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Significance Microorganisms persisting in hydraulically fractured shales must maintain osmotic balance in hypersaline fluids, gain energy in the absence of electron acceptors, and acquire carbon and nitrogen to synthesize cell building blocks. We provide evidence that that cofermentation of amino acids (Stickland reaction) meets all of these organismal needs, thus functioning as a keystone metabolism in enriched and natural microbial communities from hydraulically fractured shales. This amino acid-based metabolic network can be rationally designed to optimize biogenic methane yields and minimize undesirable chemistries in this engineered ecosystem. Our proposed ecological framework extends to the human gut and other protein-rich ecosystems, where the role of Stickland fermentations and their derived syntrophies play unrecognized roles in carbon and nitrogen turnover., 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.

Genome sequences were obtained for two isolates of the genus Arcobacter from saline fluids produced from hydraulically fractured shale gas wells in the Marcellus and Utica formations. These genomes provide insight into microbial sulfur cycles occurring in a high-salt deep terrestrial shale environment.

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.

The genomes of three Marinobacter strains, isolated from saline fluids produced from a Utica-Point Pleasant shale well, have been sequenced. These genomes provide novel information on the degradation of petroleum distillates and virulence mechanisms under microaerophilic conditions in fractured shale.

A large portion of the earth's biomass resides in the subsurface and recent studies have expanded our knowledge of indigenous microbial life. Advances in the field of metagenomics now allow analysis of microbial communities from low-biomass samples such as deep (>2.5 km) shale core samples. Here we present protocols for the best practices in contamination control, handling core material, extraction of nucleic acids, and low-input library preparation for subsequent metagenomic sequencing.

The genomes of three novel Frackibacter strains (WG11, WG12, and WG13) were sequenced. These strains were isolated from hypersaline fluid collected from a hydraulically fractured natural gas well. These genomes provide information on the mechanisms necessary for growth in these environments and offer insight into interactions with other community members.

The genomes of three novel Frackibacter strains (WG11, WG12, and WG13) were sequenced. These strains were isolated from hypersaline fluid collected from a hydraulically fractured natural gas well. These genomes provide information on the mechanisms necessary for growth in these environments and offer insight into interactions with other community members.

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.

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.

Despite being key contributors to biogeochemical processes, archaea are frequently outnumbered by bacteria, and consequently are underrepresented in combined molecular surveys. Here, we demonstrate an approach to concurrently survey the archaea alongside the bacteria with high-resolution 16S rRNA gene sequencing, linking these community data to geochemical parameters. We applied this integrated analysis to hydric soils sampled across a model methane-emitting freshwater wetland. Geochemical profiles, archaeal communities, and bacterial communities were independently correlated with soil depth and water cover. Centimeters of soil depth and corresponding geochemical shifts consistently affected microbial community structure more than hundreds of meters of lateral distance. Methanogens with diverse metabolisms were detected across the wetland, but displayed surprising OTU-level partitioning by depth. Candidatus Methanoperedens spp. archaea thought to perform anaerobic oxidation of methane linked to iron reduction were abundant. Domain-specific sequencing also revealed unexpectedly diverse non-methane-cycling archaeal members. OTUs within the underexplored Woesearchaeota and Bathyarchaeota were prevalent across the wetland, with subgroups and individual OTUs exhibiting distinct occupancy and abundance distributions aligned with environmental gradients. This study adds to our understanding of ecological range for key archaeal taxa in a model freshwater wetland, and links these taxa and individual OTUs to hypotheses about processes governing biogeochemical cycling.

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.

EA Pôle EcolDur; Microbes exist in a range of metabolic states (for example, dormant, active and growing) and analysis of ribosomal RNA (rRNA) is frequently employed to identify the 'active' fraction of microbes in environmental samples. While rRNA analyses are no longer commonly used to quantify a population's growth rate in mixed communities, due to rRNA concentration not scaling linearly with growth rate uniformly across taxa, rRNA analyses are still frequently used toward the more conservative goal of identifying populations that are currently active in a mixed community. Yet, evidence indicates that the general use of rRNA as a reliable indicator of metabolic state in microbial assemblages has serious limitations. This report highlights the complex and often contradictory relationships between rRNA, growth and activity. Potential mechanisms for confounding rRNA patterns are discussed, including differences in life histories, life strategies and non-growth activities. Ways in which rRNA data can be used for useful characterization of microbial assemblages are presented, along with questions to be addressed in future studies.

VerifyNow (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=0.95xPRU_C_20 (p0.001). Bland-Altman plots showed good agreement between PRU_H_Imm and (0.95xPRU_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.

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.

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.

There have been numerous reports in the literature on the descriptive similarities between a severe nightmare and an acute psychotic episode. Nightmares may be a prelude to psychotic decompensation, and it has been suggested that frequent lifelong nightmares may even be diagnostic of an underlying vulnerability to psychosis. In this report, we present a case study of a 40-year old female experiencing chronic paranoid schizophrenia, whose two witnessed psychotic relapses in the hospital were immediately preceded by intense and vivid nightmare attacks. Significantly, the content of these nocturnal dreams was thematically consistent with her waking hallucinations, suggesting a direct continuity between these experiences. We propose that further systematic study of the dreams and nightmares of individuals diagnosed with schizophrenia may be particularly useful in understanding their phenomenological experience.

International audience; High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

Background Microbial ecologists often employ methods from classical community ecology to analyze microbial community diversity. However, these methods have limitations because microbial communities differ from macro-organismal communities in key ways. This study sought to quantify microbial diversity using methods that are better suited for data spanning multiple domains of life and dimensions of diversity. Diversity profiles are one novel, promising way to analyze microbial datasets. Diversity profiles encompass many other indices, provide effective numbers of diversity (mathematical generalizations of previous indices that better convey the magnitude of differences in diversity), and can incorporate taxa similarity information. To explore whether these profiles change interpretations of microbial datasets, diversity profiles were calculated for four microbial datasets from different environments spanning all domains of life as well as viruses. Both similarity-based profiles that incorporated phylogenetic relatedness and naïve (not similarity-based) profiles were calculated. Simulated datasets were used to examine the robustness of diversity profiles to varying phylogenetic topology and community composition. Results Diversity profiles provided insights into microbial datasets that were not detectable with classical univariate diversity metrics. For all datasets analyzed, there were key distinctions between calculations that incorporated phylogenetic diversity as a measure of taxa similarity and naïve calculations. The profiles also provided information about the effects of rare species on diversity calculations. Additionally, diversity profiles were used to examine thousands of simulated microbial communities, showing that similarity-based and naïve diversity profiles only agreed approximately 50% of the time in their classification of which sample was most diverse. This is a strong argument for incorporating similarity information and calculating diversity with a range of emphases on rare and abundant species when quantifying microbial community diversity. Conclusions For many datasets, diversity profiles provided a different view of microbial community diversity compared to analyses that did not take into account taxa similarity information, effective diversity, or multiple diversity metrics. These findings are a valuable contribution to data analysis methodology in microbial ecology.