Coupled laboratory and field investigations resolve microbial interactions that underpin persistence in hydraulically fractured shales.

Hydraulic fracturing is one of the industrial processes behind the surging natural gas output in the United States. This technology inadvertently creates an engineered microbial ecosystem thousands of meters below Earth's surface. Here, we used laboratory reactors to perform manipulations of persisting shale microbial communities that are currently not feasible in field scenarios. Metaproteomic and metabolite findings from the laboratory were then corroborated using regression-based modeling performed on metagenomic and metabolite data from more than 40 produced fluids from five hydraulically fractured shale wells. Collectively, our findings show that Halanaerobium, Geotoga, and Methanohalophilus strain abundances predict a significant fraction of nitrogen and carbon metabolites in the field. Our laboratory findings also exposed cryptic predatory, cooperative, and competitive interactions that impact microorganisms across fractured shales. Scaling these results from the laboratory to the field identified mechanisms underpinning biogeochemical reactions, yielding knowledge that can be harnessed to potentially increase energy yields and inform management practices in hydraulically fractured shales.

Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.

About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes (MAGs). Utilizing six other previously sequenced isolate genomes and MAGs, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs.

Uncovering the Diversity and Activity of Methylotrophic Methanogens in Freshwater 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.

Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing.

The deep terrestrial biosphere harbours a substantial fraction of Earth's biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus-host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) spacers elucidated lineage-specific virus-host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.

Viral and metabolic controls on high rates of microbial sulfur and carbon cycling in wetland ecosystems.

BACKGROUND: Microorganisms drive high rates of methanogenesis and carbon mineralization in wetland ecosystems. These signals are especially pronounced in the Prairie Pothole Region of North America, the tenth largest wetland ecosystem in the world. Sulfate reduction rates up to 22 µmol cm-3 day-1 have been measured in these wetland sediments, as well as methane fluxes up to 160 mg m-2 h-1-some of the highest emissions ever measured in North American wetlands. While pore waters from PPR wetlands are characterized by high concentrations of sulfur species and dissolved organic carbon, the constraints on microbial activity are poorly understood. Here, we utilized metagenomics to investigate candidate sulfate reducers and methanogens in this ecosystem and identify metabolic and viral controls on microbial activity. RESULTS: We recovered 162 dsrA and 206 dsrD sequences from 18 sediment metagenomes and reconstructed 24 candidate sulfate reducer genomes assigned to seven phyla. These genomes encoded the potential for utilizing a wide variety of electron donors, such as methanol and other alcohols, methylamines, and glycine betaine. We also identified 37 mcrA sequences spanning five orders and recovered two putative methanogen genomes representing the most abundant taxa-Methanosaeta and Methanoregulaceae. However, given the abundance of Methanofollis-affiliated mcrA sequences, the detection of F420-dependent alcohol dehydrogenases, and millimolar concentrations of ethanol and 2-propanol in sediment pore fluids, we hypothesize that these alcohols may drive a significant fraction of methanogenesis in this ecosystem. Finally, extensive viral novelty was detected, with approximately 80% of viral populations being unclassified at any known taxonomic levels and absent from publicly available databases. Many of these viral populations were predicted to target dominant sulfate reducers and methanogens. CONCLUSIONS: Our results indicate that diversity is likely key to extremely high rates of methanogenesis and sulfate reduction observed in these wetlands. The inferred genomic diversity and metabolic versatility could result from dynamic environmental conditions, viral infections, and niche differentiation in the heterogeneous sediment matrix. These processes likely play an important role in modulating carbon and sulfur cycling in this ecosystem.

Chemical and pathogen-induced inflammation disrupt the murine intestinal microbiome.

BACKGROUND: Salmonella is one of the most significant food-borne pathogens to affect humans and agriculture. While it is well documented that Salmonella infection triggers host inflammation, the impacts on the gut environment are largely unknown. A CBA/J mouse model was used to evaluate intestinal responses to Salmonella-induced inflammation. In parallel, we evaluated chemically induced inflammation by dextran sodium sulfate (DSS) and a non-inflammation control. We profiled gut microbial diversity by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from fecal and cecal samples. These data were correlated to the inflammation marker lipocalin-2 and short-chain fatty acid concentrations. RESULTS: We demonstrated that inflammation, chemically or biologically induced, restructures the chemical and microbial environment of the gut over a 16-day period. We observed that the ten mice within the Salmonella treatment group had a variable Salmonella relative abundance, with three high responding mice dominated by >46% Salmonella at later time points and the remaining seven mice denoted as low responders. These low- and high-responding Salmonella groups, along with the chemical DSS treatment, established an inflammation gradient with chemical and low levels of Salmonella having at least 3 log-fold lower lipocalin-2 concentration than the high-responding Salmonella mice. Total short-chain fatty acid and individual butyrate concentrations each negatively correlated with inflammation levels. Microbial communities were also structured along this inflammation gradient. Low levels of inflammation, regardless of chemical or biological induction, enriched for Akkermansia spp. in the Verrucomicrobiaceae and members of the Bacteroidetes family S24-7. Relative to the control or low inflammation groups, high levels of Salmonella drastically decreased the overall microbial diversity, specifically driven by the reduction of Alistipes and Lachnospiraceae in the Bacteroidetes and Firmicutes phyla, respectively. Conversely, members of the Enterobacteriaceae and Lactobacillus were positively correlated to high levels of Salmonella-induced inflammation. CONCLUSIONS: Our results show that enteropathogenic infection and intestinal inflammation are interrelated factors modulating gut homeostasis. These findings may prove informative with regard to prophylactic or therapeutic strategies to prevent disruption of microbial communities, or promote their restoration.

Sulfide Generation by Dominant Halanaerobium Microorganisms in Hydraulically Fractured Shales.

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.

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales.

Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface.

A phase I trial of SD-9427 (progenipoietin) with a multipeptide vaccine for resected metastatic melanoma.

PURPOSE: The melanoma tumor antigen epitope peptides MART-1(26-35 (27L)), gp100(209-217 (210M)),and tyrosinase(368-376 (370D)) were emulsified with incomplete Freund's adjuvant and administered with SD-9427 (progenipoietin), an agonist of granulocyte colony-stimulating factor and the FLT-3 receptor, to evaluate the toxicities of and immune responses to this regimen as primary end points and time to relapse and survival as secondary end points. EXPERIMENTAL DESIGN: Fifteen patients with high-risk resected stage III and IV melanoma were enrolled. Each patient received peptides + incomplete Freund's adjuvant with SD-9427 at doses of either 10, 20, or 40 microg/kg s.c. for 3 days before and 7 days after each vaccination. Immunizations were administered every month for 6 months and then administered once 6 months later. A leukapheresis to obtain peripheral blood mononuclear cells for immune analyses as well as skin testing with peptides and recall antigens was performed before and after vaccination. IFN- gamma release assay, ELISPOT, and MHC-peptide tetramer analysis were performed using peripheral blood mononuclear cells collected before and after vaccination to evaluate peptide-specific cytotoxic T-cell responses. RESULTS: Local pain and granuloma formation and fatigue of grade I or II were the most common side effects. One patient developed antibody-mediated leukopenia and transient grade III neutropenia that resolved after stopping SD-9427. Six of 12 patients tested developed a positive skin test response to one or more of the peptides. Seven of 10 patients tested demonstrated an immune response to at least one peptide when evaluated by IFN-gamma release assay and ELISPOT assay after vaccination, as did 11 of 12 patients analyzed by MHC-peptide tetramer assay. Four of 15 patients have relapsed with a median follow-up of 20 months, and 1 patient in this high-risk group has died of disease. CONCLUSIONS: SD-9427 with a multipeptide vaccine was generally well tolerated, although one patient developed reversible antibody-mediated neutropenia. These data suggest that the majority of patients with resected melanoma mount an antigen-specific immune response against a multipeptide vaccine administered with SD-9427.