Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant (13)C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. (13)C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of delta-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong (13)C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant (13)C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane

Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.     

A comparison of DNA- and RNA-based clone libraries from the same marine bacterioplankton community

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by alpha-Proteobacteria in terms of repetitive DNA clones (52%), but gamma-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized if only the DNA or RNA libraries would have been analyzed alone. Of the DNA clones, 25.5% were found only in this library and no close relatives were detected in the RNA library. For clones from the RNA library, 21.5% of RNA clones did not indicate close relatives in the DNA library. Based on the comparisons between DNA and RNA libraries, our data indicate that the characterization of the bacterial community based on RNA has the potential to characterize distinct phylotypes from the marine environment, which remain undetected on the DNA level.     

Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant ¹³C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. ¹³C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of δ-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong ¹³C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant ¹³C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802-805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.     

Activity,Distribution, and Diversity of Sulfate Reducers and Other Bacteria in Sediments above Gas Hydrate (Cascadia Margin,Oregon)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. Recent investigations showed that another characteristic feature of cold seeps is the occurrence of methanotrophic archaea, which can be identified by specific biomarker lipids and 16S rDNA analysis. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH), bacterial production, enzyme activity, and sulfate reduction rates. Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6 2 10 10 cells cm m 3 ) and sulfate reduction rates (up to 8 w mol cm m 3 d m 1 ) in hydrate-bearing sediments, as well as a high bacterial diversity, especially in the group of i -proteobacteria including members of the branches Desulfosarcina/Desulfococcus , Desulforhopalus , Desulfobulbus , and Desulfocapsa . Most of the diversity of sulfate-reducing bacteria in hydrate-bearing sediments comprises seep-endemic clades, which share only low similarities with previously cultured bacteria.     

Methane production by the sulfate-reducing bacterium Desulfosarcina variabilis

The sulfate-reducing bacterium Desulfosarcina variabilis VKM B-1694 was found to produce up to 1.62 mumol methane per mg protein when grown on different substrates. The role of methanogenesis and the physicochemical factors determining this process in sulfate-reducing bacteria are discussed.     

Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin

Microbial community samples were collected from the anoxic zone of the Cariaco Basin at depths of 320, 500, and 1,310 m on a November 1996 cruise and were used to construct 16S ribosomal DNA libraries. Of 60 nonchimeric sequences in the 320-m library, 56 belonged to the epsilon subdivision of the Proteobacteria (epsilon-Proteobacteria) and 53 were closely related to ectosymbionts of Rimicaris exoculata and Alvinella pompejana, which are referred to here as epsilon symbiont relatives (ESR). The 500-m library contained sequences affiliated with the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the division Verrucomicrobia, the division Proteobacteria, and the OP3 candidate division. The Proteobacteria included members of the gamma, delta, epsilon and new candidate subdivisions, and gamma-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the epsilon-proteobacteria belonged to the ESR group. The genus Fibrobacter and its relatives were the second largest group in the library (23.6%), followed by the delta-proteobacteria and the epsilon-proteobacteria. The 1,310-m library had the greatest diversity; 59 nonchimeric clones in the library contained 30 unique sequences belonging to the planctomycetes, the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the Proteobacteria, and the OP3 and OP8 candidate divisions. The proteobacteria included members of new candidate subdivisions and the beta, gamma, delta, and epsilon-subdivisions. ESR sequences were still present in the 1,310-m library but in a much lower proportion (8.5%). One archaeal sequence was present in the 500-m library (2% of all microorganisms in the library), and eight archaeal sequences were present in the 1,310-m library (13.6%). All archaeal sequences fell into two groups; two clones in the 1,310-m library belonged to the kingdom Crenarchaeota and the remaining sequences in both libraries belonged to the kingdom Euryarchaeota. The latter group appears to be related to the Eel-TA1f2 sequence, which belongs to an archaeon suggested to be able to oxidize methane anaerobically. Based on phylogenetic inferences and measurements of dark CO(2) fixation, we hypothesized that (i) the ESR are autotrophic anaerobic sulfide oxidizers, (ii) sulfate reduction and fermentative metabolism may be carried out by a large number of bacteria in the 500- and 1,310-m libraries, and (iii) members of the Euryarchaeota found in relatively large numbers in the 1,310-m library may be involved in anaerobic methane oxidation. Overall, the composition of microbial communities from the Cariaco Basin resembles the compositions of communities from several anaerobic sediments, supporting the hypothesis that the Cariaco Basin water column is similar to anaerobic sediments.     

Aerobic and anaerobic methanotrophs in the Black Sea water column

Inputs of CH(4) from sediments, including methane seeps on the continental margin and methane-rich mud volcanoes on the abyssal plain, make the Black Sea the world's largest surface water reservoir of dissolved methane and drive a high rate of aerobic and anaerobic oxidation of methane in the water column. Here we present the first combined organic geochemical and molecular ecology data on a water column profile of the western Black Sea. We show that aerobic methanotrophs type I are responsible for methane oxidation in the oxic water column and ANME-1- and ANME-2-related organisms for anaerobic methane oxidation. The occurrence of methanotrophs type I cells in the anoxic zone suggests that inactive cells settle to deeper waters. Molecular and biomarker results suggest that a clear distinction between the occurrence of ANME-1- and ANME-2-related lineages exists, i.e. ANME-1-related organisms are responsible for anaerobic methane oxidation below 600 m water depth, whereas ANME-2-related organisms are responsible for this process in the anoxic water column above approximately 600 m water depth.     

Diversity and biogeochemical structuring of bacterial communities across the Porangahau ridge accretionary prism, New Zealand

Sediments from the Porangahau ridge, located off the northeastern coast of New Zealand, were studied to describe bacterial community structure in conjunction with differing biogeochemical regimes across the ridge. Low diversity was observed in sediments from an eroded basin seaward of the ridge and the community was dominated by uncultured members of the Burkholderiales. Chloroflexi/GNS and Deltaproteobacteria were abundant in sediments from a methane seep located landward of the ridge. Gas-charged and organic-rich sediments further landward had the highest overall diversity. Surface sediments, with the exception of those from the basin, were dominated by Rhodobacterales sequences associated with organic matter deposition. Taxa related to the Desulfosarcina/Desulfococcus and the JS1 candidates were highly abundant at the sulfate-methane transition zone (SMTZ) at three sites. To determine how community structure was influenced by terrestrial, pelagic and in situ substrates, sequence data were statistically analyzed against geochemical data (e.g. sulfate, chloride, nitrogen, phosphorous, methane, bulk inorganic and organic carbon pools) using the Biota-Environmental matching procedure. Landward of the ridge, sulfate was among the most significant structuring factors. Seaward of the ridge, silica and ammonium were important structuring factors. Regardless of the transect location, methane was the principal structuring factor on SMTZ communities.     

Diversity of 16S rDNA and Naphthalene Dioxygenase Genes from Coal-Tar-Waste-Contaminated Aquifer Waters

Microbial diversity in four wells along a groundwater flowpath in a coal-tar-waste-contaminated aquifer was examined using RFLP analysis of both 16S rDNA and naphthalene dioxygenase (NDO) genes. Amplified ribosomal DNA restriction analysis (ARDRA) relied upon eubacteria-specific primers to generate four clone libraries. From each library, 100 clones were randomly picked for analysis. Sixty percent of 400 clones contained unique ARDRA patterns. Diversity indices calculated for each community were high (Shannon-Weaver, H = 3.53 to 3.69). Clones representing ARDRA patterns found in the highest abundance were sequenced (31 total). Sequences related to aerobic bacteria (e.g., Nitrospira, Methylomonas, and Gallionella) predominated among those retrieved from the uncontaminated area of the site, whereas sequences related to facultatively aerobic and anaerobic bacteria (e.g. Azoarcus, Syntrophus, and Desulfotomaculum) predominated among those retrieved from contaminated areas of the site. Using NDO-specific primers and low-stringency PCR conditions, variability in RFLP patterns was only detected in community-derived DNA (3 of 4 wells) and not in 5 newly isolated naphthalene-degrading pure cultures. The ARDRA patterns of the pure culture isolates were not found in the clone libraries. Polymorphisms in community 16S rDNA and NDO genes found in well-water microorganisms reflected distinctive geochemical conditions across the site. Sequences related to sulfate-reducing bacteria were found in groundwater that contained sulfide, while sequences related to Gallionella, Syntrophus, and nitrate-reducing aromatic hydrocarbon-degrading bacteria were found in groundwater that contained ferrous iron, methane, and naphthalene, respectively.     

Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

Evidence for anaerobic oxidation of methane in sediments of a freshwater system (Lago di Cadagno)

Anaerobic oxidation of methane (AOM) has been investigated in sediments of a high alpine sulfate-rich lake. Hot spots of AOM could be identified based on geochemical and isotopic evidence. Very high fractionation of methane (α=1.031) during oxidation was observed in the uppermost sediment layers, where methane is oxidized most likely with sulfate-containing bottom waters. However, we could not exclude that other electron acceptors such as iron, or manganese might also be involved. Light carbon isotope values (δ13C = -10‰ vs. Vienna Pee Dee Belemnite [VPDB]) of sedimentary carbonates at 16-20 cm sediment depth are indicative of a zone where methane was oxidized and the resulting bicarbonate ions were used for carbonate precipitation. 16S rRNA gene analysis revealed the presence of sequences belonging to the marine benthic groups B, C, and D and to the recently described clade of AOM-associated archaea (AAA). Catalyzed reporter deposition-FISH analysis revealed a high abundance of Deltaproteobacteria, especially of free-living sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus branch of Deltaproteobacteria in the AOM zone. Here, loose aggregations of AAA cells were found, suggesting that AAA might be responsible for oxidation of methane in Lake Cadagno sediments.     

New insight into stratification of anaerobic methanotrophs in cold seep sediments

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions.     

小叶章生态系统根际土壤微生物及CO2、CH4、N2O动态

研究了培养 4 5 d的小叶章根际土壤微生物和二氧化碳 ,甲烷及氧化亚氮产生与消耗之间的关系。结果表明好氧微生物与厌氧微生物的空间分布与甲烷 ,二氧化碳及氧化亚氮的产生和氧化有着密切的关系。好氧微生物与甲烷产生呈负相关 ,与二氧化碳和氧化亚氮的产生呈正相关关系。厌氧微生物与甲烷的产生呈正相关 ,与二氧化碳和氧化亚氮的产生呈负相关关系     

Stratified Communities of Active Archaea in Shallow Sediments of the Pearl River Estuary, Southern China

Marine subsurface sediments represent a novel archaeal biosphere with unknown physiology. To get to know the composition and ecological roles of the archaeal communities within the sediments of the Pearl River Estuary, Southern China, the diversity and vertical distribution of active archaea in a sediment core were characterized by 16S rRNA phylogenetic analysis of clone libraries derived from RNA. In this study, the archaeal diversity above, within, and beneath the sulfate-methane transition zone (SMTZ) in the Pearl River Estuary sediment core was described. The majority of the clones obtained from the metabolically active fraction of the archaeal community were most closely related to miscellaneous crenarchaeotal group and terrestrial miscellaneous euryarchaeotal group. Notably, although the Pearl River Estuary sediment belong to high methane and high organic carbon environment, sequences affiliated with methanotrophic and methanogenic archaea were detected as minor group in 16S rRNA clone libraries. No obvious evidence suggested that these unknown archaeal phylotypes related directly to anaerobic oxidation of methane in SMTZ. This is the first phylogenetic analysis of the metabolically active fraction of the archaeal community in the coastal sediment environments.     

Molecular diversity of sulfate-reducing bacteria from two different continental margin habitats

This study examined the natural diversity and distributions of sulfate-reducing bacteria along a natural carbon gradient extending down the shelf-slope transition zone of the eastern Pacific continental margin. Dissimilatory (bi)sulfite reductase gene sequences (dsrAB) were PCR amplified and cloned from five different sampling sites, each at a discrete depth, from two different margin systems, one off the Pacific coast of Mexico and another off the coast of Washington State. A total of 1,762 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. The majority of the gene sequences recovered showed site and depth restricted distributions; however, a limited number of gene sequences were widely distributed within and between the margin systems. Cluster analysis identified 175 unique RFLP patterns, and nucleotide sequences were determined for corresponding clones. Several different continental margin DsrA sequences clustered with those from formally characterized taxa belonging to the delta subdivision of the class Proteobacteria (Desulfobulbus propionicus, Desulfosarcina variabilis) and the Bacillus-Clostridium (Desulfotomaculum putei) divisions, although the majority of the recovered sequences were phylogenetically divergent relative to all of the other DsrA sequences available for comparison. This study revealed extensive new genetic diversity among sulfate-reducing bacteria in continental margin sedimentary habitats, which appears to be tightly coupled to slope depth, specifically carbon bioavailability.     

Analysis of the sulfate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments

The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated. Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay. Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic. This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii. The Shallow Bay archaeal clone library contained clones related to the C(1)-utilizing Methanolobus and Methanococcoides and the H(2)-utilizing Methanogenium: Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal. Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments. In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster. Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments. Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.     

Characterization of Specific Membrane Fatty Acids as Chemotaxonomic Markers for Sulfate-Reducing Bacteria Involved in Anaerobic Oxidation of Methane

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as m 103. Specific fatty acids released from bacterial membranes include C 16:1 y 5c , C 17:1 y 6c , and cyC 17:0 y 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest i 13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 y 5c and cyC 17:0 y 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.     

Expanding the repertoire of electron acceptors for the anaerobic oxidation of methane in carbonates in the Atlantic and Pacific Ocean

Authigenic carbonates represent a significant microbial sink for methane, yet little is known about the microbiome responsible for the methane removal. We identify carbonate microbiomes distributed over 21 locations hosted by seven different cold seeps in the Pacific and Atlantic Oceans by carrying out a gene-based survey using 16S rRNA- and mcrA gene sequencing coupled with metagenomic analyses. Based on 16S rRNA gene amplicon analyses, these sites were dominated by bacteria affiliated to the Firmicutes, Alpha- and Gammaproteobacteria. ANME-1 and -2 archaeal clades were abundant in the carbonates yet their typical syntrophic partners, sulfate-reducing bacteria, were not significantly present. Based on mcrA amplicon analyses, the Candidatus Methanoperedens clades were also highly abundant. Our metagenome analysis indicated that methane oxidizers affiliated to the ANME-1 and -2, may be capable of performing complete methane- and potentially short-chain alkane oxidation independently using oxidized sulfur and nitrogen compounds as terminal electron acceptors. Gammaproteobacteria are hypothetically capable of utilizing oxidized nitrogen compounds and may be involved in syntrophy with methane-oxidizing archaea. Carbonate structures represent a window for a more diverse utilization of electron acceptors for anaerobic methane oxidation along the Atlantic and Pacific Margin.Subject terms: Microbiology, Biogeochemistry