Abstract
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.
Highlights
Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales
Using high-pressure growth reactors, shotgun proteomic measurements and proton nuclear magnetic resonance (1H-NMR) metabolomics analyses, we identified the potential for increased production of extracellular polymeric substances (EPS) and altered central metabolism under pressurized growth conditions
In the deep-shale environment, it is hypothesized that the degradation and fermentation of chemical additives, such as guar gum, support at least some Halanaerobium growth [7]
Summary
Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. These microorganisms occupy key roles in inferred metabolic networks that sustain microbial life in shale ecosystems, centered on the cycling of osmoprotectants and methylamine compounds [2, 8] The growth of such microorganisms in fractured shales is commonly viewed as deleterious, due to studies indicating that Halanaerobium spp. are able to catalyze thiosulfate-dependent sulfidogenesis [5], grow on additive chemicals present in input fluids [2, 7, 8], and potentially form biofilms in the subsurface. These processes could directly contribute to biofouling in the fracture network, leading to significant decreases in reservoir permeability and associated hydrocarbon recovery. While such processes are undesirable where hydrocarbons are being extracted, reductions in permeability in other geologic systems (e.g., sealing cap rock in geologic CO2 sequestration reservoirs) may be beneficial [9]
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