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Horizontal drilling and hydraulic fracturing extraction procedures have become increasingly present in Pennsylvania where the Marcellus Shale play is largely located. The potential for long-term environmental impacts to nearby headwater stream ecosystems and aquatic bacterial assemblages is still incompletely understood. Here, we perform high-throughput sequencing of the 16 S rRNA gene to characterize the bacterial community structure of water, sediment, and other environmental samples (n = 189) from 31 headwater stream sites exhibiting different histories of fracking activity in northwestern Pennsylvania over five years (2012-2016). Stream pH was identified as a main driver of bacterial changes within the streams and fracking activity acted as an environmental selector for certain members at lower taxonomic levels within stream sediment. Methanotrophic and methanogenic bacteria (i.e. Methylocystaceae, Beijerinckiaceae, and Methanobacterium) were significantly enriched in sites exhibiting Marcellus shale activity (MSA+) compared to MSA- streams. This study highlighted potential sentinel taxa associated with nascent Marcellus shale activity and some of these taxa remained as stable biomarkers across this five-year study. Identifying the presence and functionality of specific microbial consortia within fracking-impacted streams will provide a clearer understanding of the natural microbial community's response to fracking and inform in situ remediation strategies.

The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. This study investigates the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 μg/L. Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. Across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges, indicating niche-specific responses of these autochthonous populations. Altogether, our findings suggest that monitored natural attenuation is an appropriate remediation strategy for DCM contamination, and that high-throughput sequencing technologies are a robust method for assessing the potential role of biodegrading bacterial assemblages in the apparent reduction of DCM concentrations in environmental scenarios.