Methane is the most abundant hydrocarbon in the atmosphere, and it is an important greenhouse gas, which has so far contributed an estimated 20% of postindustrial global warming. A great deal of biogeochemical research has focused on the causes and effects of the variation in global fluxes of methane throughout earth's history, but the underlying microbial processes and their key agents remain poorly understood. This is a disturbing knowledge gap because 85% of the annual global methane production and about 60% of its consumption are based on microbial processes. Only three key functional groups of microorganisms of limited diversity regulate the fluxes of methane on earth, namely the aerobic methanotrophic bacteria, the methanogenic archaea, and their close relatives, the anaerobic methanotrophic archaea (ANME). The ANME represent special lines of descent within the Euryarchaeota and appear to gain energy exclusively from the anaerobic oxidation of methane (AOM), with sulfate as the final electron acceptor according to the net reaction: CH(4) + SO(42-) ---> HCO(3-) + HS(-) + H(2)O. This review summarizes what is known and unknown about AOM on earth and its key catalysts, the ANME clades and their bacterial partners.

Dense biological communities of large epifaunal taxa similar to those found along ridge crest vents at the East Pacific Rise were discovered in the abyssal Gulf of Mexico. These assemblages occur on a passive continental margin at the base of the Florida Escarpment, the interface between the relatively impermeable hemipelagic clays of the distal Mississippi Fan and the jointed Cretaceous limestone of the Florida Platform. The fauna apparently is nourished by sulfide rich hypersaline waters seeping out at near ambient temperatures onto the sea floor.

The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can be used to identify single microbial cells directly by fluorescence in situ hybridization. In this Review, we provide an update on the recent methodological improvements that have allowed more reliable quantification of microbial populations in situ in complex environmental samples, with a particular focus on the usefulness of group-specific probes in this era of ever-growing rRNA databases.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15 min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.Copyright © 2015 Elsevier GmbH. All rights reserved.

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.

Methane seeps are widespread seafloor ecosystems shaped by the emission of gas from seabed reservoirs. The microorganisms inhabiting methane seeps transform the chemical energy in methane to products that sustain rich benthic communities around the gas leaks. Despite the biogeochemical relevance of microbial methane removal at seeps, the global diversity and dispersion of seep microbiota remain unknown. Here we determined the microbial diversity and community structure of 23 globally distributed methane seeps and compared these to the microbial communities of 54 other seafloor ecosystems, including sulfate-methane transition zones, hydrothermal vents, coastal sediments, and deep-sea surface and subsurface sediments. We found that methane seep communities show moderate levels of microbial richness compared with other seafloor ecosystems and harbor distinct bacterial and archaeal taxa with cosmopolitan distribution and key biogeochemical functions. The high relative sequence abundance of ANME (anaerobic methanotrophic archaea), as well as aerobic Methylococcales, sulfate-reducing Desulfobacterales, and sulfide-oxidizing Thiotrichales, matches the most favorable microbial metabolisms at methane seeps in terms of substrate supply and distinguishes the seep microbiome from other seafloor microbiomes. The key functional taxa varied in relative sequence abundance between different seeps due to the environmental factors, sediment depth and seafloor temperature. The degree of endemism of the methane seep microbiome suggests a high local diversification in these heterogeneous but long-lived ecosystems. Our results indicate that the seep microbiome is structured according to metacommunity processes and that few cosmopolitan microbial taxa mediate the bulk of methane oxidation, with global relevance to methane emission in the ocean.

SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.

Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of (15)N(2) incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages.

Life in hypersaline environments is typically limited by bioenergetic constraints. Microbial activity at the thermodynamic edge, such as the anaerobic oxidation of methane (AOM) coupled to sulphate reduction (SR), is thus unlikely to thrive in these environments. In this study, carbon and sulphur cycling was investigated in the extremely hypersaline cold seep sediments of Mercator mud volcano. AOM activity was partially inhibited but still present at salinity levels of 292 g L(-1) (c. eightfold sea water concentration) with rates of 2.3 nmol cm(-3) day(-1) and was even detectable under saturated conditions. Methane and evaporite-derived sulphate comigrated in the ascending geofluids, which, in combination with a partial activity inhibition, resulted in AOM activity being spread over unusually wide depth intervals. Up to 79% of total cells in the AOM zone were identified by fluorescence in situ hybridization (FISH) as anaerobic methanotrophs of the ANME-1. Most ANME-1 cells formed monospecific chains without any attached partner. At all sites, AOM activity co-occurred with SR activity and sometimes significantly exceeded it. Possible causes of these unexpected results are discussed. This study demonstrates that in spite of a very low energy yield of AOM, microorganisms carrying this reaction can thrive in salinity up to halite saturation.© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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 N(5),N(10) -methylene-tetrahydromethanopterin reductase), heterodisulfide reductases and the F(420) H(2) :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.© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

ANME-1 archaea subsist on the very low energy of anaerobic oxidation of methane (AOM). Most marine sediments shift from net AOM in the sulfate methane transition zone (SMTZ) to methanogenesis in the methane zone (MZ) below it. In White Oak River estuarine sediments, ANME-1 comprised 99.5% of 16S rRNA genes from amplicons and 100% of 16S rRNA genes from metagenomes of the Methanomicrobia in the SMTZ and 99.9% and 98.3%, respectively, in the MZ. Each of the 16 ANME-1 OTUs (97% similarity) had peaks in the SMTZ that coincided with peaks of putative sulfate-reducing bacteria Desulfatiglans sp. and SEEP-SRB1. In the MZ, ANME-1, but none of the putative sulfate-reducing bacteria or cultured methanogens, increased with depth. Our meta-analysis of public data showed only ANME-1 expressed methanogenic genes during both net AOM and net methanogenesis in an enrichment culture. We conclude that ANME-1 perform AOM in the SMTZ and methanogenesis in the MZ of White Oak River sediments. This metabolic flexibility may expand habitable zones in extraterrestrial environments, since it enables greater energy yields in a fluctuating energetic landscape.© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.

Cold seeps are highly productive, fragmented marine ecosystems that form at the seafloor around hydrocarbon emission pathways. The products of microbial utilization of methane and other hydrocarbons fuel rich chemosynthetic communities at these sites, with much higher respiration rates compared with the surrounding deep-sea floor. Yet little is known as to the richness, composition and spatial scaling of bacterial communities of cold seeps compared with non-seep communities. Here we assessed the bacterial diversity across nine different cold seeps in the Eastern Mediterranean deep-sea and surrounding seafloor areas. Community similarity analyses were carried out based on automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and high-throughput 454 tag sequencing and were combined with in situ and ex situ geochemical analyses across spatial scales of a few tens of meters to hundreds of kilometers. Seep communities were dominated by Deltaproteobacteria, Epsilonproteobacteria and Gammaproteobacteria and shared, on average, 36% of bacterial types (ARISA OTUs (operational taxonomic units)) with communities from nearby non-seep deep-sea sediments. Bacterial communities of seeps were significantly different from those of non-seep sediments. Within cold seep regions on spatial scales of only tens to hundreds of meters, the bacterial communities differed considerably, sharing <50% of types at the ARISA OTU level. Their variations reflected differences in porewater sulfide concentrations from anaerobic degradation of hydrocarbons. This study shows that cold seep ecosystems contribute substantially to the microbial diversity of the deep-sea.

The anaerobic oxidation of methane (AOM) is a globally significant sink that regulates methane flux from sediments into the oceans and atmosphere. Here we examine mesophilic to thermophilic AOM in hydrothermal sediments recovered from the Middle Valley vent field, on the Juan de Fuca Ridge. Using continuous-flow sediment bioreactors and batch incubations, we characterized (i) the degree to which AOM contributes to net dissolved inorganic carbon flux, (ii) AOM and sulfate reduction (SR) rates as a function of temperature and (iii) the distribution and density of known anaerobic methanotrophs (ANMEs). In sediment bioreactors, inorganic carbon stable isotope mass balances results indicated that AOM accounted for between 16% and 86% of the inorganic carbon produced, underscoring the role of AOM in governing inorganic carbon flux from these sediments. At 90°C, AOM occurred in the absence of SR, demonstrating a striking decoupling of AOM from SR. An abundance of Fe(III)-bearing minerals resembling mixed valent Fe oxides, such as green rust, suggests the potential for a coupling of AOM to Fe(III) reduction in these metalliferous sediments. While SR bacteria were only observed in cooler temperature sediments, ANMEs allied to ANME-1 ribotypes, including a putative ANME-1c group, were found across all temperature regimes and represented a substantial proportion of the archaeal community. In concert, these results extend and reshape our understanding of the nature of high temperature methane biogeochemistry, providing insight into the physiology and ecology of thermophilic anaerobic methanotrophy and suggesting that AOM may play a central role in regulating biological dissolved inorganic carbon fluxes to the deep ocean from the organic-poor, metalliferous sediments of the global mid-ocean ridge hydrothermal vent system.© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

No microorganism capable of anaerobic growth on methane as the sole carbon source has yet been cultivated. Consequently, information about these microbes has been inferred from geochemical and microbiological observations of field samples. Stable isotope analysis of lipid biomarkers and rRNA gene surveys have implicated specific microbes in the anaerobic oxidation of methane (AOM). Here we use combined fluorescent in situ hybridization and secondary ion mass spectrometry analyses, to identify anaerobic methanotrophs in marine methane-seep sediments. The results provide direct evidence for the involvement of at least two distinct archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps. Although both archaeal groups often occurred in direct physical association with bacteria, they also were observed as monospecific aggregations and as single cells. The ANME-1 archaeal group more frequently existed in monospecific aggregations or as single filaments, apparently without a bacterial partner. Bacteria associated with both archaeal groups included, but were not limited to, close relatives of Desulfosarcina species. Isotopic analyses suggest that monospecific archaeal cells and cell aggregates were active in anaerobic methanotrophy, as were multispecies consortia. In total, the data indicate that the microbial species and biotic interactions mediating anaerobic methanotrophy are diverse and complex. The data also clearly show that highly structured ANME-2/Desulfosarcina consortia are not the sole entities responsible for AOM at marine methane seeps. Other microbial groups, including ANME-1 archaea, are capable of anaerobic methane consumption either as single cells, in monospecific aggregates, or in multispecies 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.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Marine gas and hydrocarbon seeps are hot spots of sulfate reduction which is fuelled by methane, other short-chain alkanes or a complex mixture of hydrocarbons. In this study, we investigated the global distribution and abundance of sulfate-reducing bacteria (SRB) in eight gas and hydrocarbon seeps by catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH). The majority of Deltaproteobacteria were assigned to specific SRB groups, i.e. 83 ± 14% at gas seeps and 61 ± 35% at hydrocarbon seeps, indicating that the probe set used was sufficient for classification of marine SRB. Statistical analysis showed that SRB abundance and distribution were significantly influenced by habitat type and sediment depth. Members of the Desulfosarcina/Desulfococcus (DSS) clade strongly dominated all sites. Our data indicated the presence of many diverse and highly specialized DSS species of low abundance rather than a single abundant subgroup. In addition, SEEP-SRB2, an uncultured deep-branching deltaproteobacterial group, was ubiquitously found in high abundances at all sites. SEEP-SRB2 members occurred either in a novel association with methanotrophic archaea in shell-type ANME-2/SEEP-SRB2 consortia, in association with ANME-1 archaea in Black Sea microbial mats or as single cells. Two other uncultured groups, SEEP-SRB3 and SEEP-SRB4, were preferentially detected in surface sediments from mud volcanoes.© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.