Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink

Mud volcanism is an important natural source of the greenhouse gas methane to the hydrosphere and atmosphere. Recent investigations show that the number of active submarine mud volcanoes might be much higher than anticipated (for example, see refs 3-5), and that gas emitted from deep-sea seeps might reach the upper mixed ocean. Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release are unknown. It is also unclear how efficiently methane-oxidizing microorganisms remove methane. Here we investigate the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72 degrees N, 14 degrees 44' E; 1,250 m water depth) to provide quantitative estimates of the in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities: aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a previously undescribed clade of archaea (ANME-3) associated with bacterial mats. We found that the upward flow of sulphate- and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to <40% of the total flux.     

A conspicuous nickel protein in microbial mats that oxidize methane anaerobically

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.     

Methane-consuming archaebacteria in marine sediments

Large amounts of methane are produced in marine sediments but are then consumed before contacting aerobic waters or the atmosphere. Although no organism that can consume methane anaerobically has ever been isolated, biogeochemical evidence indicates that the overall process involves a transfer of electrons from methane to sulphate and is probably mediated by several organisms, including a methanogen (operating in reverse) and a sulphate-reducer (using an unknown intermediate substrate). Here we describe studies of sediments related to a decomposing methane hydrate. These provide strong evidence that methane is being consumed by archaebacteria that are phylogenetically distinct from known methanogens. Specifically, lipid biomarkers that are commonly characteristic of archaea are so strongly depleted in carbon-13 that methane must be the carbon source, rather than the metabolic product, for the organisms that have produced them. Parallel gene surveys of small-subunit ribosomal RNA (16S rRNA) indicate the predominance of a new archael group which is peripherally related to the methanogenic orders Methanomicrobiales and Methanosarcinales.     

Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia

Aerobic methanotrophic bacteria consume methane as it diffuses away from methanogenic zones of soil and sediment. They act as a biofilter to reduce methane emissions to the atmosphere, and they are therefore targets in strategies to combat global climate change. No cultured methanotroph grows optimally below pH 5, but some environments with active methane cycles are very acidic. Here we describe an extremely acidophilic methanotroph that grows optimally at pH 2.0-2.5. Unlike the known methanotrophs, it does not belong to the phylum Proteobacteria but rather to the Verrucomicrobia, a widespread and diverse bacterial phylum that primarily comprises uncultivated species with unknown genotypes. Analysis of its draft genome detected genes encoding particulate methane monooxygenase that were homologous to genes found in methanotrophic proteobacteria. However, known genetic modules for methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses some novel methylotrophic pathways. Phylogenetic analysis of its three pmoA genes (encoding a subunit of particulate methane monooxygenase) placed them into a distinct cluster from proteobacterial homologues. This indicates an ancient divergence of Verrucomicrobia and Proteobacteria methanotrophs rather than a recent horizontal gene transfer of methanotrophic ability. The findings show that methanotrophy in the Bacteria is more taxonomically, ecologically and genetically diverse than previously thought, and that previous studies have failed to assess the full diversity of methanotrophs in acidic environments.     

Glycerol ether biomarkers and their carbon isotopic compositions in a cold seep carbonate chimney from the Shenhu area,northern South China Sea

At modern cold seeps,the anaerobic oxidation of methane(AOM)is the dominant pathway for methane consumption in marine sediments.AOM,which is mediated by a consortium of methane oxidizing archaea and sulfate reducing bacteria,is proposed to be responsible for authigenic carbonate formation.A methane-derived carbonate chimney was collected from the Shenhu area, northern South China Sea.The membrane lipids and their very low carbon isotopic compositions(?115‰to?104‰)in the Shenhu chimney suggest the presence of an AOM process.Three specific archaeal and bacterial biomarkers were detected,including Ar,DAGE 1f,and monocyclic MDGD.Their strongly depleted??13C values(?115‰to?104‰),which are lower than those of the normal marine lipids in sediments,reveal biogenic methane as their origin.The carbonate deposits exhibiting a chimney structure indicate that a vigorous methane-rich fluid expulsion may have occurred at the seafloor.We propose that the decomposition of gas hydrates at depth is the likely cause of seepage and cold seep carbonate formation in the Shenhu area.     

丹江口水库上下游古菌优势菌群落结构特征分析

采用16S rRNA Illumina Miseq高通量测序技术, 分析丹江口水库库区及汉江下游古菌物种组成, 并对大坝上、下游水体与沉积物中占优势的氨氧化古菌(Ammonia-oxidizing archaea, AOA)和产甲烷古菌(Methanogenic archaea)群落结构进行分析。结果表明, 优势种群落结构组成受到水体与沉积物样本差异的影响, 可由氨氧化古菌的好氧特性与产甲烷古菌的厌氧特性合理地解释。网络图分析表明, 丹江口水库上游氨氧化古菌与产甲烷古菌具有显著的相关关系。受丹江口水库运行的影响, 大坝下游水体及沉积物中氨氧化古菌丰度皆比大坝上游少, 而沉积物中产甲烷古菌丰度较高, 二者间相关性不明显。     

A marine microbial consortium apparently mediating anaerobic oxidation of methane

A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.     

A microbial consortium couples anaerobic methane oxidation to denitrification

Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.     

Methanotrophy below pH 1 by a new Verrucomicrobia species

Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices with significant methane fluxes to the atmosphere (10(-1) to 10(3) t y(-1)). Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes, no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 degrees C and pH down to 1.8). The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the beta-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 degrees C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, 'outside' the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8--far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated, is coupled to a geochemically relevant reaction.     

Methane formation from long-chain alkanes by anaerobic microorganisms.

Biological formation of methane is the terminal process of biomass degradation in aquatic habitats where oxygen, nitrate, ferric iron and sulphate have been depleted as electron acceptors. The pathway leading from dead biomass to methane through the metabolism of anaerobic bacteria and archaea is well understood for easily degradable biomolecules such as carbohydrates, proteins and lipids. However, little is known about the organic compounds that lead to methane in old anoxic sediments where easily degradable biomolecules are no longer available. One class of naturally formed long-lived compounds in such sediments is the saturated hydrocarbons (alkanes). Alkanes are usually considered to be inert in the absence of oxygen, nitrate or sulphate, and the analysis of alkane patterns is often used for biogeochemical characterization of sediments. However, alkanes might be consumed in anoxic sediments below the zone of sulphate reduction, but the underlying process has not been elucidated. Here we used enrichment cultures to show that the biological conversion of long-chain alkanes to the simplest hydrocarbon, methane, is possible under strictly anoxic conditions.     

Potential methane reservoirs beneath Antarctica

Once thought to be devoid of life, the ice-covered parts of Antarctica are now known to be a reservoir of metabolically active microbial cells and organic carbon. The potential for methanogenic archaea to support the degradation of organic carbon to methane beneath the ice, however, has not yet been evaluated. Large sedimentary basins containing marine sequences up to 14?kilometres thick and an estimated 21,000 petagrams (1?Pg equals 10(15)?g) of organic carbon are buried beneath the Antarctic Ice Sheet. No data exist for rates of methanogenesis in sub-Antarctic marine sediments. Here we present experimental data from other subglacial environments that demonstrate the potential for overridden organic matter beneath glacial systems to produce methane. We also numerically simulate the accumulation of methane in Antarctic sedimentary basins using an established one-dimensional hydrate model and show that pressure/temperature conditions favour methane hydrate formation down to sediment depths of about 300?metres in West Antarctica and 700?metres in East Antarctica. Our results demonstrate the potential for methane hydrate accumulation in Antarctic sedimentary basins, where the total inventory depends on rates of organic carbon degradation and conditions at the ice-sheet bed. We calculate that the sub-Antarctic hydrate inventory could be of the same order of magnitude as that of recent estimates made for Arctic permafrost. Our findings suggest that the Antarctic Ice Sheet may be a neglected but important component of the global methane budget, with the potential to act as a positive feedback on climate warming during ice-sheet wastage.     

琼东南盆地甲烷微渗漏活动地球化学示踪研究

为了深入认识南海北部琼东南盆地的甲烷微渗漏活动,通过测试位于该盆地某似海底放射（BSR）发育区内柱状样HQ-6PC和HQ-38PC的孔隙水阴阳离子浓度及δ¹³C_DIC等指标,对甲烷微渗漏活动特征进行了研究。结果显示,在柱状样HQ-6P和HQ-38PC的5.2 m以上部分,硫酸盐消耗由有机质硫酸盐还原作用（OSR）和甲烷缺氧氧化作用（AOM）共同主导,而在柱状样HQ-38PC的5.2 m以下部分主要受AOM的影响。柱状样HQ-38PC的硫酸根甲烷转换界面（SMTZ）埋深为9.9 m,甲烷向上扩散的通量约为32 mmol·m^-2·a^-1。两个柱状样孔隙水的Mg/Ca和Sr/Ca质量比随深度的变化指示其中形成的自生碳酸盐矿物主要为高镁方解石。HQ-6PC的Cl^-浓度在3.5 m以下明显降低,可能有天然气水合物分解时排放的低盐度流体加入,而HQ-38PC在4.0~5.5 m处存在较高的盐度异常,暗示其中可能混入了来自水合物形成时排放的高盐度流体。因此,两个站位浅表层发育显著的甲烷微渗漏活动,其下方可能发育水合物。     

Detection and classification of atmospheric methane oxidizing bacteria in soil

Well-drained non-agricultural soils mediate the oxidation of methane directly from the atmosphere, contributing 5 to 10% towards the global methane sink. Studies of methane oxidation kinetics in soil infer the activity of two methanotrophic populations: one that is only active at high methane concentrations (low affinity) and another that tolerates atmospheric levels of methane (high affinity). The activity of the latter has not been demonstrated by cultured laboratory strains of methanotrophs, leaving the microbiology of methane oxidation at atmospheric concentrations unclear. Here we describe a new pulse-chase experiment using long-term enrichment with 12CH4 followed by short-term exposure to 13CH4 to isotopically label methanotrophs in a soil from a temperate forest. Analysis of labelled phospholipid fatty acids (PLFAs) provided unambiguous evidence of methane assimilation at true atmospheric concentrations (1.8-3.6 p.p.m.v.). High proportions of 13C-labelled C18 fatty acids and the co-occurrence of a labelled, branched C17 fatty acid indicated that a new methanotroph, similar at the PLFA level to known type II methanotrophs, was the predominant soil micro-organism responsible for atmospheric methane oxidation.     

Anaerobic oxidation of methane: Geochemical evidence from pore-water in coastal sediments of Qi’ao Island (Pearl River Estuary), southern China

The concentrations of CH4 and SO4^2- in pore-water and the carbon isotope compositions of total dissolved inorganic （TCO2） and OH4 were determined for three coastal sedimentary cores collected from Qi＇ao Island （Pearl River Estuary）, southern China. Results show that methane concentration changes dramatically at the base of the sulfate-reducing zone and sulfate concentration gradients are linear for all stations. In addition, the carbon isotope of methane becomes heavier at the sulfate-methane transition （SMT）, which causes ∑CO2-δ^13C to become the minimum. The geochemical profiles of pore-water render indirect evidence for anaerobic oxidation of methane （AOM）. Based on numerical modeling of AOM and sulfate-reducing rates, the portion of total sulfate reduction occurring via AOM is 9.0%, 84% and 45.5%, respectively, and the percentage of TCO2 added to the pore-water is 4.7%, 72.4% and 29.45% correspondingly for three sites. Furthermore, it is found that the methane concentration, methane diffusive flux and the depth of SMT are controlled by the quantity and quality of sedimentary organic matter incorporated into the sediments. The great amount of organic material is favorable for rapid depletion of sulfate via sedimentary organic matter degradation, and on the other hand, causes the increase of the methane flux in the SMT, which results in a portion of sulfate reduction supported by AOM. Accordingly, the SMT was shifted towards the sediment surface.     

微藻生物质暗发酵产氢气和甲烷的研究

微藻生物质厌氧消化生产氢气和甲烷效率低下,本研究报道了一种新型微藻处理工艺即两段式暗发酵提高氢气和甲烷产量。结果表明微藻生物质的最佳有机负荷为10 g/L,相应的氢气产量为18.6 mL/g (每克挥发性有机质产气量)。进一步研究表明蛋白酶预处理能进一步提高水解酸化相中氢气的产量至35.5 mL/g,反应pH最低为6.0。同时,蛋白酶预处理能够提高产甲烷相中甲烷产量,并且最大产量为251 mL/g,显著高于空白对照组。机理研究表明两段式消化分别为水解酸化相和产甲烷相提供最佳环境。     

Methanotrophic symbionts provide carbon for photosynthesis in peat bogs

Wetlands are the largest natural source of atmospheric methane, the second most important greenhouse gas. Methane flux to the atmosphere depends strongly on the climate; however, by far the largest part of the methane formed in wetland ecosystems is recycled and does not reach the atmosphere. The biogeochemical controls on the efficient oxidation of methane are still poorly understood. Here we show that submerged Sphagnum mosses, the dominant plants in some of these habitats, consume methane through symbiosis with partly endophytic methanotrophic bacteria, leading to highly effective in situ methane recycling. Molecular probes revealed the presence of the bacteria in the hyaline cells of the plant and on stem leaves. Incubation with (13)C-methane showed rapid in situ oxidation by these bacteria to carbon dioxide, which was subsequently fixed by Sphagnum, as shown by incorporation of (13)C-methane into plant sterols. In this way, methane acts as a significant (10-15%) carbon source for Sphagnum. The symbiosis explains both the efficient recycling of methane and the high organic carbon burial in these wetland ecosystems.     

(ADP-ribose)n participates in DNA excision repair

Chromatin proteins are covalently modified by at least five different processes; in no case has the precise physiological function been established. One of these post-synthetic, covalent modifications is effected by the enzyme poly(ADP-ribose) polymerase, which uses the coenzyme NAD+ to ADP-ribosylate chromatin proteins. The modification consists largely of mono(ADP-ribose), but long, homopolymer chains of (ADP-ribose) are also present. Various physiological functions have been suggested for (ADP-ribose)n. Here we demonstrate that one function of (ADP-ribose)n is to participate in the cellular recovery from DNA damage. Specific inhibitors of poly(ADP-ribose) polymerase prevent rejoining of DNA strand breaks caused by dimethyl sulphate and cytotoxicity is enhanced thereby. The rejoining of strand breaks is prevented also by nutritionally depleting the cells of NAD.     

硫酸钙晶须的表面改性研究

采用硼酸酯表面活性剂SBW-181对硫酸钙晶须进行了单因素条件改性试验,分别考察了改性剂用量、改性温度、改性时间和搅拌速率等工艺因素对硫酸钙晶须表面改性的影响.试验结果表明:硼酸酯表面活性剂SBW-181对硫酸钙晶须的改性效果较好,在最佳工艺条件下,改性产品的活化指数为0.996,接触角为103.4°.建立了硼酸酯表面活性剂SBW-181与硫酸钙晶须的作用模型.得出的结论对硫酸钙晶须表面改性的工业化生产具有重要的指导意义,并为硫酸钙晶须的应用研究奠定了理论基础.     

A hydrothermal origin for isotopically anomalous cap dolostone cements from south China

The release of methane into the atmosphere through destabilization of clathrates is a positive feedback mechanism capable of amplifying global warming trends that may have operated several times in the geological past. Such methane release is a hypothesized cause or amplifier for one of the most drastic global warming events in Earth history, the end of the Marinoan 'snowball Earth' ice age, ～635?Myr ago. A key piece of evidence supporting this hypothesis is the occurrence of exceptionally depleted carbon isotope signatures (δ(13)C(PDB) down to -48‰; ref. 8) in post-glacial cap dolostones (that is, dolostone overlying glacial deposits) from south China; these signatures have been interpreted as products of methane oxidation at the time of deposition. Here we show, on the basis of carbonate clumped isotope thermometry, (87)Sr/(86)Sr isotope ratios, trace element content and clay mineral evidence, that carbonates bearing the (13)C-depleted signatures crystallized more than 1.6?Myr after deposition of the cap dolostone. Our results indicate that highly (13)C-depleted carbonate cements grew from hydrothermal fluids and suggest that their carbon isotope signatures are a consequence of thermogenic methane oxidation at depth. This finding not only negates carbon isotope evidence for methane release during Marinoan deglaciation in south China, but also eliminates the only known occurrence of a Precambrian sedimentary carbonate with highly (13)C-depleted signatures related to methane oxidation in a seep environment. We propose that the capacity to form highly (13)C-depleted seep carbonates, through biogenic anaeorobic oxidation of methane using sulphate, was limited in the Precambrian period by low sulphate concentrations in sea water. As a consequence, although clathrate destabilization may or may not have had a role in the exit from the 'snowball' state, it would not have left extreme carbon isotope signals in cap dolostones.     

Crude-oil biodegradation via methanogenesis in subsurface petroleum reservoirs

Biodegradation of crude oil in subsurface petroleum reservoirs has adversely affected the majority of the world's oil, making recovery and refining of that oil more costly. The prevalent occurrence of biodegradation in shallow subsurface petroleum reservoirs has been attributed to aerobic bacterial hydrocarbon degradation stimulated by surface recharge of oxygen-bearing meteoric waters. This hypothesis is empirically supported by the likelihood of encountering biodegraded oils at higher levels of degradation in reservoirs near the surface. More recent findings, however, suggest that anaerobic degradation processes dominate subsurface sedimentary environments, despite slow reaction kinetics and uncertainty as to the actual degradation pathways occurring in oil reservoirs. Here we use laboratory experiments in microcosms monitoring the hydrocarbon composition of degraded oils and generated gases, together with the carbon isotopic compositions of gas and oil samples taken at wellheads and a Rayleigh isotope fractionation box model, to elucidate the probable mechanisms of hydrocarbon degradation in reservoirs. We find that crude-oil hydrocarbon degradation under methanogenic conditions in the laboratory mimics the characteristic sequential removal of compound classes seen in reservoir-degraded petroleum. The initial preferential removal of n-alkanes generates close to stoichiometric amounts of methane, principally by hydrogenotrophic methanogenesis. Our data imply a common methanogenic biodegradation mechanism in subsurface degraded oil reservoirs, resulting in consistent patterns of hydrocarbon alteration, and the common association of dry gas with severely degraded oils observed worldwide. Energy recovery from oilfields in the form of methane, based on accelerating natural methanogenic biodegradation, may offer a route to economic production of difficult-to-recover energy from oilfields.