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Background:Rivers serve as a nexus for nutrient transfer between terrestrial and marine ecosystems and as such, have a significant impact on global carbon and nitrogen cycles. In river ecosystems, the sediments found within the hyporheic zone are microbial hotspots that can account for a significant portion of ecosystem respiration and have profound impacts on system biogeochemistry. Despite this, studies using genome-resolved analyses linking microbial and viral communities to nitrogen and carbon biogeochemistry are limited.Results:Here, we characterized the microbial and viral communities of Columbia River hyporheic zone sediments to reveal the metabolisms that actively cycle carbon and nitrogen. Using genome-resolved metagenomics, we created the Hyporheic Uncultured Microbial and Viral (HUM-V) database, containing a dereplicated database of 55 microbial Metagenome-Assembled Genomes (MAGs), representing 12 distinct phyla. We also sampled 111 viral Metagenome Assembled Genomes (vMAGs) from 26 distinct and novel genera. The HUM-V recruited metaproteomes from these same samples, providing the first inventory of microbial gene expression in hyporheic zone sediments. Combining this data with metabolite data, we generated a conceptual model where heterotrophic and autotrophic metabolisms co-occur to drive an integrated carbon and nitrogen cycle, revealing microbial sources and sinks for carbon dioxide and ammonium in these sediments. We uncovered the metabolic handoffs underpinning these processes including mutualistic nitrification by Thermoproteota (formerly Thaumarchaeota) and Nitrospirota, as well as identified possible cooperative and cheating behavior impacting nitrogen mineralization. Finally, by linking vMAGs to microbial genome hosts, we reveal possible viral controls on microbial nitrification and organic carbon degradation.Conclusions:Our multi-omics analyses provide new mechanistic insight into coupled carbon-nitrogen cycling in the hyporheic zone. This is a key step in developing predictive hydrobiogeochemical models that account for microbial cross-feeding and viral influences over potential and expressed microbial metabolisms. Furthermore, the publicly available HUM-V genome resource can be queried and expanded by researchers working in other ecosystems to assess the transferability of our results to other parts of the globe.

Organohalides are routinely detected in fluid produced from hydraulically fractured oil and natural-gas wells, yet the origin and fate of these compounds remain largely unknown. Because few organoh...

Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism., It is thought that polyphenols inhibit organic matter decomposition in soils devoid of oxygen. Here the authors use metabolomics and genome-resolved metaproteomics to provide experimental evidence of polyphenol biodegradation and maintained soil microbial community metabolism despite anoxia.

Produced waters from hydraulically fractured shale formations give insight into the microbial ecology and biogeochemical conditions down-well. This study explores the potential for sulfide production by persistent microorganisms recovered from produced water samples collected from the Marcellus shale formation. Hydrogen sulfide is highly toxic and corrosive, and can lead to the formation of "sour gas" which is costly to refine. Furthermore, microbial colonization of hydraulically fractured shale could result in formation plugging and a reduction in well productivity. It is vital to assess the potential for sulfide production in persistent microbial taxa, especially when considering the trend of reusing produced waters as input fluids, potentially enriching for problematic microorganisms. Using most probable number (MPN) counts and 16S rRNA gene sequencing, multiple viable strains of bacteria were identified from stored produced waters, mostly belonging to the Genus Halanaerobium, that were capable of growth via fermentation, and produced sulfide when supplied with thiosulfate. No sulfate-reducing bacteria (SRB) were detected through culturing, despite the detection of relatively low numbers of sulfate-reducing lineages by high-throughput 16S rRNA gene sequencing. These results demonstrate that sulfidogenic produced water populations remain viable for years post production and, if left unchecked, have the potential to lead to natural gas souring during shale gas extraction.

Understanding the sources and controls on microbial methane production from wetland soils is critical to global methane emission predictions, particularly in light of changing climatic conditions. Current biogeochemical models of methanogenesis consider only acetoclastic and hydrogenotrophic sources and exclude methylotrophic methanogenesis, potentially underestimating microbial contributions to methane flux. Our multi-omic results demonstrated that methylotrophic methanogens of the family Methanomassiliicoccaceae were present and active in a freshwater wetland, with metatranscripts indicating that methanol, not methylamines, was the likely substrate under the conditions measured here. However, laboratory experiments indicated the potential for other methanogens to become enriched in response to trimethylamine, revealing the reservoir of methylotrophic methanogenesis potential residing in these soils. Collectively, our approach used coupled field and laboratory investigations to illuminate metabolisms influencing the terrestrial microbial methane cycle, thereby offering direction for increased realism in predictive process-oriented models of methane flux in wetland soils., Wetland soils are one of the largest natural contributors to the emission of methane, a potent greenhouse gas. Currently, microbial contributions to methane emissions from these systems emphasize the roles of acetoclastic and hydrogenotrophic methanogens, while less frequently considering methyl-group substrates (e.g., methanol and methylamines). Here, we integrated laboratory and field experiments to explore the potential for methylotrophic methanogenesis in Old Woman Creek (OWC), a temperate freshwater wetland located in Ohio, USA. We first demonstrated the capacity for methylotrophic methanogenesis in these soils using laboratory soil microcosms amended with trimethylamine. However, subsequent field porewater nuclear magnetic resonance (NMR) analyses to identify methanogenic substrates failed to detect evidence for methylamine compounds in soil porewaters, instead noting the presence of the methylotrophic substrate methanol. Accordingly, our wetland soil-derived metatranscriptomic data indicated that methanol utilization by the Methanomassiliicoccaceae was the likely source of methylotrophic methanogenesis. Methanomassiliicoccaceae relative contributions to mcrA transcripts nearly doubled with depth, accounting for up to 8% of the mcrA transcripts in 25-cm-deep soils. Longitudinal 16S rRNA amplicon and mcrA gene surveys demonstrated that Methanomassiliicoccaceae were stably present over 2 years across lateral and depth gradients in this wetland. Meta-analysis of 16S rRNA sequences similar (>99%) to OWC Methanomassiliicoccaceae in public databases revealed a global distribution, with a high representation in terrestrial soils and sediments. Together, our results demonstrate that methylotrophic methanogenesis likely contributes to methane flux from climatically relevant wetland soils. IMPORTANCE Understanding the sources and controls on microbial methane production from wetland soils is critical to global methane emission predictions, particularly in light of changing climatic conditions. Current biogeochemical models of methanogenesis consider only acetoclastic and hydrogenotrophic sources and exclude methylotrophic methanogenesis, potentially underestimating microbial contributions to methane flux. Our multi-omic results demonstrated that methylotrophic methanogens of the family Methanomassiliicoccaceae were present and active in a freshwater wetland, with metatranscripts indicating that methanol, not methylamines, was the likely substrate under the conditions measured here. However, laboratory experiments indicated the potential for other methanogens to become enriched in response to trimethylamine, revealing the reservoir of methylotrophic methanogenesis potential residing in these soils. Collectively, our approach used coupled field and laboratory investigations to illuminate metabolisms influencing the terrestrial microbial methane cycle, thereby offering direction for increased realism in predictive process-oriented models of methane flux in wetland soils.

The current paradigm, widely incorporated in soil biogeochemical models, is that microbial methanogenesis can only occur in anoxic habitats. In contrast, here we show clear geochemical and biological evidence for methane production in well-oxygenated soils of a freshwater wetland. A comparison of oxic to anoxic soils reveal up to ten times greater methane production and nine times more methanogenesis activity in oxygenated soils. Metagenomic and metatranscriptomic sequencing recover the first near-complete genomes for a novel methanogen species, and show acetoclastic production from this organism was the dominant methanogenesis pathway in oxygenated soils. This organism, Candidatus Methanothrix paradoxum, is prevalent across methane emitting ecosystems, suggesting a global significance. Moreover, in this wetland, we estimate that up to 80% of methane fluxes could be attributed to methanogenesis in oxygenated soils. Together, our findings challenge a widely held assumption about methanogenesis, with significant ramifications for global methane estimates and Earth system modeling., Methane production is traditionally not found in oxygenated soils, a paradigm incorporated in global greenhouse gas modelling efforts. Here the authors show geochemical and biological evidence of active methanogenesis in bulk-oxic wetland soils, attributing up to 80% of the total methane budget for the site.

Here we used soil metagenomics and metatranscriptomics to uncover novel members within the genus Methylobacter. We denote these closely related genomes as members of the lineage OWC Methylobacter. Despite the incredibly high microbial diversity in soils, here we present findings that unexpectedly showed that methane cycling was primarily mediated by a single genus for both methane production (“Candidatus Methanothrix paradoxum”) and methane consumption (OWC Methylobacter). Metatranscriptomic analyses revealed that decreased methanotrophic activity rather than increased methanogenic activity possibly contributed to the greater methane emissions that we had previously observed in summer months, findings important for biogeochemical methane models. Although members of this Methylococcales order have been cultivated for decades, multi-omic approaches continue to illuminate the methanotroph phylogenetic and metabolic diversity harbored in terrestrial and marine ecosystems., Microbial carbon degradation and methanogenesis in wetland soils generate a large proportion of atmospheric methane, a highly potent greenhouse gas. Despite their potential to mitigate greenhouse gas emissions, knowledge about methane-consuming methanotrophs is often limited to lower-resolution single-gene surveys that fail to capture the taxonomic and metabolic diversity of these microorganisms in soils. Here our objective was to use genome-enabled approaches to investigate methanotroph membership, distribution, and in situ activity across spatial and seasonal gradients in a freshwater wetland near Lake Erie. 16S rRNA gene analyses demonstrated that members of the methanotrophic Methylococcales were dominant, with the dominance largely driven by the relative abundance of four taxa, and enriched in oxic surface soils. Three methanotroph genomes from assembled soil metagenomes were assigned to the genus Methylobacter and represented the most abundant methanotrophs across the wetland. Paired metatranscriptomes confirmed that these Old Woman Creek (OWC) Methylobacter members accounted for nearly all the aerobic methanotrophic activity across two seasons. In addition to having the capacity to couple methane oxidation to aerobic respiration, these new genomes encoded denitrification potential that may sustain energy generation in soils with lower dissolved oxygen concentrations. We further show that Methylobacter members that were closely related to the OWC members were present in many other high-methane-emitting freshwater and soil sites, suggesting that this lineage could participate in methane consumption in analogous ecosystems. This work contributes to the growing body of research suggesting that Methylobacter may represent critical mediators of methane fluxes in freshwater saturated sediments and soils worldwide.

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system.

Flowback/Produced fluid samples were collected from several wells from two Utica/Point Pleasant (UPP) sites (UPPW and UPPS) in Ohio, and one Marcellus (Marcellus Shale Energy and Environment Laboratory (MSEEL)) site in West Virginia over a period of approximately two years. Although these formations have different ages, depositional environments, diagenetic histories, and geochemical and mineralogical compositions (i.e. the UPP is significantly more carbonate rich than the Marcellus which is more siliceous), analysis of trends in fluid species over time shows that, overall, the TDS and major solubilized elements (Na, Ca, Cl) in the UPP and Marcellus brines are remarkably similar. Total dissolved solutes (TDS) in these brines ranged from approximately 40 to 250 g/L salt, and in general, concentrations increased with time elapsed since natural gas well completion and stimulation. The behavior of Na, Br, and Cl suggests that the produced water signatures from these formations are largely derived from the native formational brines which display evidence of originating from evaporated seawater. There is a strong correlation between Cl and Br, indicating that both species behave conservatively, and the similarity among each of these brines suggests no appreciable contribution of salt from halite dissolution because Br is excluded from the halite structure. Cl/Br ratios in the brines range from ~80 to 120 (mg/L/mg/L). Other elements, such as K, which readily reacts between fluids and ion exchange sites on clays, generally exhibit conservative behavior for an individual site, but show significant variations among each of the different well pads. The concentrations of Sr and Ba vary dramatically among well sites, and increase with respect to Cl− over time, suggesting increasing solubilization, presumably from desorption from clay minerals or dissolution of carbonates or sulfates from the source formation(s). The UPPW well site has very low Ba due to high-sulfate input fluid, which resulted in precipitation of barite/celestite in the brines. In contrast the UPPS well site had elevated Sr (~ 3500 mg/L), presumably due to the use of Sr-rich recycled brine used in hydraulic fracturing. The Marcellus site had the highest Ba concentrations (up to 10 g/L) and highest Ba/Sr ratios in the fluids, due to the high concentration of barium in the Marcellus target (~ 1000 ppm, as compared to ~200 ppm in the UPP). These observations suggest that solutes in the FP fluids are derived from native brines, water-rock interactions that have occurred over geologic time scales, as well as some contribution from contemporaneous reactions in the subsurface. The results also show that the composition of the injected fluid can influence flowback fluid chemistry and possibly production efficiency.

Hydraulic fracturing of black shale formations has greatly increased United States oil and natural gas recovery. However, the accumulation of biomass in subsurface reservoirs and pipelines is detrimental because of possible well souring, microbially induced corrosion, and pore clogging. Temporal sampling of produced fluids from a well in the Utica Shale revealed the dominance of Halanaerobium strains within the in situ microbial community and the potential for these microorganisms to catalyze thiosulfate-dependent sulfidogenesis. From these field data, we investigated biogenic sulfide production catalyzed by a Halanaerobium strain isolated from the produced fluids using proteogenomics and laboratory growth experiments. Analysis of Halanaerobium isolate genomes and reconstructed genomes from metagenomic data sets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes capable of converting thiosulfate to sulfide. Shotgun proteomics measurements using a Halanaerobium isolate verified that these proteins were more abundant when thiosulfate was present in the growth medium, and culture-based assays identified thiosulfate-dependent sulfide production by the same isolate. Increased production of sulfide and organic acids during the stationary growth phase suggests that fermentative Halanaerobium uses thiosulfate to remove excess reductant. These findings emphasize the potential detrimental effects that could arise from thiosulfate-reducing microorganisms in hydraulically fractured shales, which are undetected by current industry-wide corrosion diagnostics. IMPORTANCE Although thousands of wells in deep shale formations across the United States have been hydraulically fractured for oil and gas recovery, the impact of microbial metabolism within these environments is poorly understood. Our research demonstrates that dominant microbial populations in these subsurface ecosystems contain the conserved capacity for the reduction of thiosulfate to sulfide and that this process is likely occurring in the environment. Sulfide generation (also known as “souring”) is considered deleterious in the oil and gas industry because of both toxicity issues and impacts on corrosion of the subsurface infrastructure. Critically, the capacity for sulfide generation via reduction of sulfate was not detected in our data sets. Given that current industry wellhead tests for sulfidogenesis target canonical sulfate-reducing microorganisms, these data suggest that new approaches to the detection of sulfide-producing microorganisms may be necessary.

SummaryVerifyNow (VN) P2Y12 is a point-of-care assay used to assess response to P2Y12 inhibitors. Sodium citrate (citrate) is the standard anticoagulant used for this assay but requires a pre-incubation period. Hirudin is an alternative anticoagulant for platelet function studies that maintains physiological divalent cation levels. We investigated whether hirudin anticoagulation might allow more rapid testing of P2Y12 inhibition at the time of percutaneous coronary intervention (PCI). Blood was collected from the arterial sheath of aspirin-treated patients undergoing elective, urgent or emergency coronary angiography ± PCI and aliquots were anticoagulated with either citrate or hirudin. For each anticoagulant, VN P2Y12 was performed both immediately and after 20 minutes. A total of 98 patients were included in this study following pre-treatment with clopidogrel (n = 88), prasugrel (n = 6) or no P2Y12 inhibitor (n = 4). PRU with hirudin immediately (PRU_H_Imm) and PRU with citrate 20 minutes post sampling (PRU_C_20) were very strongly correlated (R = 0.95) though PRU_H_Imm tended to be lower than PRU_C_20 so that optimal correlation was estimated by the equation PRU_H_Imm = 0.95 x PRU_C_20 (p < 0.001). Bland-Altman plots showed good agreement between PRU_H_Imm and (0.95 x PRU_C_20). Platelet reactivity was more stable over the studied time course with hirudin as compared to citrate. We therefore conclude that VN P2Y12 with hirudin anticoagulation can be performed more rapidly and results are strongly correlated with delayed citrate measurements. Further studies are warranted to assess the utility of this method for improving clinical outcomes in patients undergoing PCI.

Bioreduction-based strategies for remediating uranium (U)-contaminated sediments face the challenge of maintaining the reduced status of U for long times. Because groundwater influxes continuously bring in oxidizing terminal electron acceptors (O2, NO3(-)), it is necessary to continue supplying organic carbon (OC) to maintain the reducing environment after U bioreduction is achieved. We tested the influence of OC supply rates on mobility of previously microbial reduced uranium U(IV) in contaminated sediments. We found that high degrees of U mobilization occurred when OC supply rates were high, and when the sediment still contained abundant Fe(III). Although 900 days with low levels of OC supply minimized U mobilization, the sediment redox potential increased with time as did extractable U(VI) fractions. Molecular analyses of total microbial activity demonstrated a positive correlation with OC supply and analyses of Geobacteraceae activity (RT-qPCR of 16S rRNA) indicated continued activity even when the effluent Fe(II) became undetectable. These data support our hypothesis on the mechanisms responsible for remobilization of U under reducing conditions; that microbial respiration caused increased (bi)carbonate concentration and formation of stable uranyl carbonate complexes, thereby shifted U(IV)/U(VI) equilibrium to more reducing potentials. The data also suggested that low OC concentrations could not sustain the reducing condition of the sediment for much longer time. Bioreduced U(IV) is not sustainable in an oxidizing environment for a very long time.

Remediation of uranium-contaminated sediments through in situ stimulation of bioreduction to insoluble UO2 is a potential treatment strategy under active investigation. Previously, we found that newly reduced U(IV) can be reoxidized under reducing conditions sustained by a continuous supply of organic carbon (OC) because of residual reactive Fe(III) and enhanced U(VI) solubilitythrough complexation with carbonate generated through OC oxidation. That finding motivated this investigation directed at identifying a range of OC supply rates that is optimal for establishing U bioreduction and immobilization in initially oxidizing sediments. The effects of OC supply rate, from 0 to 580 mmol of OC (kg of sediment)(-1) year(-1), and OC form (lactate and acetate) on U bioreduction were tested in flow-through columns containing U-contaminated sediments. An intermediate supply rate on the order of 150 mmol of OC (kg of sediment)(-1) year(-1) was determined to be most effective at immobilizing U. At lower OC supply rates, U bioreduction was not achieved, and U(VI) solubilitywas enhanced by complexation with carbonate (from OC oxidation). At the highest OC supply rate, the resulting highly carbonate-enriched solutions also supported elevated levels of U(VI), even though strongly reducing conditions were established. Lactate and acetate were found to have very similar geochemical impacts on effluent U concentrations (and other measured chemical species), when compared at equivalent OC supply rates. While the catalysts of U(VI) reduction to U(IV) are presumably bacteria, the composition of the bacterial community,the Fe-reducing community, and the sulfate-reducing community had no direct relationship with effluent U concentrations. The OC supply rate has competing effects of driving reduction of U(VI) to low-solubility U(IV) solids, as well as causing formation of highly soluble U(VI)-carbonato complexes. These offsetting influences will require careful control of OC supply rates in order to optimize bioreduction-based U stabilization.

Bioreduction of U in contaminated sediments is an attractive strategy because of its low cost, and because of short-term studies supporting its feasibility. However, any in-situ immobilization approach for U will require assurance of either permanent fixation, or of very low release rates into the biosphere. Our previous long-term (2 years) laboratory experiments have shown that organic carbon (OC) based U(VI) bioreduction to UO2 can be transient even under sustained reducing (methanogenic) conditions. The biogeochemical processes underlying this finding urgently need to be understood. The current research is designed to identify mechanisms responsible for anaerobic U oxidation, and identify conditions that will support long-term stability of bioreduced U. We are investigating: (1) effects of OC concentration and supply rate on remobilization of bioreduced U, (2) the roles of Fe- and Mn-oxides as potential U oxidants in sediments, and (3) the role of microorganisms in U reoxidation, and (4) influences of pH on U(IV)/U(VI) redox equilibrium.