Deep-Subsurface Pressure Stimulates Metabolic Plasticity in Shale-Colonizing Halanaerobium spp

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.

Evidence of sporulation capability of the ubiquitous oil reservoir microbeHalanaerobium congolense

“…H. congolense is anaerobic, has an optimal growth temperature of 42 C and optimal salinity concentration range of 10-15% (Ravot et al 1997), coinciding with many subsurface reservoir conditions where it is frequently found. The observation of this organism in unconventional hydraulically fractured reservoirs has been previously reported (Booker et al 2019;Daly et al 2016;Mouser et al 2016). Indigenous organisms are not likely in unconventional reservoirs due to the lack of nutrient flux within low permeability shale and minimal accommodation in shale organic porosity, which supports the hypothesis that these organisms are introduced by oil and gas drilling and production activities.…”

In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing

“…Although carbon was not limited in our experiments, we observed significantly higher abundances of the universal stress protein (uspA, 10238) in surfactant amended cultures ( Supplementary File 4). Moreover, in a recent study on a closely-related Halanaerobium strain isolated from the same natural-gas well (H. congolense WG8), the methylglyoxal bypass was initiated under growth at high pressures with glucose as the sole carbon source, possibly to dispose of excess reducing equivalents [48]. Here, we propose cometabolism of glycols occurs either through cellular stress and/or high concentrations of glycols within the medium, activating both the methylglyoxal bypass and the propanediol dehydratase pathways.…”

“…2a). Although there is currently no characterized enzyme for the biotransformation of PPGs under anaerobic conditions, propylene glycol, the monomer of PPGs, can be degraded by a diol dehydratase under both aerobic and anaerobic conditions, generating propionaldehyde, n-propanol, propionate, and acetone as products [33,[47][48][49]. We therefore mined metagenomes from Utica-Point Pleasant produced fluid samples for genes encoding enzymes known to degrade polyglycols to determine whether the aforementioned microbial biotransformation pathway existed in this system.…”

Identification of Persistent Sulfidogenic Bacteria in Shale Gas Produced Waters

“…Metabolic pathway analyses have revealed that Halanaerobium species have the potential for thiosulfate reduction, acid production and biofilm formation (Lipus et al, 2017), potentially making this a highly problematic microorganism during shale gas extraction. Indeed this ability has led to an emerging interest in the role that Halanaerobium species may have in the biogenesis of sulfide and corrosive organic acids, such as acetate (Booker et al, 2017(Booker et al, , 2019. It is therefore unsurprising that Halanaerobium spp.…”

“…Additionally, the formation of microbial biofilms may impede gas flow and result in fracture clogging (Bottero et al, 2010). Evidence suggests that organic chemicals used during hydraulic fracturing may stimulate these deleterious processes, for example via organic acid production from fermentation reactions, and sulfide production from sulfate and thiosulfate reduction pathways (Struchtemeyer et al, 2011;Liang et al, 2016;Booker et al, 2017Booker et al, , 2019Nixon et al, 2017;Evans et al, 2019). Sulfate-reducing bacteria (SRB) have been identified previously in production fluids through next generation sequencing but their in situ role in sulfide production is undetermined (Davis et al, 2012;Struchtemeyer and Elshahed, 2012;Liang et al, 2016), whereas TRB have been isolated from these environments and represent an alternative route to sulfide production (Ravot et al, 1997(Ravot et al, , 2005Liang et al, 2016;Booker et al, 2017;Lipus et al, 2017).…”