Bacterial Community Composition and Diversity in Methane Charged Sediments Revealed by Multitag Pyrosequencing

Bacterial diversity in sediments obtained along the Chilean margin from areas containing methane seeps, and a hydrate mound were explored by cloning and sequencing and multitag pyrosequencing (MTPS). These libraries were statistically compared to determine the robustness of taxonomic assignment derived from multiplexed pyrosequencing strategies targeting variable regions V1 and V2 of the small subunit rRNA gene for environmental studies. There was no statistical difference in the composition of the libraries, thus, MTPS was utilized to describe diversity in three geochemical zones in these environments. Unidentified Cyanobacteria isolates were abundant in the sulfate reduction zone (SRZ), Deltaproteobacteria were concentrated at the sulfate methane transition zone (SMTZ) and Chloroflexi/GNS dominated methanogenesis zone (MGZ). Although there was variation among specific groups, communities in the SRZ and MGZ did not differ significantly. However, the community dominated by Deltaproteobacteria differentiates the SMTZ from the other zones. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.     

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.     

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.     

Ocean currents shape the microbiome of Arctic marine sediments

Prokaryote communities were investigated on the seasonally stratified Alaska Beaufort Shelf (ABS). Water and sediment directly underlying water with origin in the Arctic, Pacific or Atlantic oceans were analyzed by pyrosequencing and length heterogeneity-PCR in conjunction with physicochemical and geographic distance data to determine what features structure ABS microbiomes. Distinct bacterial communities were evident in all water masses. Alphaproteobacteria explained similarity in Arctic surface water and Pacific derived water. Deltaproteobacteria were abundant in Atlantic origin water and drove similarity among samples. Most archaeal sequences in water were related to unclassified marine Euryarchaeota. Sediment communities influenced by Pacific and Atlantic water were distinct from each other and pelagic communities. Firmicutes and Chloroflexi were abundant in sediment, although their distribution varied in Atlantic and Pacific influenced sites. Thermoprotei dominated archaea in Pacific influenced sediments and Methanomicrobia dominated in methane-containing Atlantic influenced sediments. Length heterogeneity-PCR data from this study were analyzed with data from methane-containing sediments in other regions. Pacific influenced ABS sediments clustered with Pacific sites from New Zealand and Chilean coastal margins. Atlantic influenced ABS sediments formed another distinct cluster. Density and salinity were significant structuring features on pelagic communities. Porosity co-varied with benthic community structure across sites and methane did not. This study indicates that the origin of water overlying sediments shapes benthic communities locally and globally and that hydrography exerts greater influence on microbial community structure than the availability of methane.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

Stratified Community Responses to Methane and Sulfate Supplies in Mud Volcano Deposits: Insights from an In Vitro Experiment

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world''s oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30–55 cm below the sea floor (bsf); the second is at 165–205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry.     

Influence of electron acceptor availability and microbial community structure on sedimentary methane oxidation in a boreal estuary

Methane is produced microbially in vast quantities in sediments throughout the world’s oceans. However, anaerobic oxidation of methane (AOM) provides a near-quantitative sink for the produced methane and is primarily responsible for preventing methane emissions from the oceans to the atmosphere. AOM is a complex microbial process that involves several different microbial groups and metabolic pathways. The role of different electron acceptors in AOM has been studied for decades, yet large uncertainties remain, especially in terms of understanding the processes in natural settings. This study reports whole-core incubation methane oxidation rates along an estuarine gradient ranging from near fresh water to brackish conditions, and investigates the potential role of different electron acceptors in AOM. Microbial community structure involved in different methane processes is also studied in the same estuarine system using high throughput sequencing tools. Methane oxidation in the sediments was active in three distinct depth layers throughout the studied transect, with total oxidation rates increasing seawards. We find extensive evidence of non-sulphate AOM throughout the transect. The highest absolute AOM rates were observed below the sulphate-methane transition zone (SMTZ), strongly implicating the role of alternative electron acceptors (most likely iron and manganese oxides). However, oxidation rates were ultimately limited by methane availability. ANME-2a/b were the most abundant microbial phyla associated with AOM throughout the study sites, followed by ANME-2d in much lower abundances. Similarly to oxidation rates, highest abundances of microbial groups commonly associated with AOM were found well below the SMTZ, further reinforcing the importance of non-sulphate AOM in this system.

     

The archaeal lipidome in estuarine sediment dominated by members of the Miscellaneous Crenarchaeotal Group

The anoxic sediments of the White Oak River estuary comprise a distinctive sulfate–methane transition zone (SMTZ) and natural enrichment of the archaea affiliated with the Miscellaneous Crenarchaeotal Group (MCG). Archaeal biphytanes were generally depleted in ¹³C, with δ¹³C values being less than –35‰, indicative of production by active sedimentary archaeal populations. Multivariate analysis of the downcore distributions of 63 lipid biomarkers identified three major groups of lipids that were enriched in the surface, SMTZ or subsurface depths. Intact polar lipids with phosphatidylglycerol headgroups and glycerol dibiphytanyl glycerol tetraethers containing one, two or three cyclopentane rings were enriched at the base of the SMTZ and likely represent the accumulated product of a small but active ANME‐1 community. The recently identified butanetriol dibiphytanyl glycerol tetraethers (BDGT), which increased relatively to other lipids with depth, were correlated with the relative abundance of MCG in archaeal 16S rRNA clone libraries, and were ¹³C depleted throughout the depth profile, suggesting BDGT lipids as putative biomarkers of an MCG community that may either be autotrophic or feeding on ¹³C‐depleted organic substrates transported by porewater.     

Geomicrobial characterization of gas hydrate-bearing sediments along the mid-Chilean margin

Bacterial diversity in eight sediment cores from the mid-Chilean margin was studied using length heterogeneity (LH)-PCR, and described in relation to in situ geochemical conditions. DNA from the sulfate-methane transition (SMT) of three cores [one containing methane gas; two proximal to a gas hydrate mound (GHM)] was cloned and sequenced. Clones related to uncultured relatives of Desulfosarcina variabilis were found in all clone libraries and dominated one. Desulfosarcina variabilis related clones were similar to phylotypes observed at the SMT in association with anaerobic methane oxidation in the Eel River basin, Cascadia margin and the Gulf of Mexico. The LH-PCR amplicon associated with D. variabilis clones matched the amplicon that dominated most SMT samples, indicating environmental selection for D. variabilis relatives. Clones related to the Verrucomicrobia dominated the library for the methane gas-containing core. Uncultured Treponema relatives dominated the library for the core obtained on the edge of a GHM. Statistical analysis using geochemical data to describe variance in LH-PCR data revealed that stable carbon isotope ratios of dissolved inorganic carbon are the principal structuring factor on SMT communities. These data suggest that D. variabilis relatives are involved in anaerobic oxidation of methane at the SMT in Chilean margin sediments.     

Carbon cycling within the sulfate-methane-transition-zone in marine sediments from the Ulleung Basin

The significance of the various carbon cycling pathways in driving the sharp sulfate methane transition zone (SMTZ) observed at many locations along continental margins is still a topic of debate. Unraveling these processes is important to our understanding of the carbon cycle in general and to evaluate whether the location of this front can be used to infer present and past methane fluxes from deep reservoirs (e.g., gas hydrate). Here we report the pore water data from the second Ulleung Basin Gas Hydrate Expedition and on the results of a box model that balances solute fluxes among different carbon pools and satisfies the observed isotopic signatures. Our analysis identifies a secondary methanogenesis pathway within the SMTZ, whereby 25–35 % of the dissolved inorganic carbon (DIC) produced by the anaerobic oxidation of methane (AOM) is consumed by CO₂ reduction (CR). To balance this DIC consumption, a comparable rate of organic matter degradation becomes necessary, which in turn consumes a significant amount of sulfate. The fraction of sulfate consumed by AOM ranges from 70 to 90 %. Whereas a simple mass balance would suggest a one to one relationship between sulfate and methane fluxes; our isotopic considerations show that methane flux estimates based solely on sulfate data may be in error by as much as 30 %. Furthermore, the carbon cycling within the SMTZ is fueled by a significant contribution (10–40 %) of methane produced by CR just below the SMTZ. Therefore sulfate gradient cannot necessarily be used to infer methane contributions from gas hydrate reservoirs that may lay tens to hundreds of meters below the SMTZ.     

Fish growth enhances microbial sulfur cycling in aquaculture pond sediments

Microbial sulfate reduction and sulfur oxidation are vital processes to enhance organic matter degradation in sediments. However, the diversity and composition of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) and their environmental driving factors are still poorly understood in aquaculture ponds, which received mounting of organic matter. In this study, bacterial communities, SRB and SOB from sediments of aquaculture ponds with different sizes of grass carp (Ctenopharyngodon idellus) were analysed using high-throughput sequencing and quantitative real-time PCR (qPCR). The results indicated that microbial communities in aquaculture pond sediments of large juvenile fish showed the highest richness and abundance of SRB and SOB, potentially further enhancing microbial sulfur cycling. Specifically, SRB were dominated by Desulfobulbus and Desulfovibrio, whereas SOB were dominated by Dechloromonas and Leptothrix. Although large juvenile fish ponds had relatively lower concentrations of sulfur compounds (i.e. total sulfur, acid-volatile sulfide and elemental sulfur) than those of larval fish ponds, more abundant SRB and SOB were found in the large juvenile fish ponds. Further redundancy analysis (RDA) and linear regression indicated that sulfur compounds and sediment suspension are the major environmental factors shaping the abundance and community structure of SRB and SOB in aquaculture pond sediments. Findings of this study expand our current understanding of microbial driving sulfur cycling in aquaculture ecosystems and also provide novel insights for ecological and green aquaculture managements.     

Diversity of dimethylsulfide-degrading methanogens and sulfate-reducing bacteria in anoxic sediments along the Medway Estuary,UK

Methane is a powerful greenhouse gas but the microbial diversity mediating methylotrophic methanogenesis is not well-characterized. One overlooked route to methane is via the degradation of dimethylsulfide (DMS), an abundant organosulfur compound in the environment. Methanogens and sulfate-reducing bacteria (SRB) can degrade DMS in anoxic sediments depending on sulfate availability. However, we know little about the underlying microbial community and how sulfate availability affects DMS degradation in anoxic sediments. We studied DMS-dependent methane production along the salinity gradient of the Medway Estuary (UK) and characterized, for the first time, the DMS-degrading methanogens and SRB using cultivation-independent tools. DMS metabolism resulted in high methane yield (39%–42% of the theoretical methane yield) in anoxic sediments regardless of their sulfate content. Methanomethylovorans, Methanolobus and Methanococcoides were dominant methanogens in freshwater, brackish and marine incubations respectively, suggesting niche-partitioning of the methanogens likely driven by DMS amendment and sulfate concentrations. Adding DMS also led to significant changes in SRB composition and abundance in the sediments. Increases in the abundance of Sulfurimonas and SRB suggest cryptic sulfur cycling coupled to DMS degradation. Our study highlights a potentially important pathway to methane production in sediments with contrasting sulfate content and sheds light on the diversity of DMS degraders.     

Sulfate reduction and microbial processes of the methane cycle in the sediments of the Sevastopol Bay

The rates of microbial processes of sulfate reduction and of the methane cycle were measured in the bottom sediments of the Sevastopol basin, where seeps of gaseous methane have been previously found. Typically for marine environments, sulfate reduction played the major role in the terminal phase of decomposition of organic matter (OM) in reduced sediments of this area. The rate of this process depended on the amount of available OM. The rate of methanogenesis in the sediments increased with depth, peaking in the subsurface horizons, where decreased sulfate concentration was detected in the pore water. The highest rates of sulfate-dependent anaerobic methane oxidation were found close to the methane-sulfate transition zone as is typical of most investigated marine sediments. The data on the carbon isotopic composition of gaseous methane from the seeps and dissolved CH₄ from the bottom sediments, as well as on the rates of microbial methanogenesis and methane oxidation indicate that the activity of the methane seeps results from accumulation of biogenic methane in the cavities of the underlying geological structures with subsequent periodic release of methane bubbles into the water column.     

Microbial Community Structure Associated with Biogeochemical Processes in the Sulfate–Methane Transition Zone (SMTZ) of Gas-hydrate-bearing Sediment of the Ulleung Basin,East Sea

We investigated the biogeochemical constituents, microbial communities and functional genes (mcr and dsr) associated with anaerobic methane oxidation and sulfate reduction, and metabolic activities by sulfate reduction in the sulfate–methane transition zone (SMTZ) of gas-hydrate-bearing sediment of the Ulleung Basin in the East Sea. Maxima in the sulfate reduction rate (12.6 nmol cm^?3 d^?1), CO concentration (83 μM), and gene abundances of dsrA (9.1 × 10⁶ copies cm^?3) and mcrA (11.6 × 10⁶ copies cm^?3) occurred in the SMTZ. The peaks of CO consistently found in the SMTZ suggested that CO is an intermediate metabolic product related to methane oxidation. Candidate division JS1, the predominant bacterial group that comprised 59.0–63.7% of the 16S rRNA gene sequences, was recognized as an important organic carbon oxidizer. Both Marine Benthic Group D (MBGD) and Marine Benthic Group B (MBGB), which constituted 40.8–52.9 and 10.3–43.9% of the 16S rRNA gene sequences, respectively, were the dominant archaeal groups. Analysis of functional gene diversity revealed that anaerobic methanotroph-1-related phylotypes appeared to be the major CH₄ oxidizer, whereas Firmicutes-like group was a predominant sulfate reducer in the 0.8 mbsf in SMTZ with low SO₄^2? concentration. Overall results indicated that JS1 and two archaeal groups (MBGB and MBGD) seem to play a significant role in carbon and elements cycles in the gas-hydrate-bearing subsurface sediment of the Ulleung Basin.     

Microbial life in ridge flank crustal fluids

To determine the microbial community diversity within old oceanic crust, a novel sampling strategy was used to collect crustal fluids at Baby Bare Seamount, a 3.5 Ma old outcrop located in the north-east Pacific Ocean on the eastern flank of the Juan de Fuca Ridge. Stainless steel probes were driven directly into the igneous ocean crust to obtain samples of ridge flank crustal fluids. Genetic signatures and enrichment cultures of microorganisms demonstrate that these crustal fluids host a microbial community composed of species indigenous to the subseafloor, including anaerobic thermophiles, and species from other deep-sea habitats, such as seawater and sediments. Evidence using molecular techniques indicates the presence of a relatively small but active microbial population, dominated by bacteria. The microbial community diversity found in the crustal fluids may indicate habitat variability in old oceanic crust, with inputs of nutrients from seawater, sediment pore-water fluids and possibly hydrothermal sources. This report further supports the presence of an indigenous microbial community in ridge flank crustal fluids and advances our understanding of the potential physiological and phylogenetic diversity of this community.     

Bacterial diversity in organically-enriched fish farm sediments

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.     

Microbiological and isotopic-geochemical investigations of meromictic lakes in Khakasia in winter

Microbiological and isotopic-geochemical investigations of the brackish meromictic lakes Shira and Shunet were performed in the steppe region of Khakasia in winter. Measurements made with a submersed sensor demonstrated that one-meter ice transmits light in a quantity sufficient for oxygenic and anoxygenic photosynthesis. As in the summer season, in the community of phototrophic bacteria found in Lake Shira, the purple sulfur bacteria Amoebobacter purpureus dominated, whereas, in Lake Shunet, the green sulfur bacteria Pelodictyon luteolum were predominant. Photosynthetic production, measured using the radioisotopic method, was several times lower than that in summer. The rates of sulfate reduction and production and oxidation of methane in the water column and bottom sediments were also lower than those recorded in summer. The process of anaerobic methane oxidation in the sediments was an exception, being more intense in winter than in summer. The data from radioisotopic measurements of the rates of microbial processes correlate well with the results of determination of the isotopic composition of organic and mineral carbon (delta13C) and hydrogen sulfide and sulfate (delta34S) and suggest considerable seasonal variations in the activity of the microbial community in the water bodies investigated.     

海水富营养化对海洋细菌影响的研究进展

综述了海水富营养化对海洋细菌影响的研究进展。随着海水富营养化程度的增加,海洋细菌数量或生物量增加;反硝化细菌、大肠菌群尤其是厌氧性的硫酸盐还原菌和产甲烷菌等典型细菌生理群数量增加;浮游细菌群落结构随富营养化递增趋于简单,物种多样性降低;富营养化也明显导致细菌群落正常功能活性的紊乱。海水富营养化对细菌群落的结构和功能有着深远的影响。