Showing papers on "Methanogen published in 2008"

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

Abstract: Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

Cell proliferation at 122°C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation

Abstract: We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO2. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (−34‰ to−27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<−12‰), and the kinetic isotope effect at 122°C and 40 MPa was −9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.

Structure of the archaeal community of the rumen.

Abstract: Members of the domain Archaea contribute about 0.3 to 3.3% of the microbial small subunit (16S and 18S) rRNA in the rumen (22, 39, 60). Archaea have a range of different metabolisms and are found in many habitats (6), but those known to exist in the rumen are strictly anaerobic methanogens. Yanagita et al. (59) observed that 2.8 to 4.0% of ruminal microorganisms displayed autofluorescence characteristic of F420, a methanogen cofactor, able to be seen under UV illumination during microscopy. Taken together with the small subunit rRNA abundance data, this suggests that a large part of the archaeal population is made up of methanogens. Most species of methanogens can grow using H2 and often formate as their energy sources and use the electrons derived from H2 (or formate) to reduce CO2 to CH4. Some species can grow with methyl groups, oxidizing some to CO2 to produce electrons that are used to reduce further methyl groups to methane. A few species can grow with acetate, effectively dissimilating acetate to CH4 and CO2. However, acetate is not metabolized to CH4 to any significant extent in the rumen (13). This is probably because the rate of passage of rumen contents through the rumen is greater than the growth rate of acetate-utilizing methanogens (53). In a normally functioning rumen, proteins and polymeric carbohydrates, which usually make up the largest part of the incoming feed, are fermented by a mixed microbial community to volatile fatty acids (VFAs), NH4+, CO2, and H2. The hydrogen is metabolized by the methanogens. The VFAs are taken up by the animal across the rumen wall and serve as major carbon and energy sources for the ruminant. A part of the VFAs, undigested feed components, and microbial cells leave the rumen and enter the rest of the animal's digestive tract. The central role of H2 in the rumen fermentation (12) means that, although methanogenic archaea make up only a small part of the rumen microbial biomass, they play an important role in rumen function and animal nutrition. Efficient H2 removal leads to a nutritionally more favorable pattern of VFA formation and to an increased rate of fermentation by eliminating the inhibitory effect of H2 on the microbial fermentation (26, 53). The rumen can be simplistically described as an open system with discontinuous solid (feed) and liquid (saliva and drinking water) inputs and multiple fractions that have different turnover rates (53). The methanogens in the rumen are found free in the rumen fluid, attached to particulate material and rumen protozoa, associated as endosymbionts within rumen protozoa, and attached to the rumen epithelium. The methanogens associated with these different fractions can be expected to have different growth rates since they will be removed from the rumen at different rates. In addition, the animal itself and the feed also influence the rate of passage of digesta through the rumen system (25). These different habitats may allow niche division among the methanogens and may explain some of the observed phylogenetic diversity of rumen archaea.

Phylogenetic Comparison of the Methanogenic Communities from an Acidic, Oligotrophic Fen and an Anaerobic Digester Treating Municipal Wastewater Sludge

Abstract: Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.

Effect of tea saponin on methanogenesis, microbial community structure and expression of mcrA gene, in cultures of rumen micro-organisms.

Abstract: Aims: To determine the in-vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in-vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme-M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real-time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal-associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the First Cultured Anaerobe Capable of Degrading Phenol to Acetate in Obligate Syntrophic Associations with a Hydrogenotrophic Methanogen

Abstract: Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UIT, was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H-2-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 mu M) and benzoate (a maximum of 11 mu M) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 mu M) and benzoate (maximum, 92 mu M) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UIT in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UIT belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UIT, and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.

Analysis of microbial community during biofilm development in an anaerobic wastewater treatment reactor

Abstract: The formation, structure, and biodiversity of a multispecies anaerobic biofilm inside an Upflow Anaerobic Sludge Bed (UASB) reactor fed with brewery wastewater was examined using complementary microbial ecology methods such us fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and cloning. The biofilm development can be roughly divided into three stages: an initial attachment phase (0–36 h) characterized by random adhesion of the cells to the surface; a consolidation phase (from 36 h to 2 weeks) defined by the appearance of microcolonies; and maturation phase (from 2 weeks to 2 months). During the consolidation period, proteobacteria with broad metabolic capabilities, mainly represented by members of alpha-Proteobacteria class (Oleomonas, Azospirillum), predominated. Beta-, gamma-, delta- (both syntrophobacteria and sulfate-reducing bacteria) and epsilon- (Arcobacter sp.) Proteobacteria were also noticeable. Archaea first appeared during the consolidation period. A Methanospirillum-like methanogen was detected after 36 h, and this was followed by the detection of Methanosarcina, after 4 days of biofilm development. The mature biofilm displayed a hill and valley topography with cells embedded in a matrix of exopolymers where the spatial distribution of the microorganisms became well-established. Compared to the earlier phases, the biodiversity had greatly increased. Although alpha-Proteobacteria remained as predominant, members of the phyla Firmicutes, Bacteroidete, and Thermotogae were also detected. Within the domain Archaea, the acetoclastic methanogen Methanosaeta concilii become dominant. This study provides insights on the trophic web and the shifts in population during biofilm development in an UASB reactor.

Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin‐rich fractions from different plant materials

Abstract: Aims: Investigation of the effects of saponin-rich fractions on rumen fermentation, methane production and the microbial community. Methods and Results: Saponins were extracted from Carduus, Sesbania and Knautia leaves and fenugreek seeds. Two levels of saponin-rich fractions with a substrate were incubated using the Hohenheim gas method. Methane was measured using an infrared-based methane analyser and microbial communities using quantitative PCR. On addition of saponin-rich fractions, methane and short-chain fatty acid production was not affected. The protozoal counts decreased by 10–39%. Sesbania saponins decreased methanogen population by 78%. Decrease in ruminal fungal population (20–60%) and increase in Fibrobacter succinogenes (21–45%) and Ruminococcus flavefaciens (23–40%) were observed. Conclusions: The saponins evaluated possessed anti-protozoal activity; however, this activity did not lead to methane reduction. Fenugreek saponins seemed to have potential for increasing rumen efficiency. The saponins altered the microbial community towards proliferation of fibre-degrading bacteria and inhibition of fungal population. Significance and Impact of the Study: The uni-directional relationship between protozoal numbers and methanogenesis, as affected by saponins, is not obligatory. All saponins might not hold promise for decreasing methane production from ruminants.

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I.

Abstract: Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33 degrees 56'N, 102 degrees 52'E) region and under the annual temperature around 1 degrees C. Previous studies indicated that Zoige wetland was one of the CH(4) emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales, could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10(7) cells per gram of soil, accounting for about 30% of the total Archeae. By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO(2), methanol or trimethylamine was determined to increase to 10(8) cells ml(-1), but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30 degrees C and 15 degrees C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae, and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.

Methanogenesis from methanol at low temperatures by a novel psychrophilic methanogen, "Methanolobus psychrophilus" sp. nov., prevalent in Zoige wetland of the Tibetan plateau.

Abstract: The Zoige wetland of the Tibetan plateau is at permanent low temperatures and is a methane emission heartland of the plateau; however, cold-adaptive methanogens in the soil are poorly understood. In this study, a variety of methanogenic enrichments at 15 degrees C and 30 degrees C were obtained from the wetland soil. It was demonstrated that hydrogenotrophic methanogenesis was the most efficient type at 30 degrees C, while methanol supported the highest methanogenesis rate at 15 degrees C. Moreover, methanol was the only substrate to produce methane more efficiently at 15 degrees C than at 30 degrees C. A novel psychrophilic methanogen, strain R15, was isolated from the methanol enrichment at 15 degrees C. Phylogenetic analysis placed strain R15 within the genus Methanolobus, loosely clustered with Methanolobus taylorii (96.7% 16S rRNA similarity). R15 produced methane from methanol, trimethylamine, and methyl sulfide and differed from other Methanolobus species by growing and producing methane optimally at 18 degrees C (specific growth rate of 0.063 +/- 0.001 h(-1)) and even at 0 degrees C. Based on these characteristics, R15 was proposed to be a new species and named "Methanolobus psychrophilus" sp. nov. The K(m) and V(max) of R15 for methanol conversion were determined to be 87.5 +/- 0.4 microM and 0.39 +/- 0.04 mM h(-1) at 18 degrees C, respectively, indicating a high affinity and conversion efficiency for methanol. The proportion of R15 in the soil was determined by quantitative PCR, and it accounted for 17.2% +/- 2.1% of the total archaea, enumerated as 10(7) per gram of soil; the proportion was increased to 42.4% +/- 2.3% in the methanol enrichment at 15 degrees C. This study suggests that the psychrophilic methanogens in the Zoige wetland are likely to be methylotrophic and to play a role in methane emission of the wetland.

Estimates of Biogenic Methane Production Rates in Deep Marine Sediments at Hydrate Ridge, Cascadia Margin

Abstract: Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.

Characterization of the Archaeal Community in a Minerotrophic Fen and Terminal Restriction Fragment Length Polymorphism-Directed Isolation of a Novel Hydrogenotrophic Methanogen

Abstract: Minerotrophic fen peatlands are widely distributed in northern latitudes and, because of their rapid turnover of organic matter, are potentially larger sources of atmospheric methane than bog peatlands per unit area. However, studies of the archaeal community composition in fens are scarce particularly in minerotrophic sites. Several 16S rRNA-based primer sets were used to obtain a broad characterization of the archaeal community in a minerotrophic fen in central New York State. A wide archaeal diversity was observed in the site: 11 euryarchaeal and 2 crenarchaeal groups, most of which were uncultured. The E1 group, a novel cluster in the order Methanomicrobiales, and Methanosaetaceae were the codominant groups in all libraries and results of terminal restriction fragment length polymorphism (T-RFLP) analysis. Given its abundance and potential hydrogenotrophic methane contribution, the E1 group was targeted for culture attempts with a low-ionic-strength medium (PM1). Initial attempts yielded Methanospirillum-dominated cultures. However, by incorporating a T-RFLP analysis as a quick selection tool for treatments and replicates, we were able to select an enrichment dominated by E1. Further dilutions to 10(-9) and tracking with T-RFLP yielded a strain named E1-9c. E1-9c is a novel coccoid hydrogenotrophic, mesophilic, slightly acidophilic methanogen and is highly sensitive to Na(2)S concentrations (requires <0.12 mM for growth). We propose E1-9c as the first representative of a novel genus in the Methanomicrobiales order.

Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a deep-sea whale-fall in Monterey Canyon, California

Abstract: Whale-falls represent localized areas of extreme organic enrichment in an otherwise oligotrophic deep-sea environment. Anaerobic remineralization within these habitats is typically portrayed as sulfidogenic; however, we demonstrate that these systems are also favorable for diverse methane-producing archaeal assemblages, representing up to 40% of total cell counts. Chemical analyses revealed elevated methane and depleted sulfate concentrations in sediments under the whale-fall, as compared to surrounding sediments. Carbon was enriched (up to 3.5%) in whale-fall sediments, as well as the surrounding sea floor to at least 10 m, forming a ‘bulls eye’ of elevated carbon. The diversity of sedimentary archaea associated with the 2893 m whale-fall in Monterey Canyon (California) varied both spatially and temporally. 16S rRNA diversity, determined by both sequencing and terminal restriction fragment length polymorphism analysis, as well as quantitative PCR of the methyl-coenzyme M reductase gene, revealed that methanogens, including members of the Methanomicrobiales and Methanosarcinales, were the dominant archaea (up to 98%) in sediments immediately beneath the whale-fall. Temporal changes in this archaeal community included the early establishment of methylotrophic methanogens followed by development of methanogens thought to be hydrogenotrophic, as well as members related to the newly described methanotrophic lineage, ANME-3. In comparison, archaeal assemblages in ‘reference’ sediments collected 10 m from the whale-fall primarily consisted of Crenarchaeota affiliated with marine group I and marine benthic group B. Overall, these results indicate that whale-falls can favor the establishment of metabolically and phylogenetically diverse methanogen assemblages, resulting in an active near-seafloor methane cycle in the deep sea.

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California

Abstract: Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C_(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.

Methanogen genomics to discover targets for methane mitigation technologies and options for alternative H2 utilisation in the rumen

Abstract: Reducing ruminant methane emissions is an important objective for ensuring the sustainability of ruminant-based agriculture. Methane is formed in the rumen by methanogens (part of the domain Archaea), mainly from H2 and CO2. Methanogens from a wide range of habitats are being genome-sequenced to gain a better understanding of their biology and, in particular, to identify targets for inhibition technologies for gut-associated methanogens. Genome comparisons are identifying common genes that define a methanogen, while gene differences are providing an insight into adaptations that allow methanogen survival and persistence under different environmental conditions. Within the rumen microbial food web, methanogens perform the beneficial task of removing H2, which allows reduced cofactors to be reoxidised and recycled, thereby enhancing the breakdown and fermentation of plant material. Therefore, rumen methane mitigation strategies need to consider alternative routes of H2 utilisation in the absence (or decreased levels) of methanogenesis to maintain rumen function. Two main alternatives are possible: enhancing rumen microorganisms that carry out reductive acetogenesis (combining CO2 and H2 to form acetate) or promotion of organisms that consume reducing equivalents during the conversion of metabolic intermediates (malate, fumarate and crotonate) into propionate and butyrate. A better understanding of the role and scale of methane oxidation in the rumen may also lead to future options for methane mitigation.

Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” and Methanospirillum Species

Abstract: Anaerobic dechlorination of chlorinated organic compounds is an important mechanism for the remediation of common groundwater pollutants (11, 58). It is now accepted that members of the “Dehalococcoides” group play a crucial role in the remediation of compounds such as chloroethenes, chlorobenzenes, chloroalkanes, chlorophenols, dioxins, and polychlorinated biphenyls, in some cases dechlorinating these compounds to nontoxic end products (1, 2, 6, 16, 18, 26, 32, 35). While researchers have been able to isolate and perform pure culture studies of these organisms, there is significant evidence that reductive dechlorination in environmental systems and in the most robust laboratory cultures is the work of microbial consortia (4, 11). All cultured representatives of the Dehalococcoides group require hydrogen as an electron donor (often supplied by syntrophic fermentation) and a halogenated organic as an electron acceptor. In addition, Dehalococcoides grow robustly in mixed cultures, likely due to currently undetermined growth factors from other community members (11, 18, 31, 34, 44). Though reductive dechlorination is an energetically favorable process under syntrophic conditions with low hydrogen partial pressures (19, 57, 58), other, less favorable metabolic reactions such as methanogenesis and acetogenesis often occur in these communities, especially when excess hydrogen or a donor fermented at high hydrogen partial pressures is available (19, 20, 24, 38). Many methanogens depend on acetate and/or H2, which are both utilized by Dehalococcoides. This suggests that competition for resources is an important interaction within dechlorinating microbial communities containing both methanogenic and Dehalococcoides populations. Several studies have looked at dechlorinating microbial communities derived from both enrichment cultures and environmental systems (8, 12, 17, 21, 22, 28, 30, 33, 43, 54, 55, 64). Several distinct lineages of microorganisms, representing a variety of metabolic capabilities, are commonly found in these consortia, supporting the potential complexity of community dynamics. The D2 enrichment culture, which has been studied previously (19, 20, 46, 48, 52, 53), is derived from the same consortium from which “Dehalococcoides ethenogenes” strain 195 was isolated (13, 14, 44, 45). In this study, the phylogenetic community structure of the D2 enrichment culture including D. ethenogenes was assessed from phylogenetic analysis of bacterial and archaeal 16S rRNA gene libraries created from community DNA. Within this heterogeneous enrichment culture, two distinct cellular attachment phases were observed: planktonic cells (individual suspended cells) and cells associated with bioflocs (suspended cell aggregates). In the D2 culture, bioflocs (typically 10 to 100 μm in diameter) tended to contain multiple species and form around mineral precipitates from the medium (see Movie S1 in the supplemental material for a three-dimensional [3D] micrograph). Planktonic and biofloc-associated growth forms are common in environmental microbial communities (i.e., activated sludge, marine, sediments, and groundwater) (5, 10, 41, 60). In this study, a technique for physical enrichment of these two cell attachment phases via low-speed centrifugation was developed. Fluorescence in situ hybridization (FISH) with 16S rRNA-targeting probes was used to estimate the distribution of D. ethenogenes populations between plankton and bioflocs and to examine colocalization of D. ethenogenes and methanogenic Archaea within the bioflocs. Potential differences in gene expression between the two attachment forms were determined for both D. ethenogenes and the hydrogenotrophic methanogen present in the culture, Methanospirillum hungatei, using quantitative reverse transcription-PCR. This method was also used to compare expression of housekeeping and hydrogenase genes between these organisms. Understanding the distribution and difference in gene expression of the two D. ethenogenes cell attachment phases not only is important for elucidating the ecology of these organisms; it also has implications for the use of DNA and RNA as bioindicators of Dehalococcoides activity. A groundwater sample, while easier and less expensive to obtain, would predominantly sample planktonic Dehalococcoides. Therefore, it is important to establish whether the populations and activities of the planktonic phase reflect those of the community as a whole.

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

Abstract: Summary 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.

Methyl Sulfide Production by a Novel Carbon Monoxide Metabolism in Methanosarcina acetivorans

Abstract: We observed dimethyl sulfide and methanthiol production in pure incubations of the methanogen Methanosarcina acetivorans when carbon monoxide (CO) served as the only electron donor. Energy conservation likely uses sodium ion gradients for ATP synthesis. This novel metabolism permits utilization of CO by the methanogen, resulting in quantitative sulfide methylation.

Analysis of the Methanobrevibacter ruminantium draft genome: understanding methanogen biology to inhibit their action in the rumen

Abstract: Methane is produced in the foregut (rumen) of ruminants by methanogens, which act as terminal reducers of carbon in the rumen system. The multistep methanogenesis pathway is well elucidated, mainly from the study of non-rumen methanogens, but the adaptations that allow methanogens to grow and persist in the rumen are not well understood. The Pastoral Greenhouse Gas Research Consortium is sequencing the genome of Methanobrevibacter ruminantium, a prominent methanogen in New Zealand ruminants, as part of a project to mitigate greenhouse gases. The genome is ~3.0 Mb in size with a guanine–cytosine (GC) content of 33.68%. All of the components of the methanogenesis pathway have been identified and comparison of these gene sequences with those from Methanothermobacter thermoautotrophicus and Methanosphaera stadtmanae indicates that methanogenesis gene organisation is conserved within the Methanobacteriales. The genome of M. ruminantium contains a prophage sequence (designated φmru) with distinct functional modules encoding phage integration, DNA replication and packaging, capsid proteins and lysis functions. A low GC region found at the distal end of the phage sequence harbours a putative DNA restriction/modification system which might provide additional protection against foreign DNA. The genome also contains many large surface proteins with characteristics that indicate that they may mediate association with other rumen microbes. Approximately half of the genes identified within the genome have no known function. Determining the function of these new genes will assist in defining the role of M. ruminantium in methane formation in the rumen and help identify means to control methane emissions from ruminant animals.

Diversity of methanogens in ruminants in Queensland

Abstract: Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.

Identification of Archaeal population in the granular sludge of an UASB reactor treating sewage at low temperatures

Abstract: Effect of low temperature on up-flow anaerobic sludge bed (UASB) reactor performance treating raw sewage was investigated in terms of the variations in methanogenic diversity using the 16S rRNA based Fluorescence In-Situ Hybridization (FISH) technique. The diversity of microorganisms present in the anaerobic granular sludge and the structure of the granules operated at 13°C have been investigated using FISH combined with CSLM (Confocal Scanning Laser Microscopy). According to FISH results, archaeal cells representing methanogens were found intensively dominant in the bottom sampling port of the UASB reactor and acetoclastic Methanosaeta was the abundant methanogen. Other methanogens such as Methanosarcina and Methanobacterium like species were also observed. The abundance of originally mesophilic Methanosaeta-related Archaea under low temperature at all sampling days revealed the microbial adaptation to psychrophilic conditions. This might be attributed to the enzymatic alterations in Methanosaeta cells o...

Indigenous microorganism oil flooding method

Abstract: An oil extraction method driven by substrate microorganism, the first stage: aerobic fermentation stage, oxybiontic hydrocarbon-oxidizing bacterium and facultative anaerobe of hydrocarbon-oxidizing bacterium are activated at the near zone of the water injection well Because of being partial oxidation of hydrocarbons, it produces alcohol, fatty acid, surfactant, CO2, biological polysaccharide and other product; on one hand these substance is base oil releaser, on the other hand it is as nutrient source of anaerobe including methanogen; The second stage: anaerobic fermentation stage, methanogen is activated in the deep part of anoxic oil pool, producing CO4, CO2 and other matter which increases oil fluid and increases recovery after the matter dissolving in water; In the process, the proportion between isotope light methane produced by biology and general methane is increased

Process and apparatus for the microbial production of a specific product and methane

Abstract: The process according to the invention for the microbial production of a specific product and methane comprises the following steps: a) the production in a bioreactor ( 2, 10 ) of a specific product from a specific substrate using a specific organism, the producer, wherein during the production of this product metabolites such as e.g. low-molecular alcohols, aldehydes and acids, hydrogen and CO 2 , which may inhibit the production of the product, are obtained; and b) the production of methane by means of a methanogen, wherein in the production of the methane at least one metabolite is decomposed and by this means removed from the bioreactor ( 2, 10 ) and its energy is made usable.

Method for improving methane yield by organic waste water anaerobic fermentation

Abstract: The invention relates to a method used for raising the output of marsh gas by using anaerobic fermentation of organic effluent water, which pertains to the technical field of biological treatment of effluent water. The invention adopts simulated organic effluent water or practical wastewater from chemical industry as treatment objects, by adding trace metal element Co and metal ion chelating agent to an anaerobic reactor to improve the biological availability of trace metal elements which are essential for methanogen and promote the growth and activity of methanogen, thereby, methane output and transformation efficiency of contaminating material of the system is improved. The method needs only extreme micro-amount of trace metal element Co and metal ion chelating agent, therefore, the invention is not only convenient to be manipulated with high efficiency and but also low in cost which leads the invention to be extensively applicable in engineering practice.

Use of avian anti-methanogen antibodies for reduction of methane production

Abstract: Herein, it is shown that strong specific anti-methanogen avian antibodies can be produced when chickens are immunized with an optimal dose of methane producing bacterial antigen (methanogen) formulated with an appropriate adjuvant. The antibodies can in turn be used to reduce methane gas production from an animal by administering an effective amount of the anti-methanogen antibodies to the animal, thereby reducing methane gas evolved by the animal compared to an untreated or mock treated control animal of similar age and condition.