Vertical Distribution of Methanogens in the Anoxic Sediment of Rotsee (Switzerland)

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont of Plagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea and the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 ± 0.25) × 10¹⁰ to (2.62 ± 0.58) × 10¹⁰ cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteria and Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [¹⁴C]acetate or [¹⁴C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.     

Characterizing Intrinsic Bioremediation in a Petroleum Hydrocarbon-Contaminated Aquifer by Combined Chemical,Isotopic, and Biological Analyses

Chemical, isotopic, and biological parameters were evaluated over a 1-year period to characterize microbial processes associated with intrinsic bioremediation in a petroleum hydrocarbon-contaminated aquifer located in Studen, Switzerland. Chemical parameters measured included oxidants such as O₂, NO₃ ^?, and SO₄ ^2?, reduced species such as Fe²⁺ and CH₄, and dissolved inorganic carbon (DIC). Stable carbon isotope analyses of DIC were used to differentiate between different processes that contribute to DIC production. Microbial populations were identified by sequence analysis of archaeal 16S rDNA and in situ hybridization using a general DNA binding dye (DAPI) and specific probes targeting the domain Archaea (Arch915) and Bacteria (Eub338), as well as the species Methanosaeta concilii (Rotcl1) and Methanospirillum sp. (Rotcl2). Groundwater exhibited reduced conditions and elevated concentrations of DIC within the contaminated zone. Spatially distinct values of δ¹³C ranging from ?16.5l%c to ?4.44%o were found, indicating the presence of different ongoing microbial processes. Detected microbial populations (% of DAPI-stained cells) within the contaminated zone belonged to Archaea (9±2% to 31±13%) and Bacteria (13±3% to 32±13%). In wells with methanogenic activity, Methanosaeta concilii accounted for up to 26% of all DAPI-detected microorganisms. These results demonstrate that this novel combination of chemical, isotopic, and biological analysis provides valuable insights that can be used for the characterization of microbial processes in contaminated aquifers.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

The vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu

This study was conducted to characterize the vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu, which underwent a change in trophic status from oligotrophic to hypertrophic in last half of the 20th century. The results revealed that the bacterial communities in different layers of sediment sample were very similar, and were related to Alpha -, Beta -, Gamma - and Deltaproteobacteria, Nitrospira, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Chlorobi, Actinobacteria and Acidobacteria . In contrast, the archaeal communities varied greatly with depth. The archaeal communities were primarily related to Euryarchaeota and Crenarchaeota , with methanogenic Archaea accounting for approximately 2–35% of the total Archaea. Additionally, sequences related to putative ammonia-oxidizing Archaea and ammonia-oxidizing Bacteria were detected in different layers of sediment samples. The abundance of Archaea, Bacteria, methanogenic Archaea and Nitrospira was further characterized by real-time PCR.     

Archaeal communities in High Arctic wetlands at Spitsbergen, Norway (78 degrees N) as characterized by 16S rRNA gene fingerprinting

Emissions of the greenhouse gas methane from Arctic wetlands have been studied extensively, though little is known about the ecology and community structure of methanogenic archaea that catalyze the methane production. As part of a project addressing microbial transformations of methane in Arctic wetlands, we studied archaeal communities in two wetlands (Solvatnet and Stuphallet) at Spitsbergen, Norway (78 degrees N) during two summer seasons. Directly extracted peat community DNA and enrichment cultures of methanogenic archaea were analyzed by nested PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rRNA gene fragments. Sequences affiliated with Methanomicrobiales, Methanobacteriaceae, Methanosaeta and Group I.3b of the uncultured crenarchaeota were detected at both sites. Sequences affiliated with Methanosarcina were recovered only from the site Solvatnet, while sequences affiliated with the euryarchaeotal clusters Rice Cluster II and Sediment 1 were detected only at the site Stuphallet. The phylogenetic affiliation of the recovered sequences suggested a potential of both hydrogenotrophic and acetoclastic methanogenesis at both sites. At Solvatnet, there were clear temporal trends in the archaeal community structure over the Arctic summer season. The archaeal community composition was significantly affected by factors influencing the activity of the overall bacterial community, as measured by in situ emissions of CO2. Methane emissions at both sites were influenced more by peat temperatures and thaw depth than by the archaeal community structure. Enrichment cultures for methanogenic archaea determined that most of the methanogens detected directly in peat could grow in culture at 10 degrees C. Culture based biases were indicated in later enrichment steps by the abundant growth of a Methanosarcina strain that was not detected directly in peat samples.     

Microbial populations associated with treatment of an industrial dye effluent in an anaerobic baffled reactor.

Fluorescent in situ hybridization (FISH) using 16S and 23S rRNA-targeted probes together with construction of an archaeal 16S ribosomal DNA (rDNA) clone library was used to characterize the microbial populations of an anaerobic baffled reactor successfully treating industrial dye waste. Wastewater produced during the manufacture of food dyes containing several different azo and other dye compounds was decolorized and degraded under sulfidogenic and methanogenic conditions. Use of molecular methods to describe microbial populations showed that a diverse group of Bacteria and Archaea was involved in this treatment process. FISH enumeration showed that members of the gamma subclass of the class Proteobacteria and bacteria in the Cytophaga-Flexibacter-Bacteroides phylum, together with sulfate-reducing bacteria, were prominent members of a mixed bacterial population. A combination of FISH probing and analysis of 98 archaeal 16S rDNA clone inserts revealed that together with the bacterial population, a methanogenic population dominated by Methanosaeta species and containing species of Methanobacterium and Methanospirillum and a relatively unstudied methanogen, Methanomethylovorans hollandica, contributed to successful anaerobic treatment of the industrial waste. We suggest that sulfate reducers, or more accurately sulfidogenic bacteria, together with M. hollandica contribute considerably to the treatment process through metabolism of dye-associated sulfonate groups and subsequent conversion of sulfur compounds to carbon dioxide and methane.     

Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO(4). 2H(2)O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 microM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels ( approximately 0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 microM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Temporal change of composition and potential activity of the thermophilic archaeal community during the composting of organic material

To date, composting has been regarded as an aerobic process but it has been shown that composting piles are often sources of atmospheric methane. In order to gain a more comprehensive view on the diversity of methanogenic Archaea in compost, gas chromatographical methods and molecular cloning were used to study relationships of thermophilic archaeal communities and changes in methane production potential during compost maturation. According to the thermophilic methane production potential, wide differences could be detected between differently aged compost materials. In material derived from 3- and 4-week-old piles, low and no thermophilic methane production potential, respectively, was observed at 50 degrees C. Material from a 6-week-old pile showed the maximum methane production. With compost maturation, the production slowly decreased again with 6 weeks, 8 weeks, and mature compost showing an optimum methane production potential at 60 degrees C. At 70 degrees C, only 6-week-old material showed a comparable high production of methane. The 16S rRNA-based phylogenetic surveys revealed an increase of archaeal diversity with compost maturation. In the 6-week-old material, 86% of the sequences in the archaeal 16S rRNA library had the highest sequence similarities to Methanothermobacter spp. and the remaining 14% of the clones were related to Methanosarcina thermophila. Quantification of methanogens in 6-week-old material, on the basis of the methane production rate, resulted in values of about 2x10(7) cells per gram fresh weight. In 8-week-old and mature compost material, the proportion of sequences similar to Methanothermobacter spp. decreased to 34% and 0%, respectively. The mature compost material showed the highest variation in identified sequences, although 33% could be assigned to as yet uncultured Archaea (e.g. Rice cluster I, III, and IV). Our results indicate that compost harbours a diverse community of thermophilic methanogens, with changing composition during the maturation process, presumably due to altered pile conditions. Likewise, compost may act as a potential carrier for thermophilic methanogens in temperate soils because it is widely used as a soil amendment.     

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.     

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.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchst?dt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.     

Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Small Thaw Ponds: An Unaccounted Source of Methane in the Canadian High Arctic

Thawing permafrost in the Canadian Arctic tundra leads to peat erosion and slumping in narrow and shallow runnel ponds that surround more commonly studied polygonal ponds. Here we compared the methane production between runnel and polygonal ponds using stable isotope ratios, ¹⁴C signatures, and investigated potential methanogenic communities through high-throughput sequencing archaeal 16S rRNA genes. We found that runnel ponds had significantly higher methane and carbon dioxide emissions, produced from a slightly larger fraction of old carbon, compared to polygonal ponds. The methane stable isotopic signature indicated production through acetoclastic methanogenesis, but gene signatures from acetoclastic and hydrogenotrophic methanogenic Archaea were detected in both polygonal and runnel ponds. We conclude that runnel ponds represent a source of methane from potentially older C, and that they contain methanogenic communities able to use diverse sources of carbon, increasing the risk of augmented methane release under a warmer climate.     

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

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

Identification of acetate-utilizing Bacteria and Archaea in methanogenic profundal sediments of Lake Kinneret (Israel) by stable isotope probing of rRNA

Acetate is an important intermediate in the decomposition of organic matter in anoxic freshwater sediments. Here, we identified distinct microorganisms active in its oxidation and transformation to methane in the anoxic methanogenic layers of Lake Kinneret (Israel) profundal sediment by rRNA-based stable isotope probing (RNA-SIP). After 18 days of incubation with amended [U-(13)C]acetate we found that archaeal 16S rRNA was (13)C-labelled to a far greater extent than bacterial rRNA. We identified acetoclastic methanogens related to Methanosaeta concilii as being most active in the degradation and assimilation of acetate. Oxidation of the acetate-methyl group played only a minor role, but nevertheless 'heavy'(13)C-labelled bacterial rRNA templates were identified. 'Heavy' bacteria were mainly affiliated with the Betaproteobacteria (mostly Rhodocyclales and Nitrosomonadales), the Nitrospira phylum (related to 'Magnetobacterium bavaricum' and Thermodesulfovibrio yellowstonii), and also with the candidate phylum 'Endomicrobia'. However, the mode of energy gain that allowed for the assimilation of (13)C-acetate by these bacterial groups remains unknown. It may have involved syntrophic oxidation of acetate, reduction of chlorinated compounds, reduction of humic substances, fermentation of organic compounds, or even predation of (13)C-labelled Methanosaeta spp. In summary, this SIP experiment shows that acetate carbon was predominantly consumed by acetoclastic methanogens in profundal Lake Kinneret sediment, while it was also utilized by a small and heterogeneous community of bacteria.     

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.     

产甲烷分离物中Clostridium spp.与Methanosarcina barkeri潜在的种间直接电子传递

【目的】革兰氏阴性菌Geobacter metallireducens可以与乙酸型产甲烷菌Methanosaeta harundinacea或Methanosarcina barkeri通过种间直接电子传递(DIET)还原CO2产甲烷。本实验室前期的研究发现Methanosarcina mazei和Geobacteraceae在铁还原富集培养中形成团聚体,可能存在直接电子传递。然而,革兰氏阳性菌(如Clostridium spp.)与产甲烷菌是否存在种间直接电子传递尚不明确。【方法】采用Hungate厌氧滚管法,以乙醇为唯一电子供体从铁还原富集培养体系中获得产甲烷分离物(S6)。通过T-RFLP及克隆文库分析群落多样性,结合循环伏安法等电化学方法研究产甲烷分离物的电活性。【结果】Clostridium spp.(与C.tunisiense相似性最高)和M.barkeri分别在S6细菌和古菌群落中占优势。S6与G.metallireducens共培养后铁还原和产甲烷能力未明显增加,Clostridium spp.可能与G.metallireducens类似,将电子直接传递给产甲烷菌M.barkeri产甲烷。此外,电化学检测发现,在用透析袋包裹电极阻碍微生物与电极表面通过直接接触形成生物膜的条件下,电流密度显著降低,并且循环伏安扫描无明显氧化还原峰。【结论】产甲烷分离物S6中存在直接电子传递途径。本工作提出在产甲烷分离物中占优势的革兰氏阳性菌Clostridium spp.和M.barkeri之间可能存在种间直接电子传递。     

Aceticlastic and NaCl-requiring methanogen "Methanosaeta pelagica" sp. nov., isolated from marine tidal flat sediment

Among methanogens, only 2 genera, Methanosaeta and Methanosarcina, are known to contribute to methanogenesis from acetate, and Methanosaeta is a specialist that uses acetate specifically. However, Methanosaeta strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q(T), from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and mcrA genes revealed that the isolate belongs to the genus Methanosaeta. Unlike the other known Methanosaeta species, this isolate grows at Na(+) concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus Methanosaeta in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of Methanosaeta organisms increased with increasing depth and was much higher than that of Methanosarcina organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine Methanosaeta species, and based on phylogenetic and characteristic studies, the name "Methanosaeta pelagica" sp. nov. is proposed for this novel species, with type strain 03d30q.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.