Microbial activity in the produced water from hydraulically fractured oil and gas wells may potentially interfere with hydrocarbon production and cause damage to the well and surface infrastructure via corrosion, sulfide release, and fouling. In this study, we surveyed the microbial abundance and community structure of produced water sampled from 42 Marcellus Shale wells in southwestern Pennsylvania (well age ranged from 150 to 1,846 days) to better understand the microbial diversity of produced water. We sequenced the V4 region of the 16S rRNA gene to assess taxonomy and utilized quantitative PCR (qPCR) to evaluate the microbial abundance across all 42 produced water samples. Bacteria of the order were found to be the most abundant organisms in the majority of the produced water samples, emphasizing their previously suggested role in hydraulic fracturing-related microbial activity. Statistical analyses identified correlations between well age and biocide formulation and the microbial community, in particular, the relative abundance of We further investigated the role of members of the order in produced water by reconstructing and annotating a draft genome (named MDAL1), using shotgun metagenomic sequencing and metagenomic binning. The recovered draft genome was found to be closely related to the species , an oil field isolate, and sp. strain T82-1, also recovered from hydraulic fracturing produced water. Reconstruction of metabolic pathways revealed sp. strain MDAL1 to have the potential for acid production, thiosulfate reduction, and biofilm formation, suggesting it to have the ability to contribute to corrosion, souring, and biofouling events in the hydraulic fracturing infrastructure. There are an estimated 15,000 unconventional gas wells in the Marcellus Shale region, each generating up to 8,000 liters of hypersaline produced water per day throughout its lifetime (K. Gregory, R. Vidic, and D. Dzombak, Elements 7:181-186, 2011, https://doi.org/10.2113/gselements.7.3.181; J. Arthur, B. Bohm, and M. Layne, Gulf Coast Assoc Geol Soc Trans 59:49-59, 2009; https://www.marcellusgas.org/index.php). Microbial activity in produced waters could lead to issues with corrosion, fouling, and souring, potentially interfering with hydraulic fracturing operations. Previous studies have found microorganisms contributing to corrosion, fouling, and souring to be abundant across produced water samples from hydraulically fractured wells; however, these findings were based on a limited number of samples and well sites. In this study, we investigated the microbial community structure in produced water samples from 42 unconventional Marcellus Shale wells, confirming the dominance of the genus in produced water and its metabolic potential for acid and sulfide production and biofilm formation.

The Bakken Shale has become one of the United States' most important oil and gas producing regions. This study examined the microbiology and geochemical characteristics of Bakken region produced water from 17 well sites sampled from the three-phase separator and produced water holding tank over a 6-month time frame. Produced water samples had high total dissolved solids (220 000-350 000 mg/L) and low dissolved organic carbon concentrations (41-132 mg/L). Microbial abundances varied between 101 and 104 16S rRNA gene copies/mL, approximately four orders of magnitude below those observed for produced waters from other hydraulic fracturing regions. The most abundant bacterial orders found in produced water samples were Bacillales, Halanaerobiales and Pseudomonadales, consistent with observations from other unconventional resource plays. Our observations suggest temporal community structuring, as produced waters sampled early in our sampling period were dominated by Halanaerobiales, and produced waters sampled at the remaining winter sampling time points were characterized by high relative abundances of Bacillales and Pseudomonadales. Data from this study extends the current available knowledge of the microbiology and chemistry associated with produced water from the Bakken region and provides insights into microbial community dynamics in hypersaline subsurface fluids.

Microbial activity during the holding and reuse of wastewater from hydraulic fracturing operations, termed produced water, may lead to issues with corrosion, sulfide release, and fouling. Biocides are applied to control biological activity, often with limited efficacy, which is typically attributed to chemical interactions with the produced water. However, it is unknown whether there is a biologically driven mechanism to biocide tolerance in produced water. Here, we demonstrate that produced water exposure results in an enhanced tolerance against the typically used biocide glutaraldehyde and increased susceptibility to the oxidative biocide hypochlorite in a native and a model bacteria and that this altered resistance is due to the salinity of the produced water. In addition, we elucidate the genetic response of the model organism Pseudomonas fluorescens to produced water exposure to provide a mechanistic interpretation of the altered biocide resistance. The RNA-seq data demonstrated the induction of genes involved in osmotic stress, energy production and conversion, membrane integrity, and protein transport following produced water exposure, which facilitates bacterial survival and alters biocide tolerance. Efforts to fundamentally understand biocide resistance mechanisms, which enable the optimization of biocide application, hold significant implications for greening of the fracturing process through encouraging produced water recycling. Specifically, these results suggest the necessity of optimizing biocide application at the level of individual shale plays, rather than historical experience, based upon produced water characteristics and salinity.

Microbial activity in produced water from hydraulic fracturing operations can lead to undesired environmental impacts and increase gas production costs. However, the metabolic profile of these microbial communities is not well understood. Here, for the first time, we present results from a shotgun metagenome of microbial communities in both hydraulic fracturing source water and wastewater produced by hydraulic fracturing. Taxonomic analyses showed an increase in anaerobic/facultative anaerobic classes related to Clostridia, Gammaproteobacteria, Bacteroidia and Epsilonproteobacteria in produced water as compared to predominantly aerobic Alphaproteobacteria in the fracturing source water. The metabolic profile revealed a relative increase in genes responsible for carbohydrate metabolism, respiration, sporulation and dormancy, iron acquisition and metabolism, stress response and sulfur metabolism in the produced water samples. These results suggest that microbial communities in produced water have an increased genetic ability to handle stress, which has significant implications for produced water management, such as disinfection.