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.     

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.     

亚硝酸盐型甲烷厌氧氧化及其功能微生物研究进展

亚硝酸盐型甲烷厌氧氧化（nitrite-dependent anaerobic methane oxidation,N-DAMO）是耦合氮循环和碳循环的关键环节,主要是由亚硝酸盐型甲烷厌氧氧化菌（Candidatus Methylomirabilis oxyfera）介导完成,对于研究全球氮和碳元素的生物地球化学循环具有重要意义。本文首先总结了国内外N-DAMO的影响因素和在不同自然生态系统中的分布;然后阐述了N-DAMO菌的生理生化特性及其富集培养优化实验和检测技术,最后探讨了N-DAMO技术的应用现状。本综述不仅有助于揭示全球碳氮循环的耦合作用机制,也为N-DAMO反应耦合其他厌氧生物处理过程应用到污水的除碳脱氮上提供了理论依据。     

微生物介导的甲烷厌氧氧化过程及其影响因子研究进展

温室气体甲烷减排是全球变化领域的研究热点,甲烷厌氧氧化(anaerobic methane oxidation,AOM)过程是一个以前被忽视的甲烷汇,在调控全球甲烷收支平衡及减缓温室效应等方面扮演着十分重要的角色。AOM微生物以甲烷为唯一电子供体,与硫酸盐(SO42-)、亚硝酸盐(NO2-)/硝酸盐(NO3-)、金属离子(Fe3+、Mn4+、Cr6+)等结合完成氧化还原过程,该过程是耦合碳、氮、硫循环的关键环节。本文系统整理分析了不同AOM类型、发生机理、相关功能微生物类群(ANME-1、ANME-2、ANME-3、NC10、MBG-D)及影响AOM过程的关键调控因子的最新研究进展。结果发现,目前80%以上研究都集中在对最常见电子受体类型(SO42-/NO3-/NO2-/Fe3+/Mn4+)的AOM相关过程,而忽视了潜在的新型电子受体(AQDS/HAs O42-/Cr6+/ClO4-等)的耦合作用过程和相对应的微生物类型及作用机理。对未来AOM研究方向提出展望,以期为研究甲烷厌氧氧化菌在不同生态系统中的生态分布及减缓全球温室气体排放提供新的思路。     

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.     

Thermophilic anaerobic microbial communities that transform cellulose into methane (biogas)

The project is devoted to the screening of active anaerobic microbial communities which produce biogas via the decomposition of cellulose in thermophilic conditions (+55°C). Twenty-four samples were isolated from different natural and anthropogenic sources that contain desired microbial organisms. Growth medium was chosen to optimize the conditions for proliferation and selection of cellulolytic and methanogenic microorganisms. During the study of biogas formation dynamics, the most productive communities that remain active during five passages were selected. The biogas composition (methane, carbon dioxide, hydrogen) was investigated by gas chromatography. On average, the methane content in the gas mixture reached 60%. Microscopic studies revealed the presence of various morphotypes of microbial cells; their ratio varied during the stabilization of communities. The significance of the research on the transformation of cellulose into biogas is discussed.     

Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities

The anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing-bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethanesulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.     

Geochemical characteristics of the carbonate constructions formed during microbial oxidation of methane under anaerobic conditions

The aragonite constructions of the Black Sea are formed in a stable anaerobic zone and are a perfect object to study the natural mechanism of anaerobic methane oxidation. The most probable pathway of methane oxidation is its methanogen-mediated reaction with bicarbonates, dissolved in seawater, with the formation of water and acetate, which is then consumed by other components of the anaerobic community. Comparison of the delta 13C values of carbonate minerals and organic matter once more demonstrated that the formation of the organic matter of biomass is accompanied by intense fractionation of carbon isotopes, as a result of which the total organic matter of biomass acquires an extremely light isotopic composition, characterized by delta 13C values as low as -83.8@1000.     

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.     

Assimilation of carbon dioxide and oxidation of methane in various zones of the Rainbow hyperthermophilic field zones

Rates of carbon dioxide assimilation and methane oxidation were determined in various zones of the Rainbow Hydrothermal Field (36 degrees N) of the Mid-Atlantic Ridge. In the plume above the hydrothermal field, anomalously high methane content was recorded; the microbial population density (up to 10(5) cells/ml) was an order of magnitude higher than the background values; and the CO2 assimilation rate varied from 0.01 to 1.1 micrograms C/(1 day). Based on the data on CO2 assimilation, the production of organic carbon due to bacterial chemosynthesis in the plume was calculated to be 930 kg/day or 340 tons/year (about 29% of the organic carbon production in the photic zone). In the black smoke above active smokers, the microbial population density was as high as 10(6) cells/ml; the rate of CO2 assimilation made up 5-10 micrograms C/(1 day); the methane oxidation rate varied from 0.15 to 12.7 mu/(1 day); and the methane concentration ranged from 1.05 to 70.6 mu/l. In bottom sediments enriched with sulfides, the rate of CO2 assimilation was at least an order of magnitude higher than in oxidized metal-bearing sediments. At the base of an active construction site, whitish sediment was found, which was characterized by a methane high content (92 mu/dm3) and a high rate of oxidation (1.7 mu/(dm3 day)).     

An inorganic geochemical argument for coupled anaerobic oxidation of methane and iron reduction in marine sediments

Here, we present results from sediments collected in the Argentine Basin, a non‐steady state depositional marine system characterized by abundant oxidized iron within methane‐rich layers due to sediment reworking followed by rapid deposition. Our comprehensive inorganic data set shows that iron reduction in these sulfate and sulfide‐depleted sediments is best explained by a microbially mediated process—implicating anaerobic oxidation of methane coupled to iron reduction (Fe‐AOM) as the most likely major mechanism. Although important in many modern marine environments, iron‐driven AOM may not consume similar amounts of methane compared with sulfate‐dependent AOM. Nevertheless, it may have broad impact on the deep biosphere and dominate both iron and methane cycling in sulfate‐lean marine settings. Fe‐AOM might have been particularly relevant in the Archean ocean, >2.5 billion years ago, known for its production and accumulation of iron oxides (in iron formations) in a biosphere likely replete with methane but low in sulfate. Methane at that time was a critical greenhouse gas capable of sustaining a habitable climate under relatively low solar luminosity, and relationships to iron cycling may have impacted if not dominated methane loss from the biosphere.     

New perspectives on anaerobic methane oxidation

Anaerobic methane oxidation is a globally important but poorly understood process. Four lines of evidence have recently improved our understanding of this process. First, studies of recent marine sediments indicate that a consortium of methanogens and sulphate-reducing bacteria are responsible for anaerobic methane oxidation; a mechanism of 'reverse methanogenesis' was proposed, based on the principle of interspecies hydrogen transfer. Second, studies of known methanogens under low hydrogen and high methane conditions were unable to induce methane oxidation, indicating that 'reverse methanogenesis' is not a widespread process in methanogens. Third, lipid biomarker studies detected isotopically depleted archaeal and bacterial biomarkers from marine methane vents, and indicate that Archaea are the primary consumers of methane. Finally, phylogenetic studies indicate that only specific groups of Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms.     

Spatial separation of anaerobic ammonium oxidation and nitrite-dependent anaerobic methane oxidation in permeable riverbeds

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) play important roles in nitrogen and carbon cycling in fresh waters but we do not know how these two processes compete for their common electron acceptor, nitrite. Here, we investigated the spatial distribution of anammox and n-damo across a range of permeable riverbed sediments. Anammox activity and gene abundance were detected in both gravel and sandy riverbeds and showed a simple, common vertical distribution pattern, while the patterns in n-damo were more complex and n-damo activity was confined to the more reduced, sandy riverbeds. Anammox was most active in surficial sediment (0–2 cm), coincident with a peak in hzsA gene abundance and nitrite. In contrast, n-damo activity peaked deeper down (4–8 cm) in the sandy riverbeds, coincident with a peak in n-damo 16S rRNA gene abundance and higher methane concentration. Pore water nitrite, methane and oxygen were key factors influencing the distribution of these two processes in permeable riverbeds. Furthermore, both anammox- and n-damo- activity were positively correlated with denitrification activity, suggesting a role for denitrification in supplying both processes with nitrite. Our data reveal spatial separation between anammox and n-damo in permeable riverbed sediments that potentially avoids them competing for nitrite.     

Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms

CH4 oxidation capacities and microbial community structures developed in response to the presence of CH4 were investigated in two types of landfill cover soil microcosms, waste soil (fine material in stabilized waste) and clay soil. CH4 emission fluxes were lower in the waste soil cover over the course of the experiment. After exposure to CH4 flow for 120 days, the waste soil developed CH4 oxidation capacity from 0.53 to 11.25-13.48micromol CH4gd.w.(-1)h(-1), which was ten times higher than the clay soil. The topsoils of the two soil covers were observed dried and inhibited CH4 oxidation. The maximum CH4 oxidation rate occurred at the depth of 10-20cm in the waste soil cover (the middle layer), whereas it took place mainly at the depth of 20-30cm in the clay soil cover (the bottom layer). The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1omega8c and 18:1omega8c for type I and II methanotrophs, respectively, showed that type I methanotrophic bacteria predominated in the clay soil, while the type II methanotrophic bacteria were abundant in the waste soil, and the highest population in the middle layer. The results also indicated that a greater active methanotrophic community was developed in the waste soil relative to the clay soil.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

High‐pressure systems for gas‐phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Novel high‐pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high‐pressure continuous incubation system (HP‐CI system) and high‐pressure manifold‐incubation system (HP‐MI system), allow for batch, fed‐batch, and continuous gas‐phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long‐term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas‐enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high‐pressure systems, we present data from continuous and fed‐batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP‐CI system initial methane‐dependent sulfide production was enhanced 10‐ to 15‐fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L^?1 day^?1 was observed over 14 days. In fed‐batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L^?1 day^?1 when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L^?1 and 45 mmol L^?1. A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed‐batch incubation in the HP‐MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L^?1 day^?1 within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed‐batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods. Biotechnol. Bioeng. 2010; 105: 524–533. © 2009 Wiley Periodicals, Inc.     

Combined anaerobic ammonium and methane oxidation for nitrogen and methane removal

Anammox (anaerobic ammonium oxidation) is an environment-friendly and cost-efficient nitrogen-removal process currently applied to high-ammonium-loaded wastewaters such as anaerobic digester effluents. In these wastewaters, dissolved methane is also present and should be removed to prevent greenhouse gas emissions into the environment. Potentially, another recently discovered microbial pathway, n-damo (nitrite-dependent anaerobic methane oxidation) could be used for this purpose. In the present paper, we explore the feasibility of simultaneously removing methane and ammonium anaerobically, starting with granules from a full-scale anammox bioreactor. We describe the development of a co-culture of anammox and n-damo bacteria using a medium containing methane, ammonium and nitrite. The results are discussed in the context of other recent studies on the application of anaerobic methane- and ammonia-oxidizing bacteria for wastewater treatment.     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.