Microbial Adaptations to High Salinity in Hydraulically Fractured Shale Enabled through Integrated `Omics' Analysis

Black shales are increasingly exploited for their petroleum resources using horizontal drilling and hydraulic fracturing technologies. This engineered process involves injection of high volumes of fluids containing water, chemical additives, and microorganisms into low permeability rock to extend fractures in the deep subsurface for increased oil and gas production. A portion of injected fluids returns to the surface, becoming increasingly saline as they equilibrate with rock brines. In this system, salinity and oxygen availability are major drivers of microbial community composition and function, with produced fluid geochemistry approaching anaerobic brine levels during the first 3-6 months of natural gas production. Here, we investigated the mechanisms for osmotolerance in microbial metagenomes from hydraulically fractured shale wells in the Marcellus Shale region of the Appalachian Basin. With increasing salinity and decreased microbial diversity, we identified three osmotolerance adaptations. Firstly, genes for sodium and potassium ion (Na⁺/K⁺) transport increase in relative abundance with time after fracturing as do genes for osmolyte import/biosynthesis. Therefore, bacteria in this system use both salt-in strategy, where cells maintain osmotic balance by K⁺ importation coupled to Na⁺ export, and compatible-solute strategy, where cells gain osmotolerance via importation/synthesis of organic osmolytes for osmotolerance. Next, as salinity increases, a higher sodium ion (Na⁺) gradient outside the cell results in sodium-dependent ATP synthesis. Microorganisms also modify their membrane lipids, including the polar lipid headgroups as well as the fatty acid length and saturation, which results in a reduction of membrane permeability at higher salinity. These processes work in tandem to aid in the osmotolerance of the shale microflora population. This work provides an improved fundamental understanding of microbial physiological adaptations to rapid changes in salinity in deep engineered shales, with implications on halotolerant microbial energetics in this and other hypersaline environments.