Temporal Changes in Microbial Metagenomic Signatures and Lipid Profiles After Fracturing in the Marcellus Shale

Abstract:

Shale gas formations represent understudied deep biosphere ecosystems with important implications to terrestrial biogeochemical cycles and global energy resources. Recent 16S rRNA gene studies examining temporal microbial community dynamics of returned fluids from hydraulically fractured wells in the Marcellus Shale indicate ecosystem changes from aerobic, low-salt associated microbes in injected fluids to anaerobic, halophilic taxa in produced fluids several months after fracturing. To further characterize changes in the ecology, functional potential and biosignatures of observed taxa, we sequenced genomic DNA from three key time points after fracturing (T7, T82, and T328; Tn, n = days) and analyzed their lipid signatures. The metagenomic profiles verify 16S rRNA gene trends, revealing strain-type changes in dominant Bacteria of Marinobacter, Halomonas, and Halanaerobium and the Archaeal genus Methanolobus through time. Novel species within the γ-Proteobacteria were also observed. Reconstructed genomes show as bioavailable N decreases through time, genes associated with N₂ fixing and obtaining N from organic pools (ncd2, nit1, and eutCB) increase in T82 and T328 samples after oxidized nitrogen species (NO₃) are depleted. Further, S oxidizing genes were only detected in the T7 sample with incomplete pathways for dissimilatory sulfate reduction (DSR). Later time points showed an increase in abundance of sulfonate importer genes and the anaerobic DSR gene, asrA, suggesting the use of sulfite and sulfonates for S acquisition after sulfate is depleted. Lipid analyses confirmed distinct profiles between T82 and T328 and revealed differences in 16 and 18 C monounsaturated fatty acids, indicative of gram (-) bacteria. The lipid profile from T328 was markedly less diverse than that of T82 and indicated a very limited community, as supported by the 16S rRNA gene and metagenomic data. This research integrates metagenomic data with lipid profiles to characterize temporal changes in biosignatures and the functional potential of N and S metabolic genes of deep shale microbes.