Potential Repercussions Associated with Halanaerobium Colonization of Hydraulically Fractured Shales

Hydraulic fracturing of black shale formations has greatly increased U.S. oil and natural gas recovery. Bacterial Halanaerobium strains become the dominant microbial community member in produced fluids from many fractured shales, regardless of their geographic location. Halanaerobium is not native to the subsurface, but is inadvertently introduced during the drilling and fracturing process. The accumulation of biomass in pipelines and reservoirs is detrimental due to possible well souring, microbially-induced corrosion, and pore clogging. Here, we used Halanaerobium strains isolated from a hydraulically fractured well in the Utica Shale, proteogenomics, isotopic and geochemical field observations, and laboratory growth experiments to identify detrimental effects associated with Halanaerobium growth. Analysis of Halanaerobium isolate genomes and reconstructed genomes from metagenomic datasets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes that can convert thiosulfate to sulfide. Furthermore, laboratory growth curves confirmed the capability of Halanaerobium to grow across a wide range of pressures (14-7000 PSI). Shotgun proteomic measurements were used to track the higher abundance of rhodanese and anaerobic sulfite reductase enzymes present when thiosulfate was available in the growth media. This technique also identified a higher abundance of proteins associated with the production of extracellular polymeric substances when Halanaerobium was grown under increasing pressures. Halanaerobium culture based assays identified thiosulfate-dependent sulfide production, while pressure incubations revealed higher cellular attachment to quartz surfaces. Increased production of sulfide and organic acids during stationary growth phase suggests that fermentative Halanaerobium use thiosulfate to remove excess reductant, aiding in NAD+ recovery. Additionally, the increased cellular attachment to surfaces under pressure indicates Halanaerobium has the capability of forming cellular clusters that could clog the shale fracture network and limit natural gas recovery. These findings bring awareness to the detrimental effects that could arise from Halanaerobium growth in hydraulically fractured shales throughout the U.S.