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Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. Halanaerobium is believed to be inadvertently introduced into this environment during the drilling and fracturing process and must therefore tolerate large changes in pressure, temperature, and salinity. Here, we used a Halanaerobium strain isolated from a natural gas well in the Utica Point Pleasant formation to investigate metabolic and physiological responses to growth under high-pressure subsurface conditions. Laboratory incubations confirmed the ability of Halanaerobium congolense strain WG8 to grow under pressures representative of deep shale formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological shifts were identified, including higher abundances of proteins associated with the production of extracellular polymeric substances. Confocal laser scanning microscopy indicated that extracellular polymeric substance (EPS) production was associated with greater cell aggregation when biomass was cultured at high pressure. Changes in Halanaerobium central carbon metabolism under the same conditions were inferred from nuclear magnetic resonance (NMR) and gas chromatography measurements, revealing large per-cell increases in production of ethanol, acetate, and propanol and cessation of hydrogen production. These metabolic shifts were associated with carbon flux through 1,2-propanediol in response to slower fluxes of carbon through stage 3 of glycolysis. Together, these results reveal the potential for bioclogging and corrosion (via organic acid fermentation products) associated with persistent Halanaerobium growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes. IMPORTANCE The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative Halanaerobium species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause "clumping" of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems., (Copyright © 2019 Booker et al.)

Microbial activity in the produced water from hydraulically fractured oil and gas wells may potentially interfere with hydrocarbon production and cause damage to the well and surface infrastructure via corrosion, sulfide release, and fouling. In this study, we surveyed the microbial abundance and community structure of produced water sampled from 42 Marcellus Shale wells in southwestern Pennsylvania (well age ranged from 150 to 1,846 days) to better understand the microbial diversity of produced water. We sequenced the V4 region of the 16S rRNA gene to assess taxonomy and utilized quantitative PCR (qPCR) to evaluate the microbial abundance across all 42 produced water samples. Bacteria of the order Halanaerobiales were found to be the most abundant organisms in the majority of the produced water samples, emphasizing their previously suggested role in hydraulic fracturing-related microbial activity. Statistical analyses identified correlations between well age and biocide formulation and the microbial community, in particular, the relative abundance of Halanaerobiales We further investigated the role of members of the order Halanaerobiales in produced water by reconstructing and annotating a Halanaerobium draft genome (named MDAL1), using shotgun metagenomic sequencing and metagenomic binning. The recovered draft genome was found to be closely related to the species H. congolense , an oil field isolate, and Halanaerobium sp. strain T82-1, also recovered from hydraulic fracturing produced water. Reconstruction of metabolic pathways revealed Halanaerobium sp. strain MDAL1 to have the potential for acid production, thiosulfate reduction, and biofilm formation, suggesting it to have the ability to contribute to corrosion, souring, and biofouling events in the hydraulic fracturing infrastructure. IMPORTANCE There are an estimated 15,000 unconventional gas wells in the Marcellus Shale region, each generating up to 8,000 liters of hypersaline produced water per day throughout its lifetime (K. Gregory, R. Vidic, and D. Dzombak, Elements 7:181-186, 2011, https://doi.org/10.2113/gselements.7.3.181; J. Arthur, B. Bohm, and M. Layne, Gulf Coast Assoc Geol Soc Trans 59:49-59, 2009; https://www.marcellusgas.org/index.php). Microbial activity in produced waters could lead to issues with corrosion, fouling, and souring, potentially interfering with hydraulic fracturing operations. Previous studies have found microorganisms contributing to corrosion, fouling, and souring to be abundant across produced water samples from hydraulically fractured wells; however, these findings were based on a limited number of samples and well sites. In this study, we investigated the microbial community structure in produced water samples from 42 unconventional Marcellus Shale wells, confirming the dominance of the genus Halanaerobium in produced water and its metabolic potential for acid and sulfide production and biofilm formation., (Copyright © 2017 American Society for Microbiology.)