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New detections of thermophiles in psychrobiotic (i.e., bearing cold-tolerant life forms) marine and terrestrial habitats including Arctic marine sediments, Antarctic accretion ice, permafrost, and elsewhere are continually being reported. These microorganisms present great opportunities for microbial ecologists to examine biogeographical processes for spore-formers and non-spore-formers alike, including dispersal histories connecting warm and cold biospheres. In this review, we examine different examples of thermophiles in cryobiotic locations, and highlight exploration of thermophiles at cold temperatures under laboratory conditions. The survival of thermophiles in psychrobiotic environments provokes novel considerations of physiological and molecular mechanisms underlying natural cryopreservation of microorganisms. Cultures of thermophiles maintained at low temperature may serve as a non-sporulating laboratory model for further exploration of metabolic potential of thermophiles at psychrobiotic temperatures, as well as for elucidating molecular mechanisms behind natural preservation and adaptation to psychrobiotic environments. These investigations are highly relevant for the search for life on other cold and icy planets in the Solar System, such as Mars, Europa and Enceladus.

Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments. 13C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to 13CO2. SIP probing of DNA revealed diverse ‘Candidatus Izemoplasma’, Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived 13C-carbon. NanoSIMS confirmed incorporation of 13C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the 13C-labelled taxa all encoded enzymatic repertoires for catabolism of DNA or subcomponents of DNA. Comparative genomics indicated that diverse ‘Candidatus Izemoplasmatales’ (former Tenericutes) are exceptional because they encode multiple (up to five) predicted extracellular nucleases and are probably specialized DNA-degraders. Analyses of additional sediment metagenomes revealed extracellular nuclease genes are prevalent among Bacteroidota at diverse sites. Together, our results reveal the identities and functional properties of microorganisms that may contribute to the key ecosystem function of degrading and recycling DNA in the seabed., Using microcosms, stable isotope probing, genome-resolved metagenomics and NanoSIMS, the authors identify diverse bacterial taxa that can degrade extracellular DNA in marine sediments, including ‘Candidatus Izemoplasma’, which encode numerous extracellular nucleases.

In marine ecosystems, viruses exert control on the composition and metabolism of microbial communities, thus influencing overall biogeochemical cycling. Deep sea sediments associated with cold seeps are known to host taxonomically diverse microbial communities, but little is known about viruses infecting these microorganisms. Here, we probed metagenomes from seven geographically diverse cold seeps across global oceans, to assess viral diversity, virus-host interaction, and virus-encoded auxiliary metabolic genes (AMGs). Gene-sharing network comparisons with viruses inhabiting other ecosystems reveal that cold seep sediments harbour considerable unexplored viral diversity. Most cold seep viruses display high degrees of endemism with seep fluid flux being one of the main drivers of viral community composition. In silico predictions linked 14.2% of the viruses to microbial host populations, with many belonging to poorly understood candidate bacterial and archaeal phyla. Lysis was predicted to be a predominant viral lifestyle based on lineage-specific virus/host abundance ratios. Metabolic predictions of prokaryotic host genomes and viral AMGs suggest that viruses influence microbial hydrocarbon biodegradation at cold seeps, as well as other carbon, sulfur and nitrogen cycling via virus-induced mortality and/or metabolic augmentation. Overall, these findings reveal the global diversity and biogeography of cold seep viruses and indicate how viruses may manipulate seep microbial ecology and biogeochemistry.

Summary Endospore‐forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non‐reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6‐pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC‐fluorescence method for DPA quantification using a single 10‐min run with pre‐column Tb3+ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore‐specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA‐based endospore quantification depends on accurate spore‐specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.

Significance The marine subsurface is one of the largest habitats on Earth composed exclusively of microorganisms and harboring on the order of 1029 microbial cells. It is unclear if deep subsurface life impacts overlying seafloor diversity and biogeochemical cycling in the deep ocean. We analyzed the microbial communities of 172 seafloor surface sediment samples, including gas and oil seeps as well as sediments not subject to upward fluid flow. A strong correlation between typical subsurface clades and active geofluid seepage suggests that subsurface life is injected into the deep ocean floor at hydrocarbon seeps, a globally widespread hydrogeological phenomenon. This supply of subsurface-derived microbial populations, biomass, and metabolic potential thus increases biodiversity and impacts carbon cycling in the deep ocean., Marine cold seeps transmit fluids between the subseafloor and seafloor biospheres through upward migration of hydrocarbons that originate in deep sediment layers. It remains unclear how geofluids influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. Here we analyzed 172 marine surficial sediments from the deep-water Eastern Gulf of Mexico to assess whether hydrocarbon fluid migration is a mechanism for upward microbial dispersal. While 132 of these sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well-known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the uncultivated bacterial phyla Atribacteria and Aminicenantes and the archaeal order Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum, in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of nonrespiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. These results point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.

Marine sediments can contain large amounts of alkanes and methylated aromatic hydrocarbons that are introduced by natural processes or anthropogenic activities. These compounds can be biodegraded by anaerobic microorganisms via enzymatic addition of fumarate. However, the identity and ecological roles of a significant fraction of hydrocarbon degraders containing fumarate-adding enzymes (FAE) in various marine sediments remains unknown. By combining phylogenetic reconstructions, protein homolog modelling, and functional profiling of publicly available metagenomes and genomes, 61 draft bacterial and archaeal genomes encoding anaerobic hydrocarbon degradation via fumarate addition were obtained. Besides Desulfobacterota (previously known as Deltaproteobacteria) that are well-known to catalyze these reactions, Chloroflexi are dominant FAE-encoding bacteria in hydrocarbon-impacted sediments, potentially coupling sulfate reduction or fermentation to anaerobic hydrocarbon degradation. Among Archaea, besides Archaeoglobi previously shown to have this capability, genomes of Heimdallarchaeota, Lokiarchaeota, Thorarchaeota and Thermoplasmata also suggest fermentative hydrocarbon degradation using archaea-type FAE. These bacterial and archaeal hydrocarbon degraders occur in a wide range of marine sediments, including high abundances of FAE-encoding Asgard archaea associated with natural seeps and subseafloor ecosystems. Our results expand the knowledge of diverse archaeal and bacterial lineages engaged in anaerobic degradation of alkanes and methylated aromatic hydrocarbons.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ. These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer-term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80-90°C for 6, 12 or 463 days. Sediments were then cooled by 10-40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore-forming Clostridia. Different Desulfofundulus, Desulfohalotomaculum, Desulfallas, Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat-resistant endospore-forming sulfate-reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S0 ) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY1 Z1 AB and soxCDY2 Z2 H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY2 Z2 H cluster and not soxXY1 Z1 AB. Despite the absence of the soxXY1 Z1 AB cluster, strain CVO oxidized S0 to thiosulfate and sulfate, demonstrating that soxCDY2 Z2 H genes alone are sufficient for S0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY2 Z2 H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant.

Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments.

Dormant endospores of anaerobic, thermophilic bacteria found in cold marine sediments offer a useful model for studying microbial biogeography, dispersal, and survival. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to be isolated and studied independently of other factors such as environmental selection. To study the resilience of thermospores to conditions relevant for survival in extreme cold conditions, their viability following different freezing treatments was tested. Marine sediment was frozen at either -80 degrees C or -20 degrees C for 10 days prior to pasteurization and incubation at +50 degrees C for 21 days to assess thermospore viability. Sulfate reduction commenced at +50 degrees C following both freezing pretreatments indicating persistence of thermophilic endospores of sulfate-reducing bacteria. The onset of sulfate reduction at +50 degrees C was delayed in -80 degrees C pretreated microcosms, which exhibited more variability between triplicates, compared to -20 degrees C pretreated microcosms and parallel controls that were not frozen in advance. Microbial communities were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative sequence abundance of thermophilic endospore-forming Firmicutes in all microcosms. Different freezing pretreatments (-80 degrees C and -20 degrees C) did not appreciably influence the shift in overall bacterial community composition that occurred during the +50 degrees C incubations. Communities that had been frozen prior to +50 degrees C incubation showed an increase in the relative sequence abundance of operational taxonomic units (OTUs) affiliated with the class Bacilli, relative to unfrozen controls. These results show that freezing impacts but does not obliterate thermospore populations and their ability to germinate and grow under appropriate conditions. Indeed the majority of the thermospore OTUs detected in this study (21 of 22) could be observed following one or both freezing treatments. These results are important for assessing thermospore viability in frozen samples and following cold exposure such as the very low temperatures that would be encountered during panspermia.

Selection by the local, contemporary environment plays a prominent role in shaping the biogeography of microbes. However, the importance of historical factors in microbial biogeography is more debatable. Historical factors include past ecological and evolutionary circumstances that may have influenced present-day microbial diversity, such as dispersal and past environmental conditions. Diverse thermophilic sulfate-reducing Desulfotomaculum are present as dormant endospores in marine sediments worldwide where temperatures are too low to support their growth. Therefore, they are dispersed to here from elsewhere, presumably a hot, anoxic habitat. While dispersal through ocean currents must influence their distribution in cold marine sediments, it is not clear whether even earlier historical factors, related to the source habitat where these organisms were once active, also have an effect. We investigated whether these historical factors may have influenced the diversity and distribution of thermophilic endospores by comparing their diversity in 10 Arctic fjord surface sediments. Although community composition varied spatially, clear biogeographic patterns were only evident at a high level of taxonomic resolution (>97% sequence similarity of the 16S rRNA gene) achieved with oligotyping. In particular, the diversity and distribution of oligotypes differed for the two most prominent OTUs (defined using a standard 97% similarity cutoff). One OTU was dominated by a single ubiquitous oligotype, while the other OTU consisted of ten more spatially localized oligotypes that decreased in compositional similarity with geographic distance. These patterns are consistent with differences in historical factors that occurred when and where the taxa were once active, prior to sporulation. Further, the influence of history on biogeographic patterns was only revealed by analyzing microdiversity within OTUs, suggesting that populations within standard OTU-level groupings do not necessarily share a common ecological and evolutionary history.

Souring is the undesirable production of hydrogen sulfide (H2S) in oil reservoirs by sulfate-reducing bacteria (SRB). Souring is a common problem during secondary oil recovery via water flooding, especially when seawater with its high sulfate concentration is introduced. Nitrate injection into these oil reservoirs can prevent and remediate souring by stimulating nitrate-reducing bacteria (NRB). Two conceptually different mechanisms for NRB-facilitated souring control have been proposed: nitrate-sulfate competition for electron donors (oil-derived organics or H2) and nitrate driven sulfide oxidation. Thermodynamics can facilitate predictions about which nitrate-driven mechanism is most likely to occur in different scenarios. From a thermodynamic perspective the question "Which reaction yields more energy, nitrate driven oxidation of sulfide or nitrate driven oxidation of organic compounds?" can be rephrased as: "Is acetate driven sulfate reduction to sulfide exergonic or endergonic?" Our analysis indicates that under conditions encountered in oil fields, sulfate driven oxidation of acetate (or other SRB organic electron donors) is always more favorable than sulfide oxidation to sulfate. That predicts that organotrophic NRB that oxidize acetate would outcompete lithotrophic NRB that oxidize sulfide. However, sulfide oxidation to elemental sulfur is different. At low acetate HS- oxidation is more favorable than acetate oxidation. Incomplete oxidation of sulfide to S0 is likely to occur when nitrate levels are low, and is favored by low temperatures; conditions that can be encountered at oil field above-ground facilities where intermediate sulfur compounds like S0 may cause corrosion. These findings have implications for reservoir management strategies and for assessing the success and progress of nitrate-based souring control strategies and the attendant risks of corrosion associated with souring and nitrate injection.

Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to normal expectation that PCR efficiency might be lower in environmental DNA, due to the presence of PCR inhibitors, we report the counterintuitive observation that PCR efficiency of pure standards can be lower than for environmental DNA. This can lead to overestimation of gene abundances if not corrected. SYBR green-based qPCR assays of 16S rRNA genes targeting Bacteria, Syntrophus and Smithella spp., Marinobacter spp., Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae in samples from methanogenic crude oil biodegradation enrichments were tested. In five out of the six assays, PCR efficiency was lower with pure standards than with environmental DNA samples. We developed a solution to this problem based on amending pure clone standards with a background of non-target environmental 16S rRNA genes which significantly improved PCR efficiency of standards in the qPCR assays that exhibited this phenomenon. Overall this method of qPCR standard preparation achieved a more reliable and robust quantification of 16S rRNA genes. We believe this may be a potentially common issue in microbial ecology that often goes unreported, as intuitively one would not expect standards to have poorer PCR efficiency than samples.

Terrestrial mud volcanoes (TMVs) represent geochemically diverse habitats with varying sulfur sources and yet sulfur cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in four TMVs in Azerbaijan. A combination of geochemical analyses, biological rate measurements and molecular diversity surveys (targeting metabolic genes aprA and dsrA and SSU ribosomal RNA) supported the presence of active sulfur-oxidizing and sulfate-reducing guilds in all four TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. The TMVs varied in potential sulfate reduction rates (SRR) by up to four orders of magnitude with highest SRR observed in sediments where in situ sulfate concentrations were highest. Maximum temperatures at which SRR were measured was 60°C in two TMVs. Corresponding with these trends in SRR, members of the potentially thermophilic, spore-forming, Desulfotomaculum were detected in these TMVs by targeted 16S rRNA analysis. Additional sulfate-reducing bacterial lineages included members of the Desulfobacteraceae and Desulfobulbaceae detected by aprA and dsrA analyses and likely contributing to the mesophilic SRR measured. Phylotypes affiliated with sulfide-oxidizing Gamma- and Betaproteobacteria were abundant in aprA libraries from low sulfate TMVs, while the highest sulfate TMV harboured 16S rRNA phylotypes associated with sulfur-oxidizing Epsilonproteobacteria. Altogether, the biogeochemical and microbiological data indicate these unique terrestrial habitats support diverse active sulfur-cycling microorganisms reflecting the in situ geochemical environment.

Libraries of 16S rRNA genes cloned from methanogenic oil degrading microcosms amended with North Sea crude oil and inoculated with estuarine sediment indicated that bacteria from the genera Smithella (Deltaproteobacteria, Syntrophaceace) and Marinobacter sp. (Gammaproteobacteria) were enriched during degradation. Growth yields and doubling times (36 days for both Smithella and Marinobacter) were determined using qPCR and quantitative data on alkanes, which were the predominant hydrocarbons degraded. The growth yield of the Smithella sp. [0.020 g(cell-C)/g(alkane-C)], assuming it utilized all alkanes removed was consistent with yields of bacteria that degrade hydrocarbons and other organic compounds in methanogenic consortia. Over 450 days of incubation predominance and exponential growth of Smithella was coincident with alkane removal and exponential accumulation of methane. This growth is consistent with Smithella's occurrence in near surface anoxic hydrocarbon degrading systems and their complete oxidation of crude oil alkanes to acetate and/or hydrogen in syntrophic partnership with methanogens in such systems. The calculated growth yield of the Marinobacter sp., assuming it grew on alkanes, was [0.0005 g(cell-C)/g(alkane-C)] suggesting that it played a minor role in alkane degradation. The dominant methanogens were hydrogenotrophs (Methanocalculus spp. from the Methanomicrobiales). Enrichment of hydrogen-oxidizing methanogens relative to acetoclastic methanogens was consistent with syntrophic acetate oxidation measured in methanogenic crude oil degrading enrichment cultures. qPCR of the Methanomicrobiales indicated growth characteristics consistent with measured rates of methane production and growth in partnership with Smithella.

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO2-reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense – an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.

Summary Marine sediments harbour diverse populations of dormant thermophilic bacterial spores that become active in sediment incubation experiments at much higher than in situ temperature. This response was investigated in the presence of natural complex organic matter in sediments of two Arctic fjords, as well as with the addition of freeze-dried Spirulina or individual high-molecular-weight polysaccharides. During 50°C incubation experiments, Arctic thermo- philes catalysed extensive mineralization of the organic matter via extracellular enzymatic hydrolysis, fermentation and sulfate reduction. This high temperature-induced food chain mirrors sediment microbial processes occurring at cold in situ tem- peratures (near 0°C), yet it is catalysed by a com- pletely different set of microorganisms. Using sulfate reduction rates (SRR) as a proxy for organic matter mineralization showed that differences in organic matter reactivity determined the extent of the thermo- philic response. Fjord sediments with higher in situ SRR also supported higher SRR at 50°C. Amendment with Spirulina significantly increased volatile fatty acids production and SRR relative to unamended sediment in 50°C incubations. Spirulina amendment also revealed temporally distinct sulfate reduction phases, consistent with 16S rRNA clone library detec- tion of multiple thermophilic Desulfotomaculum spp. enriched at 50°C. Incubations with four different fluo- rescently labelled polysaccharides at 4°C and 50°C showed that the thermophilic population in Arctic sediments produce a different suite of polymer- hydrolysing enzymes than those used in situ by the cold-adapted microbial community. Over time, dormant marine microorganisms like these are buried in marine sediments and might eventually encounter warmer conditions that favour their activation. Dis- tinct enzymatic capacities for organic polymer degra- dation could allow specific heterotrophic populations like these to play a role in sustaining microbial metabolism in the deep, warm, marine biosphere.

Temperature has a fundamental impact on the metabolic rates of microorganisms and strongly influences microbial ecology and biogeochemical cycling in the environment. In this study, we examined the catabolic temperature response of natural communities of sulfate-reducing microorganisms (SRM) in polar, temperate and tropical marine sediments. In short-term sediment incubation experiments with (35)S-sulfate, we demonstrated how the cardinal temperatures for sulfate reduction correlate with mean annual sediment temperatures, indicating specific thermal adaptations of the dominant SRM in each of the investigated ecosystems. The community structure of putative SRM in the sediments, as revealed by pyrosequencing of bacterial 16S rRNA gene amplicons and phylogenetic assignment to known SRM taxa, consistently correlated with in situ temperatures, but not with sediment organic carbon concentrations or C:N ratios of organic matter. Additionally, several species-level SRM phylotypes of the class Deltaproteobacteria tended to co-occur at sites with similar mean annual temperatures, regardless of geographic distance. The observed temperature adaptations of SRM imply that environmental temperature is a major controlling variable for physiological selection and ecological and evolutionary differentiation of microbial communities.

It is a challenge to quantitatively distinguish active from dormant microbial populations in environmental samples. Here we present an approach for estimating the abundance of dormant sulfate-reducing bacteria (SRB), present as viable endospores in environmental samples. This is achieved by inducing endospores to germinate and grow exponentially. We demonstrate this approach for thermophilic SRB in temperate sediment from Aarhus Bay, Denmark. The approach is based on measuring bulk sulfate reduction rates in pasteurized sediment and calculating associated cell-specific sulfate reduction rates based on average values for SRB growth yield and cell size known from exponentially growing pure cultures. The method presented is a faster bioassay than most probable number enumerations and has the potential to distinguish between slow- and fast-growing SRB populations in the same sample. This bioassay is attractive given the challenges posed by endospores with respect to cell permeabilization and lysis, which are prerequisite in quantitative microscopy- and nucleic acid extraction-based strategies. These molecular approaches additionally rely on designing target-appropriate oligonucleotide probes, whereas the method presented here considers the trait of interest more broadly. This strategy thus presents a useful complement to other methods in ecological investigations of microbial biogeography and for specific industrial applications such as reservoir souring and corrosion risk assessments in the oil and gas sector.

The High Arctic is dominated by polar desert habitats whose microbial communities are poorly understood. In this study, we used next generation sequencing to describe the α- and β-diversity of microbial communities in polar desert soils from the Kongsfjorden region of Svalbard. Ten phyla dominated the soils and accounted for 95% of all sequences, with the Proteobacteria, Actinobacteria, and Chloroflexi being the major lineages. In contrast to previous investigations of Arctic soils, relative Acidobacterial abundances were found to be very low as were the Archaea throughout the Kongsfjorden polar desert landscape. Lower Acidobacterial abundances were attributed to characteristic circumneutral soil pHs in this region, which has resulted from the weathering of underlying carbonate bedrock. In addition, we compared previously measured geochemical conditions as possible controls on soil microbial communities. Phosphorus, pH, nitrogen, and calcium levels all significantly correlated with β-diversity, indicating landscape-scale lithological control of available nutrients, which in turn, significantly influenced soil community composition. In addition, soil phosphorus and pH significantly correlated with α-diversity, particularly with the Shannon diversity and Chao 1 richness indices.

International audience; Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 1C) maximum in situ temperatures (B23 1C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 1C and at 60 1C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii T (B96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii T and D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 1C for 15 min; D. kuznetsovii, 135 1C for 15 min, maximum of 154 1C reached) and high-temperature conditions in an oil bath (C1A60, 1301 for 30 min, D. kuznetsovii 140 1C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (B30 Â higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.

The effects of trichloroethylene (TCE) on microbial community composition were analyzed by reverse sample genome probing. Soil enrichments were incubated in dessicators containing an organic phase of either 1 or 10% (w/w) toluene in vacuum pump oil, delivering constant equilibrium aqueous concentrations of 16 and 143 mg/l, respectively. Increasing the equilibrium aqueous concentration of TCE from 0 to 10 mg/l led to shifts in community composition at 16, but not at 143 mg/l of toluene. In closed system co-degradation studies, TCE at an aqueous concentration of 1 mg/l was effectively degraded by toluene-degrading soil enrichments once the aqueous toluene concentration dropped below 25 mg/l. Little TCE degradation was observed at higher toluene concentrations (50-250 mg/l). The results indicate that TCE changes microbial community composition under conditions where it is being actively metabolized.

Patterns of microbial biogeography result from a combination of dispersal, speciation and extinction, yet individual contributions exerted by each of these mechanisms are difficult to isolate and distinguish. The influx of endospores of thermophilic microorganisms to cold marine sediments offers a natural model for investigating passive dispersal in the ocean. We investigated the activity, diversity and abundance of thermophilic endospore-forming sulfate-reducing bacteria (SRB) in Aarhus Bay by incubating pasteurized sediment between 28 and 85°C, and by subsequent molecular diversity analyses of 16S rRNA and of the dissimilatory (bi)sulfite reductase (dsrAB) genes within the endospore-forming SRB genus Desulfotomaculum. The thermophilic Desulfotomaculum community in Aarhus Bay sediments consisted of at least 23 species-level 16S rRNA sequence phylotypes. In two cases, pairs of identical 16S rRNA and dsrAB sequences in Arctic surface sediment 3000km away showed that the same phylotypes are present in both locations. Radiotracer-enhanced most probable number analysis revealed that the abundance of endospores of thermophilic SRB in Aarhus Bay sediment was ca. 104 per cm3 at the surface and decreased exponentially to 100 per cm3 at 6.5m depth, corresponding to 4500 years of sediment age. Thus, a half-life of ca. 300 years was estimated for the thermophilic SRB endospores deposited in Aarhus Bay sediments. These endospores were similarly detected in the overlying water column, indicative of passive dispersal in water masses preceding sedimentation. The sources of these thermophiles remain enigmatic, but at least one source may be common to both Aarhus Bay and Arctic sediments.

Background Mangrove forests are coastal wetlands that provide vital ecosystem services and serve as barriers against natural disasters like tsunamis, hurricanes and tropical storms. Mangroves harbour a large diversity of organisms, including microorganisms with important roles in nutrient cycling and availability. Due to tidal influence, mangroves are sites where crude oil from spills farther away can accumulate. The relationship between mangrove bacterial diversity and oil degradation in mangrove sediments remains poorly understood. Results Mangrove sediment was sampled from 0–5, 15–20 and 35–40 cm depth intervals from the Suruí River mangrove (Rio de Janeiro, Brazil), which has a history of oil contamination. DGGE fingerprinting for bamA, dsr and 16S rRNA encoding fragment genes, and qPCR analysis using dsr and 16S rRNA gene fragment revealed differences with sediment depth. Conclusions Analysis of bacterial 16S rRNA gene diversity revealed changes with depth. DGGE for bamA and dsr genes shows that the anaerobic hydrocarbon-degrading community profile also changed between 5 and 15 cm depth, and is similar in the two deeper sediments, indicating that below 15 cm the anaerobic hydrocarbon-degrading community appears to be well established and homogeneous in this mangrove sediment. qPCR analysis revealed differences with sediment depth, with general bacterial abundance in the top layer (0–5 cm) being greater than in both deeper sediment layers (15–20 and 35–40 cm), which were similar to each other.

Insight into the effects of repeated freezing and thawing on microbial processes in sediments and soils is important for understanding sediment carbon cycling at high latitudes acutely affected by global warming. Microbial responses to repeated freeze-thaw conditions were studied in three complementary experiments using arctic sediment collected from an intertidal flat that is exposed to seasonal freeze-thaw conditions (Ymerbukta, Svalbard, Arctic Ocean). The sediment was subjected to oscillating freeze-thaw incubations, either gradual, from -5 to 4 degrees C, or abrupt, from -20 to 10 degrees C. Concentrations of low-molecular weight carboxylic acids (volatile fatty acids) were measured and sulfate reduction was assessed by measuring (35)S sulfate reduction rates (SRRs). Gradual freeze-thaw incubation decreased microbial activity in the frozen state to 0.25 % of initial levels at 4 degrees C, but activity resumed rapidly reaching >60 % of initial activity in the thawed state. Exposure of sediments to successive large temperature changes (-20 versus 10 degrees C) decreased SRR by 80% of the initial activity, suggesting that a fraction of the bacterial community recovered rapidly from extreme temperature fluctuations. This is supported by 16S rRNA gene-based denaturing gradient gel electrophoresis profiles that revealed persistence of the dominant microbial taxa under repeated freeze-thaw cycles. The fast recovery of the SRRs suggests that carbon mineralization in thawing arctic sediment can resume without delay or substantial growth of microbial populations.

Cold surface sediments host a seedbank of functionally diverse thermophilic bacteria. These thermophiles are present as endospores, which are widely dispersed in aquatic environments. Here, we investigated the functional potential of endospore populations in cold surface sediments heated to 80°C. Microbial production of acetate was observed at 80°C and could be enhanced by supplying additional organic carbon substrates. Comparison of 16S rRNA gene amplicon libraries from 80°C enrichments to sediments heated to lower temperatures (50-70°C) showed that temperature selects for distinct populations of endospore-forming bacteria. Whereas sulfate-reducing thermophiles were enriched in 50-70°C incubations, 80°C exceeds their thermal tolerance and selects for hyperthermophilic organotrophic bacteria that are similarly detected in amplicon libraries from sediments heated to 90°C. Genome-resolved metagenomics revealed novel carbon cycling members of Symbiobacteriales, Thermosediminibacteraceae, Thermanaeromonas and Calditerricola with the genomic potential for the degradation of carbohydrates, sugars, amino acids and nucleotides. Endospores of thermophilic bacteria are deposited on seabed sediments worldwide where they remain dormant as they are buried in the accumulating sediments. Our results suggest that endospore populations could be activated by temperature increases encountered during burial and show the potential for organotrophic metabolic activity contributing to acetate generation in deep hot sediments.

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas . An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.