Cold seep formation from salt diapir-controlled deep biosphere oases.

Deep sea cold seeps are sites where hydrogen sulfide, methane, and other hydrocarbon-rich fluids vent from the ocean floor. They are an important component of Earth's carbon cycle in which subsurface hydrocarbons form the energy source for highly diverse benthic micro- and macro-fauna in what is otherwise vast and spartan sea scape. Passive continental margin cold seeps are typically attributed to the migration of hydrocarbons generated from deeply buried source rocks. Many of these seeps occur over salt tectonic provinces, where the movement of salt generates complex fault systems that can enable fluid migration or create seals and traps associated with reservoir formation. The elevated advective heat transport of the salt also produces a chimney effect directly over these structures. Here, we provide geophysical and geochemical evidence that the salt chimney effect in conjunction with diapiric faulting drives a subsurface groundwater circulation system that brings dissolved inorganic carbon, nutrient-rich deep basinal fluids, and potentially overlying seawater onto the crests of deeply buried salt diapirs. The mobilized fluids fuel methanogenic archaea locally enhancing the deep biosphere. The resulting elevated biogenic methane production, alongside the upward heat-driven fluid transport, represents a previously unrecognized mechanism of cold seep formation and regulation.

Wastewater-based surveillance as a tool for public health action: SARS-CoV-2 and beyond.

Wastewater-based surveillance (WBS) has undergone dramatic advancement in the context of the coronavirus disease 2019 (COVID-19) pandemic. The power and potential of this platform technology were rapidly realized when it became evident that not only did WBS-measured SARS-CoV-2 RNA correlate strongly with COVID-19 clinical disease within monitored populations but also, in fact, it functioned as a leading indicator. Teams from across the globe rapidly innovated novel approaches by which wastewater could be collected from diverse sewersheds ranging from wastewater treatment plants (enabling community-level surveillance) to more granular locations including individual neighborhoods and high-risk buildings such as long-term care facilities (LTCF). Efficient processes enabled SARS-CoV-2 RNA extraction and concentration from the highly dilute wastewater matrix. Molecular and genomic tools to identify, quantify, and characterize SARS-CoV-2 and its various variants were adapted from clinical programs and applied to these mixed environmental systems. Novel data-sharing tools allowed this information to be mobilized and made immediately available to public health and government decision-makers and even the public, enabling evidence-informed decision-making based on local disease dynamics. WBS has since been recognized as a tool of transformative potential, providing near-real-time cost-effective, objective, comprehensive, and inclusive data on the changing prevalence of measured analytes across space and time in populations. However, as a consequence of rapid innovation from hundreds of teams simultaneously, tremendous heterogeneity currently exists in the SARS-CoV-2 WBS literature. This manuscript provides a state-of-the-art review of WBS as established with SARS-CoV-2 and details the current work underway expanding its scope to other infectious disease targets.

A Bayesian framework for modeling COVID-19 case numbers through longitudinal monitoring of SARS-CoV-2 RNA in wastewater.

Wastewater-based surveillance has become an important tool for research groups and public health agencies investigating and monitoring the COVID-19 pandemic and other public health emergencies including other pathogens and drug abuse. While there is an emerging body of evidence exploring the possibility of predicting COVID-19 infections from wastewater signals, there remain significant challenges for statistical modeling. Longitudinal observations of viral copies in municipal wastewater can be influenced by noisy datasets and missing values with irregular and sparse samplings. We propose an integrative Bayesian framework to predict daily positive cases from weekly wastewater observations with missing values via functional data analysis techniques. In a unified procedure, the proposed analysis models severe acute respiratory syndrome coronavirus-2 RNA wastewater signals as a realization of a smooth process with error and combines the smooth process with COVID-19 cases to evaluate the prediction of positive cases. We demonstrate that the proposed framework can achieve these objectives with high predictive accuracies through simulated and observed real data.

Surveillance for SARS-CoV-2 and its variants in wastewater of tertiary care hospitals correlates with increasing case burden and outbreaks.

Wastewater-based SARS-CoV-2 surveillance enables unbiased and comprehensive monitoring of defined sewersheds. We performed real-time monitoring of hospital wastewater that differentiated Delta and Omicron variants within total SARS-CoV-2-RNA, enabling correlation to COVID-19 cases from three tertiary-care facilities with >2100 inpatient beds in Calgary, Canada. RNA was extracted from hospital wastewater between August/2021 and January/2022, and SARS-CoV-2 quantified using RT-qPCR. Assays targeting R203M and R203K/G204R established the proportional abundance of Delta and Omicron, respectively. Total and variant-specific SARS-CoV-2 in wastewater was compared to data for variant specific COVID-19 hospitalizations, hospital-acquired infections, and outbreaks. Ninety-six percent (188/196) of wastewater samples were SARS-CoV-2 positive. Total SARS-CoV-2 RNA levels in wastewater increased in tandem with total prevalent cases (Delta plus Omicron). Variant-specific assessments showed this increase to be mainly driven by Omicron. Hospital-acquired cases of COVID-19 were associated with large spikes in wastewater SARS-CoV-2 and levels were significantly increased during outbreaks relative to nonoutbreak periods for total SARS-CoV2, Delta and Omicron. SARS-CoV-2 in hospital wastewater was significantly higher during the Omicron-wave irrespective of outbreaks. Wastewater-based monitoring of SARS-CoV-2 and its variants represents a novel tool for passive COVID-19 infection surveillance, case identification, containment, and potentially to mitigate viral spread in hospitals.

Hydrocarbon seepage in the deep seabed links subsurface and seafloor biospheres.

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.

Tracking Emergence and Spread of SARS-CoV-2 Omicron Variant in Large and Small Communities by Wastewater Monitoring in Alberta, Canada.

Wastewater monitoring of SARS-CoV-2 enables early detection and monitoring of the COVID-19 disease burden in communities and can track specific variants of concern. We determined proportions of the Omicron and Delta variants across 30 municipalities covering >75% of the province of Alberta (population 4.5 million), Canada, during November 2021-January 2022. Larger cities Calgary and Edmonton exhibited more rapid emergence of Omicron than did smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a medium-sized northern community that has many workers who fly in and out regularly. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden by late December, before the peak in newly diagnosed clinical cases throughout Alberta in mid-January. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of SARS-CoV-2 variants.

Hyperthermophilic endospores germinate and metabolize organic carbon in sediments heated to 80°C.

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.

The Critical Role of Environmental Synergies in the Creation of Bionanohybrid Microbes.

A wide range of bacteria can synthesize surface-associated nanoparticles (SANs) through exogenous metal ions reacting with sulfide produced via cysteine metabolism, resulting in the emergence of a biological-nanoparticle hybrid (bionanohybrid). The attached nanoparticles may couple to extracellular electron transfer, facilitating de novo photoelectrochemical processes. While SAN-cell coupling in hybrid organisms is opening a range of biotechnological possibilities, observation of bionanohybrids in nature is not commonly reported and their lab-based behavior remains difficult to control. We describe the critical role environmental synergy (microbial growth stage, cell densities, cysteine, and exogenous metal concentrations) plays in controlling the form and occurrence of Escherichia coli and Moorella thermoacetica bionanohybrids. SAN development depends on an appropriate cell density to metal ratio, with too few cells resulting in nanoparticle suppression through cytotoxicity or inhibition of cysteine conversion, and with too many cells diluting the number and size of particles produced. This cell number is governed by the concentration of cysteine present, which acts to protect the cells from metal ion toxicity. Exposing cells to metal and cysteine during the lag phase leads to SAN development, whereas cells in the exponential growth phase predominantly produce dispersed nanoparticles. Applying these principles more broadly, E. coli is shown to biosynthesize composite Bi/Cu sulfide SANs, and Clostridioides difficile can be coaxed into a bionanohybrid lifestyle by fine-tuning the cysteine dosage. Bionanohybrids maintain a remarkable ability for binary fission and sustained growth, opening doors to the production of SANs tailored to specific technological functions. IMPORTANCE Some bacteria can produce nanoscale-sized particles, which remain attached to the surface of the organism. The surface association of these nanoparticles creates a new mode of interaction between the microbe's environment and its internal cellular function, giving rise to a new hybrid lifeform, a biological nanoparticle hybrid (bionanohybrid). These hybrid organisms gain new or enhanced biological functions, and thus their creation opens a wide range of biotechnological possibilities. Despite this potential, the fundamental controls on bionanohybrid formation and occurrence remain poorly constrained. In this study, Escherichia coli K-12, Moorella thermoacetica, and Clostridioides difficile were used to test the combined influences of the growth phase, cell density, cysteine dose, and metal concentration in determining single and composite metal sulfide surface-associated nanoparticle production. The significance of this study is that it defined the critical synergies controlling nanoparticle formation on bacterial cell surfaces, unlocking the potential for bionanohybrid applications in a range of organisms.

Inhibition of Sulfate Reduction and Cell Division by Desulfovibrio desulfuricans Coated in Palladium Metal.

The growth of sulfate-reducing bacteria (SRB) and associated hydrogen sulfide production can be problematic in a range of industries such that inhibition strategies are needed. A range of SRB can reduce metal ions, a strategy that has been utilized for bioremediation, metal recovery, and synthesis of precious metal catalysts. In some instances, the metal remains bound to the cell surface, and the impact of this coating on bacterial cell division and metabolism has not previously been reported. In this study, Desulfovibrio desulfuricans cells (1g dry weight) enabled the reduction of up to 1500 mmol (157.5 g) palladium (Pd) ions, resulting in cells being coated in approximately 1 µm of metal. Thickly coated cells were no longer able to metabolize or divide, ultimately leading to the death of the population. Increasing Pd coating led to prolonged inhibition of sulfate reduction, which ceased completely after cells had been coated with 1200 mmol Pd g-1 dry cells. Less Pd nanoparticle coating permitted cells to carry out sulfate reduction and divide, allowing the population to recover over time as surface-associated Pd diminished. Overcoming inhibition in this way was more rapid using lactate as the electron donor, compared to formate. When using formate as an electron donor, preferential Pd(II) reduction took place in the presence of 100 mM sulfate. The inhibition of important metabolic pathways using a biologically enabled casing in metal highlights a new mechanism for the development of microbial control strategies. IMPORTANCE Microbial reduction of sulfate to hydrogen sulfide is highly undesirable in several industrial settings. Some sulfate-reducing bacteria are also able to transform metal ions in their environment into metal phases that remain attached to their outer cell surface. This study demonstrates the remarkable extent to which Desulfovibrio desulfuricans can be coated with locally generated metal nanoparticles, with individual cells carrying more than 100 times their mass of palladium metal. Moreover, it reveals the effect of metal coating on metabolism and replication for a wide range of metal loadings, with bacteria unable to reduce sulfate to sulfide beyond a specific threshold. These findings present a foundation for a novel means of modulating the activity of sulfate-reducing bacteria.

Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments.

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.

Diesel and Crude Oil Biodegradation by Cold-Adapted Microbial Communities in the Labrador Sea.

Oil spills in the subarctic marine environment off the coast of Labrador, Canada, are increasingly likely due to potential oil production and increases in ship traffic in the region. To understand the microbiome response and how nutrient biostimulation promotes biodegradation of oil spills in this cold marine setting, marine sediment microcosms amended with diesel or crude oil were incubated at in situ temperature (4°C) for several weeks. Sequencing of 16S rRNA genes following these spill simulations revealed decreased microbial diversity and enrichment of putative hydrocarbonoclastic bacteria that differed depending on the petroleum product. Metagenomic sequencing revealed that the genus Paraperlucidibaca harbors previously unrecognized capabilities for alkane biodegradation, which were also observed in Cycloclasticus. Genomic and amplicon sequencing together suggest that Oleispira and Thalassolituus degraded alkanes from diesel, while Zhongshania and the novel PGZG01 lineage contributed to crude oil alkane biodegradation. Greater losses in PAHs from crude oil than from diesel were consistent with Marinobacter, Pseudomonas_D, and Amphritea genomes exhibiting aromatic hydrocarbon biodegradation potential. Biostimulation with nitrogen and phosphorus (4.67 mM NH4Cl and 1.47 mM KH2PO4) was effective at enhancing n-alkane and PAH degradation following low-concentration (0.1% [vol/vol]) diesel and crude oil amendments, while at higher concentrations (1% [vol/vol]) only n-alkanes in diesel were consumed, suggesting toxicity induced by compounds in unrefined crude oil. Biostimulation allowed for a more rapid shift in the microbial community in response to petroleum amendments, more than doubling the rates of CO2 increase during the first few weeks of incubation. IMPORTANCE Increases in transportation of diesel and crude oil in the Labrador Sea will pose a significant threat to remote benthic and shoreline environments, where coastal communities and wildlife are particularly vulnerable to oil spill contaminants. Whereas marine microbiology has not been incorporated into environmental assessments in the Labrador Sea, there is a growing demand for microbial biodiversity evaluations given the pronounced impact of climate change in this region. Benthic microbial communities are important to consider given that a fraction of spilled oil typically sinks such that its biodegradation occurs at the seafloor, where novel taxa with previously unrecognized potential to degrade hydrocarbons were discovered in this work. Understanding how cold-adapted microbiomes catalyze hydrocarbon degradation at low in situ temperature is crucial in the Labrador Sea, which remains relatively cold throughout the year.

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments.

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.

Sediment cooling triggers germination and sulfate reduction by heat-resistant thermophilic spore-forming bacteria.

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.

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

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.

Metabolites of an Oil Field Sulfide-Oxidizing, Nitrate-Reducing Sulfurimonas sp. Cause Severe Corrosion.

Oil reservoir souring and associated material integrity challenges are of great concern to the petroleum industry. The bioengineering strategy of nitrate injection has proven successful for controlling souring in some cases, but recent reports indicate increased corrosion in nitrate-treated produced water reinjection facilities. Sulfide-oxidizing, nitrate-reducing bacteria (soNRB) have been suggested to be the cause of such corrosion. Using the model soNRB Sulfurimonas sp. strain CVO obtained from an oil field, we conducted a detailed analysis of soNRB-induced corrosion at initial nitrate-to-sulfide (N/S) ratios relevant to oil field operations. The activity of strain CVO caused severe corrosion rates of up to 0.27 millimeters per year (mm y-1) and up to 60-µm-deep pitting within only 9 days. The highest corrosion during the growth of strain CVO was associated with the production of zero-valent sulfur during sulfide oxidation and the accumulation of nitrite, when initial N/S ratios were high. Abiotic corrosion tests with individual metabolites confirmed biogenic zero-valent sulfur and nitrite as the main causes of corrosion under the experimental conditions. Mackinawite (FeS) deposited on carbon steel surfaces accelerated abiotic reduction of both sulfur and nitrite, exacerbating corrosion. Based on these results, a conceptual model for nitrate-mediated corrosion by soNRB is proposed.IMPORTANCE Ambiguous reports of corrosion problems associated with the injection of nitrate for souring control necessitate a deeper understanding of this frequently applied bioengineering strategy. Sulfide-oxidizing, nitrate-reducing bacteria have been proposed as key culprits, despite the underlying microbial corrosion mechanisms remaining insufficiently understood. This study provides a comprehensive characterization of how individual metabolic intermediates of the microbial nitrogen and sulfur cycles can impact the integrity of carbon steel infrastructure. The results help explain the dramatic increases seen at times in corrosion rates observed during nitrate injection in field and laboratory trials and point to strategies for reducing adverse integrity-related side effects of nitrate-based souring mitigation.

Distribution of thermophilic endospores in a temperate estuary indicate that dispersal history structures sediment microbial communities.

Endospores of thermophilic bacteria are found in cold and temperate sediments where they persist in a dormant state. As inactive endospores that cannot grow at the low ambient temperatures, they are akin to tracer particles in cold sediments, unaffected by factors normally governing microbial biogeography (e.g., selection, drift, mutation). This makes thermophilic endospores ideal model organisms for studying microbial biogeography since their spatial distribution can be directly related to their dispersal history. To assess dispersal histories of estuarine bacteria, thermophilic endospores were enriched from sediments along a freshwater-to-marine transect of the River Tyne in high temperature incubations (50°C). Dispersal histories for 75 different taxa indicated that the majority of estuarine endospores were of terrestrial origin; most closely related to bacteria from warm habitats associated with industrial activity. A subset of the taxa detected were marine derived, with close relatives from hot deep marine biosphere habitats. These patterns are consistent with the sources of sediment in the River Tyne being predominantly terrestrial in origin. The results point to microbial communities in estuarine and marine sediments being structured by bi-directional currents, terrestrial run-off and industrial effluent as vectors of passive dispersal and immigration.

Environmental Selection and Biogeography Shape the Microbiome of Subsurface Petroleum Reservoirs.

Petroleum reservoirs within the deep biosphere are extreme environments inhabited by diverse microbial communities and represent biogeochemical hot spots in the subsurface. Despite the ecological and industrial importance of oil reservoir microbiomes, systematic study of core microbial taxa and their associated genomic attributes spanning different environmental conditions is limited. Here, we compile and compare 343 16S rRNA gene amplicon libraries and 25 shotgun metagenomic libraries from oil reservoirs in different parts of the world to test for the presence of core taxa and functions. These oil reservoir libraries do not share any core taxa at the species, genus, family, or order levels, and Gammaproteobacteria was the only taxonomic class detected in all samples. Instead, taxonomic composition varies among reservoirs with different physicochemical characteristics and with geographic distance highlighting environmental selection and biogeography in these deep biosphere habitats. Gene-centric metagenomic analysis reveals a functional core of metabolic pathways including carbon acquisition and energy-yielding strategies consistent with biogeochemical cycling in other subsurface environments. Genes for anaerobic hydrocarbon degradation are observed in a subset of the samples and are therefore not considered to represent core functions in oil reservoirs despite hydrocarbons representing an abundant source of carbon in these deep biosphere settings. Overall, this work reveals common and divergent features of oil reservoir microbiomes that are shaped by and responsive to environmental factors, highlighting controls on subsurface microbial community assembly. IMPORTANCE This comprehensive analysis showcases how environmental selection and geographic distance influence the microbiome of subsurface petroleum reservoirs. We reveal substantial differences in the taxonomy of the inhabiting microbes but shared metabolic function between reservoirs with different in situ temperatures and between reservoirs separated by large distances. The study helps understand and advance the field of deep biosphere science by providing an ecological framework and footing for geologists, chemists, and microbiologists studying these habitats to elucidate major controls on deep biosphere microbial ecology.

Potential for natural attenuation of crude oil hydrocarbons in benthic microbiomes near coastal communities in Kivalliq, Nunavut, Canada.

Oil spilled in marine environments can settle to the seafloor through aggregation and sedimentation processes. This has been predicted to be especially relevant in the Arctic due to plankton blooms initiated by melting sea ice. These conditions exist in the Kivalliq region in Nunavut, Canada, where elevated shipping traffic has increased the risk of accidental spills. Experimental microcosms combining surface sediment and crude oil were incubated at 4 °C over 21 weeks to evaluate the biodegradation potential of seabed microbiomes. Sediments sampled near the communities of Arviat and Chesterfield Inlet were assessed for biodegradation capabilities by combining hydrocarbon geochemistry with 16S rRNA gene and metagenomic sequencing, revealing decreased microbial diversity but enrichment of oil-degrading taxa. Alkane and aromatic hydrocarbon losses corresponded to detection of genes and genomes that encode enzymes for aerobic biodegradation of these compounds, pointing to the utility of marine microbiome surveys for predicting the fate of oil released into Arctic marine environments.

SituSeq: an offline protocol for rapid and remote Nanopore 16S rRNA amplicon sequence analysis.

Microbiome analysis through 16S rRNA gene sequencing is a crucial tool for understanding the microbial ecology of any habitat or ecosystem. However, workflows require large equipment, stable internet, and extensive computing power such that most of the work is performed far away from sample collection in both space and time. Performing amplicon sequencing and analysis at sample collection would have positive implications in many instances including remote fieldwork and point-of-care medical diagnoses. Here we present SituSeq, an offline and portable workflow for the sequencing and analysis of 16S rRNA gene amplicons using Nanopore sequencing and a standard laptop computer. SituSeq was validated by comparing Nanopore 16S rRNA gene amplicons, Illumina 16S rRNA gene amplicons, and Illumina metagenomes, sequenced using the same environmental DNA. Comparisons revealed consistent community composition, ecological trends, and sequence identity across platforms. Correlation between the abundance of taxa in each taxonomic level in Illumina and Nanopore data sets was high (Pearson's r > 0.9), and over 70% of Illumina 16S rRNA gene sequences matched a Nanopore sequence with greater than 97% sequence identity. On board a research vessel on the open ocean, SituSeq was used to analyze amplicon sequences from deep sea sediments less than 2 h after sequencing, and 8 h after sample collection. The rapidly available results informed decisions about subsequent sampling in near real-time while the offshore expedition was still underway. SituSeq is a portable and user-friendly workflow that helps to bring the power of microbial genomics and diagnostics to many more researchers and situations.