MICROBES LIVING IN UNCONVENTIONAL SHALE DURING ENERGY EXTRACTION HAVE DIVERSE HYDROCARBON DEGRADATION PATHWAY

Diverse microorganisms have been identified in produced fluids from unconventional shale wells, but little is know of their hydrocarbon degrading capacity as it relates to shale organic matter or organic additives used in hydraulic fracturing operations. We sequenced the metagenomes from five temporal samples recovered from injected and flowback fluids for Marcellus shale wells and reconstructed 34 near-complete genomes. Hydrocarbon degrading pathways changed with time after flowback and varied for strains detected at different time points. Three genera, including Pseudomonas, Marinobacter, and Marinobacterium showed complete pathways for aerobic benzene, toluene, ethylbenzene, xylene, or naphthalene (BTEX-N) degradation in early time points, (injected fluids and samples collected during two weeks of flowback). These genera employed oxygenase enzymes to transform aromatic compounds to phenolic, benzoate, or catechol structures. Catechol/methylcatechol compounds were further degraded to small carboxylic acids or –CoA compounds for entry into glyoxylate, glycolysis, propanoate or TCA systems. Two other genera, Vibrio and Halomonas, could not catalyze the first steps of BTEX-N degradation, but were capable of degrading hydrocarbon metabolites from benzoate compounds through the TCA cycle. Interestingly, we failed to recover genes for aerobic or anaerobic degradation from partially-complete Marinobacter and Halomonas strains detected in samples collected 328 days into production, suggesting active in situ hydrocarbon degradation may be sustained by input materials rather than shale-derived hydrocarbons. All five genomes contained alcohol and aldehyde dehydrogenases, important for degradation of surfactant additives. To put these data in context with detected compounds, we found BTEX and cycloaromatics in injected and produced fluids, along with methylated benzenes, glycols, ketones, and ethoxylated alcohol xenobiotics. Identified pathways are therefore consistent with detected metabolites, and suggest that microorganisms play an important role in degrading organic compounds used for well completions via aerobic respiration during early phases, changing to fermentative metabolism during later production phases when oxidative power has been depleted within the well.