Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Gene expression and ultrastructure of meso‐ and thermophilic methanotrophic consortia

The sulfate‐dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon‐rich environments under different temperature regimes: ANME‐1a/HotSeep‐1 (60°C), ANME‐1a/Seep‐SRB2 (37°C) and ANME‐2c/Seep‐SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME‐2c encodes all enzymes, while ANME‐1a lacks the gene for N5,N10‐methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c‐type cytochromes, some predicted to be extracellular. ANME‐2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME‐1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire‐like structures and heme‐rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate‐dependent AOM, and that both partners synthesize molecules to enable it.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

Enrichment of ANME‐2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with ¹³C labeled methane, ¹³C labeled inorganic carbon was produced and the AOM rate based on ¹³C‐inorganic carbon was 1.2 μmol/(g_dw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME‐2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM.     

Thermophilic anaerobic oxidation of methane by marine microbial consortia

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.     

Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.     

Anaerobic oxidation of methane by sulfate in hypersaline groundwater of the Dead Sea aquifer

Geochemical and microbial evidence points to anaerobic oxidation of methane (AOM) likely coupled with bacterial sulfate reduction in the hypersaline groundwater of the Dead Sea (DS) alluvial aquifer. Groundwater was sampled from nine boreholes drilled along the Arugot alluvial fan next to the DS. The groundwater samples were highly saline (up to 6300 mm chlorine), anoxic, and contained methane. A mass balance calculation demonstrates that the very low δ¹³C_DIC in this groundwater is due to anaerobic methane oxidation. Sulfate depletion coincident with isotope enrichment of sulfur and oxygen isotopes in the sulfate suggests that sulfate reduction is associated with this AOM. DNA extraction and 16S amplicon sequencing were used to explore the microbial community present and were found to be microbial composition indicative of bacterial sulfate reducers associated with anaerobic methanotrophic archaea (ANME) driving AOM. The net sulfate reduction seems to be primarily controlled by the salinity and the available methane and is substantially lower as salinity increases (2.5 mm sulfate removal at 3000 mm chlorine but only 0.5 mm sulfate removal at 6300 mm chlorine). Low overall sulfur isotope fractionation observed (³⁴ε = 17 ± 3.5‰) hints at high rates of sulfate reduction, as has been previously suggested for sulfate reduction coupled with methane oxidation. The new results demonstrate the presence of sulfate‐driven AOM in terrestrial hypersaline systems and expand our understanding of how microbial life is sustained under the challenging conditions of an extremely hypersaline environment.     

Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.     

Integrated metagenomic and metaproteomic analyses of an ANME-1-dominated community in marine cold seep sediments

Sulfate‐reducing methanotrophy by anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) is a major biological sink of methane in anoxic methane‐enriched marine sediments. The physiology of a microbial community dominated by free‐living ANME‐1 at 14–16 cm below the seafloor in the G11 pockmark at Nyegga was investigated by integrated metagenomic and metaproteomic approaches. Total DNA was subjected to 454‐pyrosequencing (829 527 reads), and 16.6 Mbp of sequence information was assembled into 27352 contigs. Taxonomic analysis supported a high abundance of Euryarchaea (70%) with 66% of the assembled metagenome belonging to ANME‐1. Extracted sediment proteins were separated in two dimensions and subjected to mass spectrometry (LTQ‐Orbitrap XL). Of 356 identified proteins, 245 were expressed by ANME‐1. These included proteins for cold‐adaptation and production of gas vesicles, reflecting both the adaptation of the ANME‐1 community to a permanently cold environment and its potential for positioning in specific sediment depths respectively. In addition, key metabolic enzymes including the enzymes in the reverse methanogenesis pathway (except N⁵,N¹⁰‐methylene‐tetrahydromethanopterin reductase), heterodisulfide reductases and the F₄₂₀H₂:quinone oxidoreductase (Fqo) complex were identified. A complete dissimilatory sulfate reduction pathway was expressed by sulfate‐reducing Deltaproteobacteria. Interestingly, an APS‐reductase comprising Gram‐positive SRB and related sequences were identified in the proteome. Overall, the results demonstrated that our approach was effective in assessing in situ metabolic processes in cold seep sediments.     

Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.

The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.     

A distinct freshwater-adapted subgroup of ANME-1 dominates active archaeal communities in terrestrial subsurfaces in Japan

Anaerobic methane-oxidizing archaea (ANME) are known to play an important role in methane flux, especially in marine sediments. The 16S rRNA genes of ANME have been detected in terrestrial freshwater subsurfaces. However, it is unclear whether ANME are actively involved in methane oxidation in these environments. To address this issue, Holocene sediments in the subsurface of the Kanto Plain in Japan were collected for biogeochemical and molecular analysis. The potential activity of the anaerobic oxidation of methane (AOM) (0.38-3.54 nmol cm?3 day?1) was detected in sediment slurry incubation experiments with a (13) CH(4) tracer. Higher AOM activity was observed in low-salinity treatment compared with high-salinity condition (20‰), which supports the adaptation of ANME in freshwater habitats. The 16S rRNA sequence analysis clearly revealed the presence of a distinct subgroup of ANME-1, designated ANME-1a-FW. Phylogenetic analysis of the mcrA genes also implied the presence of the distinct subgroup in ANME-1. ANME-1a-FW was found to be the most dominant active group in the archaeal communities on the basis of 16S rRNA analysis (75.0-93.8% of total archaeal 16S rRNA clones). Sulfate-reducing bacteria previously known as the syntrophic bacterial partners of ANME-1 was not detected. Our results showed that ANME-1a-FW is adapted to freshwater habitats and is responsible for AOM in terrestrial freshwater subsurface environments.     

A thermodynamic analysis of the anaerobic oxidation of methane in marine sediments

Anaerobic oxidation of methane (AOM) in anoxic marine sediments is a significant process in the global methane cycle, yet little is known about the role of bulk composition, temperature and pressure on the overall energetics of this process. To better understand the biogeochemistry of AOM, we have calculated and compared the energetics of a number of candidate reactions that microorganisms catalyse during the anaerobic oxidation of methane in (i) a coastal lagoon (Cape Lookout Bight, USA), (ii) the deep Black Sea, and (iii) a deep-sea hydrothermal system (Guaymas basin, Gulf of California). Depending on the metabolic pathway and the environment considered, the amount of energy available to the microorganisms varies from 0 to 184 kJ mol(-1). At each site, the reactions in which methane is either oxidized to HCO3(-), acetate or formate are generally only favoured under a narrow range of pressure, temperature and solution composition--particularly under low (10(-10 )m) hydrogen concentrations. In contrast, the reactions involving sulfate reduction with H2, formate and acetate as electron donors are nearly always thermodynamically favoured. Furthermore, the energetics of ATP synthesis was quantified per mole of methane oxidized. Depending on depth, between 0.4 and 0.6 mol of ATP (mol CH4(-1) was produced in the Black Sea sediments. The largest potential productivity of 0.7 mol of ATP (mol CH4(-1) was calculated for Guaymas Basin, while the lowest values were predicted at Cape Lookout Bight. The approach used in this study leads to a better understanding of the environmental controls on the energetics of AOM.     

Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME‐1 group

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate‐reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME‐1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82–90% of a composite ANME‐1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME‐1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron‐accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]‐hydrogenases are lacking in the dataset, genes for an [FeFe]‐hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c‐type cytochromes were identified, which have not yet been correlated with methanogenesis‐related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME‐1 to the bacterial sulfate‐reducing partner.     

Consumption of methane and CO2 by methanotrophic microbial mats from gas seeps of the anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Biochemistry of methanogenesis.

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH3-S-CoM) to CH4. Seminal studies on the CO2-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO2 to the methyl level (CH3-S-CoM) with electrons from H2 or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH3-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO2 by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH3-S-CoM; electrons for reduction of CH3-S-CoM to CH4 are provided by the oxidation of methyl groups to CO2.     

Insights into the genomes of archaea mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.     

The synthesis of acetyl-CoA by Clostridium thermoaceticum from carbon dioxide,hydrogen, coenzyme A and methyltetrahydrofolate

It has been demonstrated that enzymes from Clostridium thermoaceticum catalyze the following reaction in which Fd is ferredoxin and CH3THF is methyltetrahydrofolate. (for formula see text). The system involves hydrogenase, CO dehydrogenase, a methyltransferase, a corrinoid enzyme and other unknown components. Hydrogenase catalyzes the reduction of ferredoxin by H2; CO dehydrogenase then uses the reduced ferredoxin to reduce CO2 to a one-carbon intermediate that combines with CoASH and with a methyl group originating from CH3THF to form acetyl-CoA. It is proposed that these reactions are part of the mechanism which enables certain acetogenic autotrophic bacteria to grow on CO2 and H2.     

Identification of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rod-shaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.     

水生生态系统中金属依赖型甲烷厌氧氧化过程的研究进展

甲烷是一种比CO_2更活跃的温室气体,微生物驱动的甲烷厌氧氧化(anaerobicoxidationof methane,AOM)过程对于降低全球甲烷的排放有着重要意义。参与AOM反应的最终电子受体主要分为三类,即硫酸盐、亚硝酸盐/硝酸盐以及以Fe(III)、Mn(IV)等为代表的金属离子。可溶性金属物质和不溶性金属矿物都可以被用作AOM的电子受体,这大大提高了参与金属依赖型甲烷厌氧氧化(metal-dependent anaerobic oxidation of methane,Metal-AOM)微生物的生态价值。目前研究聚焦在功能菌群、生态分布等方面。部分甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)具有直接或间接参与Metal-AOM过程的能力。但由于功能菌群纯化富集和分离具有一定难度,有关其生理生化和生态学等特征的研究受到限制。同时,随着Metal-AOM被发现存在于不同水生生境中,其在污染治理领域的应用也被广泛讨论,但是河口生境尚缺乏深入研究。本文从Metal-AOM的发现入手,阐述了参与该过程的主要微生物及其在水域环境下的生态分布,并介绍了Metal-AOM的反应机制和在实际应用中的机遇与挑战。最后,根据现有研究结果,提出对功能菌群、机制及环保应用的研究展望,包括微生物分离纯化和影响因素、菌群代谢活性和作用机制的解析以及新型生产工艺的设计和发展应用,以期为今后的环境污染治理和工业应用提供借鉴意义。