Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the 'BIG' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel 'ANME-2c Sonora' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.

Subsurface petroleum reservoirs are an important component of the deep biosphere where indigenous microorganisms live under extreme conditions and in isolation from the Earth’s surface for millions of years. However, unlike the bulk of the deep biosphere, the petroleum reservoir deep biosphere is subject to extreme anthropogenic perturbation, with the introduction of new electron acceptors, donors and exogenous microbes during oil exploration and production. Despite the fundamental and practical significance of this perturbation, there has never been a systematic evaluation of the ecological changes that occur over the production lifetime of an active offshore petroleum production system. Analysis of the entire Halfdan oil field in the North Sea (32 producing wells in production for 1–15 years) using quantitative PCR, multigenic sequencing, comparative metagenomic and genomic bins reconstruction revealed systematic shifts in microbial community composition and metabolic potential, as well as changing ecological strategies in response to anthropogenic perturbation of the oil field ecosystem, related to length of time in production. The microbial communities were initially dominated by slow growing anaerobes such as members of the Thermotogales and Clostridiales adapted to living on hydrocarbons and complex refractory organic matter. However, as seawater and nitrate injection (used for secondary oil production) delivered oxidants, the microbial community composition progressively changed to fast growing opportunists such as members of the Deferribacteres, Delta-, Epsilon- and Gammaproteobacteria, with energetically more favorable metabolism (for example, nitrate reduction, H2S, sulfide and sulfur oxidation). This perturbation has profound consequences for understanding the microbial ecology of the system and is of considerable practical importance as it promotes detrimental processes such as reservoir souring and metal corrosion. These findings provide a new conceptual framework for understanding the petroleum reservoir biosphere and have consequences for developing strategies to manage microbiological problems in the oil industry.

e Offshore oil production facilities are frequently victims of internal piping corrosion, potentially leading to human and environmental risks and significant economic losses. Microbially influenced corrosion (MIC) is believed to be an important factor in this major problem for the petroleum industry. However, knowledge of the microbial communities and metabolic processes leading to corrosion is still limited. Therefore, the microbial communities from three anaerobic biofilms recovered from the inside of a steel pipe exhibiting high corrosion rates, iron oxide deposits, and substantial amounts of sulfur, which are characteristic of MIC, were analyzed in detail. Bacterial and archaeal community structures were investigated by automated ribosomal intergenic spacer analysis, multigenic (16S rRNA and functional genes) high-throughput Illumina MiSeq sequencing, and quantitative PCR analysis. The microbial community analysis indicated that bacteria, particularly Desulfovibrio species, dominated the biofilm microbial communities. However, other bacteria, such as Pelobacter, Pseudomonas, and Geotoga, as well as various methanogenic archaea, previously detected in oil facilities were also detected. The microbial taxa and functional genes identified suggested that the biofilm communities harbored the potential for a number of different but complementary metabolic processes and that MIC in oil facilities likely involves a range of microbial metabolisms such as sulfate, iron, and elemental sulfur reduction. Furthermore, extreme corrosion leading to leakage and exposure of the biofilms to the external environment modify the microbial community structure by promoting the growth of aerobic hydrocarbon-degrading organisms.

Permafrost thawing results in the formation of thermokarst lakes, which are biogeochemical hotspots in northern landscapes and strong emitters of greenhouse gasses to the atmosphere. Most studies of thermokarst lakes have been in summer, despite the predominance of winter and ice-cover over much of the year, and the microbial ecology of these waters under ice remains poorly understood. Here we first compared the summer versus winter microbiomes of a subarctic thermokarst lake using DNA- and RNA-based 16S rRNA amplicon sequencing and qPCR. We then applied comparative metagenomics and used genomic bin reconstruction to compare the two seasons for changes in potential metabolic functions in the thermokarst lake microbiome. In summer, the microbial community was dominated by Actinobacteria and Betaproteobacteria, with phototrophic and aerobic pathways consistent with the utilization of labile and photodegraded substrates. The microbial community was strikingly different in winter, with dominance of methanogens, Planctomycetes, Chloroflexi and Deltaproteobacteria, along with various taxa of the Patescibacteria/Candidate Phyla Radiation (Parcubacteria, Microgenomates, Omnitrophica, Aminicenantes). The latter group was underestimated or absent in the amplicon survey, but accounted for about a third of the metagenomic reads. The winter lineages were associated with multiple reductive metabolic processes, fermentations and pathways for the mobilization and degradation of complex organic matter, along with a strong potential for syntrophy or cross-feeding. The results imply that the summer community represents a transient stage of the annual cycle, and that carbon dioxide and methane production continue through the prolonged season of ice cover via a taxonomically distinct winter community and diverse mechanisms of permafrost carbon transformation.

dNext-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.

We describe an inexpensive, reliable, and easily executed improvement for the extraction of DNA from SterivexTM filter units, that involves the separation of the SterivexTM filter from its casing. Our study demonstrates that our modification of the original extraction protocol significantly increased DNA yields, with an average increase of 4.1‐fold more DNA than with the standard approach. A comparison of the diversity after Illumina MiSeq sequencing of bacterial communities extracted with both the standard approach and the proposed one indicated that our modified protocol has no or little impact on the results. This protocol provides a relatively straight forward means to achieve higher yields of DNA from the extraction of SterivexTM cartridges without altering the community composition and will likely be of interest to a wide range of scientists that use techniques based on the recovery of DNA from filters.

Bacteria are key players in biogeochemical cycles and control water quality in freshwater ecosystems. Nevertheless, little is known about the identity and ecology of riverine bacteria, especially during ice‐covered periods that are often mistakenly perceived as periods with negligible biological activities. Here, we analyzed in detail the effects of environmental and climatic conditions on freshwater bacterial community structure and diversity over a 2‐yr sampling campaign, targeting a seasonally ice‐covered river of the Quebec City (Canada) area, the Saint‐Charles River. Quantitative polymerase chain reaction and 16S ribosomal RNA gene high‐throughput sequencing demonstrated a strong seasonal cycle of the bacterial community composition with rapid successions of bacterial lineages, reflecting the harsh climatic condition of the region. During the summer, the bacterial community was dominated by typical freshwater microorganisms such as Limnohabitans, Sporichthyaceae hgcI/acI clade, and Pseudarcicella. In contrast, the results suggest that during the cold season, the low water temperatures, combined with other prevailing conditions such as reduced light availability and minimal particulate inputs from the catchment, created various environmental niches for potential methanotrophic Gammaproteobacteria, such as Crenothrix and Methylobacter, other Betaproteobacteria, such as Candidatus Methylopumilus, Candidatus Nitrotoga, and Rhodoferax, as well as Verrucomicrobia and Parcubacteria. The presence of these taxa in the winter suggests active carbon, iron, and nitrogen cycling under ice, whereas summer lineages are dormant or in a phase of reduced activity. These results increase our understanding of bacterial dynamic and potential metabolic processes occurring in seasonally ice‐covered inland waters, providing evidence that winter can be an especially important period for freshwater ecological processes.

Background The sulfur cycle encompasses a series of complex aerobic and anaerobic transformations of S-containing molecules and plays a fundamental role in cellular and ecosystem-level processes, influencing biological carbon transfers and other biogeochemical cycles. Despite their importance, the microbial communities and metabolic pathways involved in these transformations remain poorly understood, especially for inorganic sulfur compounds of intermediate oxidation states (thiosulfate, tetrathionate, sulfite, polysulfides). Isolated and highly stratified, the extreme geochemical and environmental features of meromictic ice-capped Lake A, in the Canadian High Arctic, provided an ideal model ecosystem to resolve the distribution and metabolism of aquatic sulfur cycling microorganisms along redox and salinity gradients. Results Applying complementary molecular approaches, we identified sharply contrasting microbial communities and metabolic potentials among the markedly distinct water layers of Lake A, with similarities to diverse fresh, brackish and saline water microbiomes. Sulfur cycling genes were abundant at all depths and covaried with bacterial abundance. Genes for oxidative processes occurred in samples from the oxic freshwater layers, reductive reactions in the anoxic and sulfidic bottom waters and genes for both transformations at the chemocline. Up to 154 different genomic bins with potential for sulfur transformation were recovered, revealing a panoply of taxonomically diverse microorganisms with complex metabolic pathways for biogeochemical sulfur reactions. Genes for the utilization of sulfur cycle intermediates were widespread throughout the water column, co-occurring with sulfate reduction or sulfide oxidation pathways. The genomic bin composition suggested that in addition to chemical oxidation, these intermediate sulfur compounds were likely produced by the predominant sulfur chemo- and photo-oxidisers at the chemocline and by diverse microbial degraders of organic sulfur molecules. Conclusions The Lake A microbial ecosystem provided an ideal opportunity to identify new features of the biogeochemical sulfur cycle. Our detailed metagenomic analyses across the broad physico-chemical gradients of this permanently stratified lake extend the known diversity of microorganisms involved in sulfur transformations over a wide range of environmental conditions. The results indicate that sulfur cycle intermediates and organic sulfur molecules are major sources of electron donors and acceptors for aquatic and sedimentary microbial communities in association with the classical sulfur cycle.