Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface. S hale gas accounts for one-third of natural gas energy resources worldwide. It has been estimated that shale gas will provide half of the natural gas in the USA, annually, by 2040, with the Marcellus shale in the Appalachian Basin projected to produce three times more than any other formation 1 . Recovery of these hydrocarbons is dependent on hydraulic fracturing technologies, where the high-pressure injection of water and chemical additives generates extensive fractures in the shale matrix. Hydrocarbons trapped in tiny pore spaces are subsequently released and collected at the wellpad surface, together with a portion of the injected fluids that have reacted with the shale formation. The mixture of injected fluids and hydrocarbons collected is referred to as 'produced fluids'.Microbial metabolism and growth in hydrocarbon reservoirs has both positive and negative impacts on energy recovery. Whereas stimulation of methanogens in coal beds enhances energy recovery 2 , bacterial hydrogen sulfide production ('reservoir souring') decreases profits and contributes to corrosion and the risk of environmental contamination 3 . Additionally, biomass accumulation within newly generated fractures may reduce their permeability, decreasing natural gas recovery. Despite these potential microbial impacts, little is known about the function and activity of microorganisms in hydraulically fractured shale.Initial work by our group and others 4-9 used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing. To assign functional roles to these organisms, we conducted metagenomic and metabolite analyses on input and produced fluids up to a year after hydraulic fracturing (HF) from two Appala...
