of propionic acid, butyric acid, acetic acid and ethanol) was used to provide substrates representing the later stages of AD. Community structure was monitored using 16S rRNA gene clone libraries, as well as DNA and cDNA-based DGGE analysis, while the abundance of relevant methanogens was followed using qPCR. In addition, metaproteomics, microautoradiography-fluorescence in situ hybridization, and methanogenic activity measurements were employed to investigate microbial activities and functions. Methanomicrobiales abundance increased at low temperature, which correlated with an increased contribution of CH4 production from hydrogenotrophic methanogenesis at 15°C. Methanosarcinales utilized acetate and H2/CO2 as CH4 precursors at both temperatures and a partial shift from acetoclastic to hydrogenotrophic methanogenesis was observed for this archaeal population at 15°C. An upregulation of protein expression was reported at low temperature as well as the detection of chaperones indicating that mesophilic communities experienced stress during long-term exposure to 15°C. Overall, changes in microbial community structure and function were found to underpin the adaptation of mesophilic sludge to psychrophilic AD.

Substrate sources regulate spatial variation of metabolically active methanogens from two contrasting freshwater wetlands.

There is ample evidence that methane (CH4) emissions from natural wetlands exhibit large spatial variations at a field scale. However, little is known about the metabolically active methanogens mediating these differences. We explored the spatial patterns in active methanogens of summer inundated Calamagrostis angustifolia marsh with low CH4 emissions and permanently inundated Carex lasiocarpa marsh with high CH4 emissions in Sanjiang Plain, China. In C. angustifolia marsh, the addition of (13)C-acetate significantly increased the CH4 production rate, and Methanosarcinaceae methanogens were found to participate in the consumption of acetate. In C. lasiocarpa marsh, there was no apparent increase in the CH4 production rate and no methanogen species were labeled with (13)C. When (13)CO2-H2 was added, however, CH4 production was found to be due to Fen Cluster (Methanomicrobiales) in C. angustifolia marsh and Methanobacterium Cluster B (Methanobacteriaceae) together with Fen Cluster in C. lasiocarpa marsh. These results suggested that CH4 was produced primarily by hydrogenotrophic methanogens using substrates mainly derived from plant litter in C. lasiocarpa marsh and by both hydrogenotrophic and acetoclastic methanogens using substrates mainly derived from root exudate in C. angustifolia marsh. The significantly lower CH4 emissions measured in situ in C. angustifolia marsh was primarily due to a deficiency of substrates compared to C. lasiocarpa marsh. Therefore, we speculate that the substrate source regulates both the type of active methanogens and the CH4 production pathway and consequently contributes to the spatial variations in CH4 productions observed in these freshwater marshes.

Changes in bacterial and archaeal communities in anaerobic digesters treating different organic wastes.

The goal of this study was to characterize microbial communities in anaerobic batch digesters treating different representative organic sources (sewage sludge, food waste, septage). Among the digesters, the anaerobic digester of food waste had the highest methanogen density, producing a peak value methane yield of 813.2mLCH4/gVS. In all the digesters, acetoclastic Methanosarcinales and hydrogenotrophic Methanomicrobiales were the most dominant methanogen groups, but their proportion among the methanogens varied depending on the organic sources. The bacteria community in the anaerobic digestion (AD) of food waste and septage was distinctly different from that found in the AD of sewage sludge (primary sludge and waste activated sludge). Shifts in both bacterial and archaeal community structures could be related to differences in chemical properties, production, and accumulation of intermediates digested from organic wastes having different characteristics. These findings could prove useful in optimizing the microbial community to enhance AD process treating organic wastes.

Electrochemical nutrient recovery enables ammonia toxicity control and biogas desulfurization in anaerobic digestion.

Organic waste streams can be valorized and reduced in volume with anaerobic digestion (AD). An often-encountered key issue however is the high ammonium (NH4(+)) content of certain waste streams. Ammonia (NH3), in equilibrium with NH4(+), is a toxic compound to the methanogenic community, which limits the organic loading rate and endangers process stability. An electrochemical system (ES) linked to a digester could, besides recovering this nutrient, decrease NH3 toxicity through electrochemical extraction. Therefore, two digesters with and without ES attached in the recirculation loop were operated to test whether the ES could control NH3 toxicity. During periods of high ammonium loading rates, the methane (CH4) production of the ES-coupled reactor was up to 4.5 times higher compared to the control, which could be explained through simultaneous NH4(+) extraction and electrochemical pH control. A nitrogen flux of 47 g N m(­2) membrane d(­1) could be obtained in the ES-coupled reactor, resulting in a current and removal efficiency of 38 ± 5% and 28 ± 2%, respectively, at an electrochemical power input of 17 ± 2 kWh kg(­1) N. The anode also oxidized sulfide, resulting in a significantly lower H2S emission via the biogas. Lastly, limited methanogenic community dynamics pointed to a nonselective influence of the different operational conditions.

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal.

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.

Evaluation of sludge properties in a pilot-scale UASB reactor for sewage treatment in a temperate region.

In this study, continuous operation of a pilot-scale upflow anaerobic sludge blanket (UASB) reactor for sewage treatment was conducted for 630 days to investigate the physical and microbial characteristics of the retained sludge. The UASB reactor with a working volume of 20.2 m(3) was operated at ambient temperature (16-29 °C) and seeded with digested sludge. After 180 days of operation, when the sewage temperature had dropped to 20 °C or lower, the removal efficiency of both total suspended solids (TSS) and total biochemical oxygen demand (BOD) deteriorated due to washout of retained sludge. At low temperature, the cellulose concentration of the UASB sludge increased owing to the rate limitation of the hydrolytic reaction of suspended solids in the sewage. However, after an improvement in sludge retention (settleability and concentration) in the UASB reactor, the process performance stabilized and gave sufficient results (68% of TSS removal, 75% of total BOD removal) at an hydraulic retention time (HRT) of 9.7 h. The methanogenic activity of the retained sludge significantly increased after day 246 due to the accumulation of Methanosaeta and Methanobacterium following the improvement in sludge retention in the UASB reactor. Acid-forming bacteria from phylum Bacteroidetes were detected at high frequency; thus, these bacteria may have an important role in suspended solids degradation.

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique.

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.

Determination of methanogenic Archaea abundance in a mesophilic biogas plant based on 16S rRNA gene sequence analysis.

Energy production of renewable raw material is of increasing importance for sustainable energy production. As an indispensable prerequirement for further upgrading of technical equipment and operation modes of biogas plants, a deeper knowledge of the microbial community responsible for methane formation is crucial. To overcome these limitations a mesophilic biogas plant converting pig manure, maize silage, and grains of crop was sampled and subsequently analysed by construction of a methanogenic Archaea specific 16S rRNA gene clone library combined with PCR-RFLP analysis and group-specific quantitative real-time PCR (qPCR). Seventy percent of all analysed clones belonged to the order Methanomicrobiales, whereas 13% belonged to Methanosarcinales, 6% belonged to the Methanobacteriales group, and 11% of all detected clones were assigned to the CA11 and Arch1 cluster. Comparable percentages were obtained with qPCR: 84% of all detected 16S rRNA gene copy numbers were affiliated with the Methanomicrobiales, while only 14% belonged to the Methanosarcinales and 2% to the Methanobacteriales order. In conclusion, both approaches detected similar archaeal groups and revealed nearly the same abundance, pointing to a predominance of hydrogenotrophic methanogens in the biogas plant.

Enrichment of hydrogenotrophic methanogens in coupling with methane production using an electrochemical bioreactor.

Anaerobic digestion sludge was cultivated in an electrochemical bioreactor (ECB) to enrich the hydrogenotrophic methanogens. A modified graphite felt cathode with neutral red (NR-cathode) was charged with electrochemical reducing power generated from a solar cell. The methane and carbon dioxide collected in a Teflon bag from the ECB were more than 80 ml/l of reactant/day and less than 20 ml/l of reactant/day, respectively, whereas the methane and carbon dioxide collected from a conventional bioreactor (CB) was around 40 ml/l of reactant/day, respectively. Moreover, the maximal volume ratios of methane to carbon dioxide (M/C ratio) collected in the Teflon bag from the ECB and CB were 7 and 1, respectively. The most predominant methanogens isolated from the CB on the 20th, 80th, and 150th days of incubation were hydrogenotrophs. The methanogenic diversity analyzed by temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region was higher in the ECB than in the CB. The DNA extracted from the TGGE bands was more than 95% homologous with hydrogenotrophic methanogens in the CB. In conclusion, the ECB was demonstrated as a useful system for enriching hydrogenotrophic methanogens and increasing the M/C ratio of the gas product.

Shifts in methanogen community structure and function associated with long-term manipulation of sulfate and salinity in a hypersaline microbial mat.

Methanogenesis was characterized in hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico both in situ and after long-term manipulation in a greenhouse environment. Substrate addition experiments indicate methanogenesis to occur primarily through the catabolic demethylation of non-competitive substrates, under field conditions. However, evidence for the coexistence of other metabolic guilds of methanogens was obtained during a previous manipulation of sulfate concentrations. To fully characterize methanogenesis in these mats, in the absence of competition for reducing equivalents with sulfate-reducing microorganisms, we maintained microbial mats for longer than 1 year under conditions of lowered sulfate and salinity levels. The goal of this study was to assess whether observed differences in methane production during sulfate and salinity manipulation were accompanied by shifts in the composition of methanogen communities. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogen assemblages. Clone libraries from mats sampled in situ or maintained at field-like conditions in the greenhouse were exclusively composed of sequences related to methylotrophic members of the Methanosarcinales. Increases in pore water methane concentrations under conditions of low sulfate correlated with an observed increase in the abundance of putatively hydrogenotrophic mcrA, related to Methanomicrobiales. Geochemical and molecular data provide evidence of a significant shift in the metabolic pathway of methanogenesis from a methylotroph-dominated system in high-sulfate environments to a mixed community of methylotrophic and hydrogenotrophic methanogens under low sulfate conditions.

Phylogenetic diversity of the archaeal community in a continental high-temperature, water-flooded petroleum reservoir.

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.

Phylogenetic analysis of methanogenic enrichment cultures obtained from Lonar Lake in India: isolation of Methanocalculus sp. and Methanoculleus sp.

The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH approximately 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H(2):CO(2), sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.

The effect of sulfide on alpha-glucosidases: implications for starch degradation in anaerobic bioreactors.

Membrane associated alpha-glucosidase activity was investigated in a methanogenic bioreactor (MR) and a biosulfidogenic bioreactor (SR). Temperature and pH optima studies showed temperature optima of 50 degrees C and pH optima of 8.0 for the alpha-glucosidases from both the MR and SR. Sulfide (at a concentration of 150 mg l(-1)) resulted in the complete loss of all alpha-glucosidase activity in both the MR and SR. beta-Glucosidase activities in our bioreactors were previously shown to be stimulated in the presence of sulfide. alpha-Glucosidases, in contrast, are inhibited by sulfide. This differential effect of sulfide on alpha-glucosidase and beta-glucosidase activities is highlighted and is of crucial consequence to the respective degradation and utilization of starch and cellulose substrates in natural anaerobic environments and anaerobic bioreactors specifically designed for the accelerated digestion of wastewater sludge under biosulfidogenic conditions.

Vertical distribution of structure and function of the methanogenic archaeal community in Lake Dagow sediment.

Detailed studies on the relation of structure and function of microbial communities in a sediment depth profile scarcely exist. We determined as functional aspect the vertical distribution of the acetotrophic and hydrogenotrophic CH4 production activity by measuring production rates and stable 13C/12C-isotopic signatures of CH4 in the profundal sediment of Lake Dagow. The structural aspect was determined by the composition of the methanogenic community by quantifying the abundance of different archaeal groups using 'real-time' polymerase chain reaction and analysis of terminal restriction fragment length polymorphism (T-RFLP). Methane production rates in the surface sediment (0-3 cm depth) were higher in August than in May, but strongly decreased with depth (down to 20 cm). The delta13C of the produced CH4 and CO2 indicated an increase in isotopic fractionation with sediment depth. The relative contribution of hydrogenotrophic to total methanogenesis, which was calculated from the isotopic signatures, increased with depth from about 22% to 38%. Total numbers of microorganisms were higher in August than in May, but strongly decreased with depth. The increase of microorganisms from May to August mainly resulted from Bacteria. The Archaea, on the other hand, exhibited a rather constant abundance, but also decreased with depth from about 1 x 10(8) copies of the archaeal 16S rRNA gene per gram of dry sediment at the surface to 4 x 10(7) copies per gram at 15-20 cm depth. T-RFLP analysis combined with phylogenetic analysis of cloned sequences of the archaeal 16S rRNA genes showed that the methanogenic community consisted mainly of Methanomicrobiales and Methanosaetaceae. The relative abundance of Methanosaetaceae decreased with depth, whereas that of Methanomicrobiales slightly increased. Hence, the vertical distribution of the functional characteristics (CH4 production from acetate versus H2/CO2) was reflected in the structure of the community consisting of acetotrophic (Methanosaetaceae) versus hydrogenotrophic (Methanomicrobiales) phenotypes.

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing.

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.

Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat.

The emission of methane (1.3 mmol of CH(4) m(-2) day(-1)), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH(4)-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.

7-Mercaptoheptanoylthreonine phosphate substitutes for heat-stable factor (mobile factor) for growth of Methanomicrobium mobile.

Methanomicrobium mobile requires a heat-stable factor present in ruminal fluid and in boiled cell extract from Methanobacterium thermoautotrophicum for growth. By comparing the growth of M. mobile with boiled cell extract with that observed with various methanogenic cofactors, we found that 7-mercaptoheptanoylthreonine phosphate (HS-HTP) supported sustained growth of M. mobile, at an optimal concentration of 100 microM. No derivatives or possible biosynthetic precursors of HS-HTP could replace HS-HTP as the sole source of growth factor. Results suggest that the growth requirement might be satisfied by 7-mercaptoheptanoic acid plus a second, unidentified heat-stable factor.