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Produced waters from hydraulically fractured shale formations give insight into the microbial ecology and biogeochemical conditions down-well. This study explores the potential for sulfide production by persistent microorganisms recovered from produced water samples collected from the Marcellus shale formation. Hydrogen sulfide is highly toxic and corrosive, and can lead to the formation of “sour gas” which is costly to refine. Furthermore, microbial colonization of hydraulically fractured shale could result in formation plugging and a reduction in well productivity. It is vital to assess the potential for sulfide production in persistent microbial taxa, especially when considering the trend of reusing produced waters as input fluids, potentially enriching for problematic microorganisms. Using most probable number (MPN) counts and 16S rRNA gene sequencing, multiple viable strains of bacteria were identified from stored produced waters, mostly belonging to the Genus Halanaerobium, that were capable of growth via fermentation, and produced sulfide when supplied with thiosulfate. No sulfate-reducing bacteria (SRB) were detected through culturing, despite the detection of relatively low numbers of sulfate-reducing lineages by high-throughput 16S rRNA gene sequencing. These results demonstrate that sulfidogenic produced water populations remain viable for years post production and, if left unchecked, have the potential to lead to natural gas souring during shale gas extraction.

Hydraulic fracturing is the prevailing method for enhancing recovery of hydrocarbon resources from unconventional shale formations, yet little is understood regarding the microbial impact on biogeochemical cycling in natural-gas wells. Although the metabolisms of certain fermentative bacteria and methanogenic archaea that dominate in later produced fluids have been well studied, few details have been reported on microorganisms prevelant during the early flowback period, when oxygen and other surface-derived oxyanions and nutrients become depleted. Here, we report the isolation, genomic and phenotypic characterization of Marinobacter and Arcobacter bacterial species from natural-gas wells in the Utica-Point Pleasant and Marcellus Formations coupled to supporting geochemical and metagenomic analyses of produced fluid samples. These unconventional hydrocarbon system-derived Marinobacter sp. are capable of utilizing a diversity of organic carbon sources including aliphatic and aromatic hydrocarbons, amino acids, and carboxylic acids. Marinobacter and Arcobacter can metabolize organic nitrogen sources and have the capacity for denitrification and dissimilatory nitrate reduction to ammonia (DNRA) respectively; with DNRA and ammonification processes partially explaining high concentrations of ammonia measured in produced fluids. Arcobacter is capable of chemosynthetic sulfur oxidation, which could fuel metabolic processes for other heterotrophic, fermentative, or sulfate-reducing community members. Our analysis revealed mechanisms for growth of these taxa across a broad range of salinities (up to 15% salt), which explains their enrichment during early natural-gas production. These results demonstrate the prevalence of Marinobacter and Arcobacter during a key maturation phase of hydraulically fractured natural-gas wells, and highlight the significant role these genera play in biogeochemical cycling for this economically important energy system.