Hydraulic fracturing offers view of microbial life in the deep terrestrial subsurface.

Horizontal drilling and hydraulic fracturing are increasingly used for recovering energy resources in black shales across the globe. Although newly drilled wells are providing access to rocks and fluids from kilometer depths to study the deep biosphere, we have much to learn about microbial ecology of shales before and after 'fracking'. Recent studies provide a framework for considering how engineering activities alter this rock-hosted ecosystem. We first provide data on the geochemical environment and microbial habitability in pristine shales. Next, we summarize data showing the same pattern across fractured shales: diverse assemblages of microbes are introduced into the subsurface, eventually converging to a low diversity, halotolerant, bacterial and archaeal community. Data we synthesized show that the shale microbial community predictably shifts in response to temporal changes in geochemistry, favoring conservation of key microorganisms regardless of inputs, shale location or operators. We identified factors that constrain diversity in the shale and inhibit biodegradation at the surface, including salinity, biocides, substrates and redox. Continued research in this engineered ecosystem is required to assess additive biodegradability, quantify infrastructure biocorrosion, treat wastewaters that return to the surface and potentially enhance energy production through in situ methanogenesis.

Temporal changes in microbial ecology and geochemistry in produced water from hydraulically fractured Marcellus shale gas wells.

Microorganisms play several important roles in unconventional gas recovery, from biodegradation of hydrocarbons to souring of wells and corrosion of equipment. During and after the hydraulic fracturing process, microorganisms are subjected to harsh physicochemical conditions within the kilometer-deep hydrocarbon-bearing shale, including high pressures, elevated temperatures, exposure to chemical additives and biocides, and brine-level salinities. A portion of the injected fluid returns to the surface and may be reused in other fracturing operations, a process that can enrich for certain taxa. This study tracked microbial community dynamics using pyrotag sequencing of 16S rRNA genes in water samples from three hydraulically fractured Marcellus shale wells in Pennsylvania, USA over a 328-day period. There was a reduction in microbial richness and diversity after fracturing, with the lowest diversity at 49 days. Thirty-one taxa dominated injected, flowback, and produced water communities, which took on distinct signatures as injected carbon and electron acceptors were attenuated within the shale. The majority (>90%) of the community in flowback and produced fluids was related to halotolerant bacteria associated with fermentation, hydrocarbon oxidation, and sulfur-cycling metabolisms, including heterotrophic genera Halolactibacillus, Vibrio, Marinobacter, Halanaerobium, and Halomonas, and autotrophs belonging to Arcobacter. Sequences related to halotolerant methanogenic genera Methanohalophilus and Methanolobus were detected at low abundance (<2%) in produced waters several months after hydraulic fracturing. Five taxa were strong indicators of later produced fluids. These results provide insight into the temporal trajectory of subsurface microbial communities after "fracking" and have important implications for the enrichment of microbes potentially detrimental to well infrastructure and natural gas fouling during this process.

Role of norEF in denitrification, elucidated by physiological experiments with Rhodobacter sphaeroides.

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (Vmax and Ks), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in Vmax but no change in apparent Ks. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.

Microbial community changes in hydraulic fracturing fluids and produced water from shale gas extraction.

Microbial communities associated with produced water from hydraulic fracturing are not well understood, and their deleterious activity can lead to significant increases in production costs and adverse environmental impacts. In this study, we compared the microbial ecology in prefracturing fluids (fracturing source water and fracturing fluid) and produced water at multiple time points from a natural gas well in southwestern Pennsylvania using 16S rRNA gene-based clone libraries, pyrosequencing, and quantitative PCR. The majority of the bacterial community in prefracturing fluids constituted aerobic species affiliated with the class Alphaproteobacteria. However, their relative abundance decreased in produced water with an increase in halotolerant, anaerobic/facultative anaerobic species affiliated with the classes Clostridia, Bacilli, Gammaproteobacteria, Epsilonproteobacteria, Bacteroidia, and Fusobacteria. Produced water collected at the last time point (day 187) consisted almost entirely of sequences similar to Clostridia and showed a decrease in bacterial abundance by 3 orders of magnitude compared to the prefracturing fluids and produced water samplesfrom earlier time points. Geochemical analysis showed that produced water contained higher concentrations of salts and total radioactivity compared to prefracturing fluids. This study provides evidence of long-term subsurface selection of the microbial community introduced through hydraulic fracturing, which may include significant implications for disinfection as well as reuse of produced water in future fracturing operations.

Microbial communities in flowback water impoundments from hydraulic fracturing for recovery of shale gas.

Hydraulic fracturing for natural gas extraction from shale produces waste brine known as flowback that is impounded at the surface prior to reuse and/or disposal. During impoundment, microbial activity can alter the fate of metals including radionuclides, give rise to odorous compounds, and result in biocorrosion that complicates water and waste management and increases production costs. Here, we describe the microbial ecology at multiple depths of three flowback impoundments from the Marcellus shale that were managed differently. 16S rRNA gene clone libraries revealed that bacterial communities in the untreated and biocide-amended impoundments were depth dependent, diverse, and most similar to species within the taxa γ-proteobacteria, α-proteobacteria, δ-proteobacteria, Clostridia, Synergistetes, Thermotogae, Spirochetes, and Bacteroidetes. The bacterial community in the pretreated and aerated impoundment was uniform with depth, less diverse, and most similar to known iodide-oxidizing bacteria in the α-proteobacteria. Archaea were identified only in the untreated and biocide-amended impoundments and were affiliated to the Methanomicrobia class. This is the first study of microbial communities in flowback water impoundments from hydraulic fracturing. The findings expand our knowledge of microbial diversity of an emergent and unexplored environment and may guide the management of flowback impoundments.

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025).

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.

Mechanisms of oxygen inhibition of nirK expression in Rhodobacter sphaeroides.

R. sphaeroides strain 2.4.3, when lacking the cbb(3) oxidase, is unable to transition from aerobic respiration to denitrification using cellular respiration as a means of reducing oxygen levels. This is due to an inability to express nirK, the gene encoding nitrite reductase. Under certain photosynthetic conditions this strain can transition from aerobic to nitrate respiration, demonstrating that nirK expression can occur in the absence of a functional cbb(3) oxidase. If oxygen levels are reduced under non-photosynthetic conditions using low-oxygen gas mixes, nitrite reductase activity is detected at wild-type levels in the strain lacking the oxidase. In addition, co-culture experiments show that incubation of the cbb(3) deficient strain 2.4.3 with R. sphaeroides 2.4.1, which is nirK deficient but has the high-affinity cbb(3) oxidase, restores denitrification in sealed-vessel experiments. Taken together these results indicate that high end-point O(2) levels are the reason why the strain lacking the cbb(3) oxidase cannot transition from aerobic respiration to denitrification under certain conditions. The protein probably being affected by these O(2) levels is the transcriptional regulator NnrR.

Identification, functional studies, and genomic comparisons of new members of the NnrR regulon in Rhodobacter sphaeroides.

Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain. nnrV, under control of a previously identified NnrR site, was also identified. paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase. paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome c(2) together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions.

Agrobacterium tumefaciens C58 uses ActR and FnrN to control nirK and nor expression.

Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression.