Temporal Changes in Microbial Ecology and Geochemistry in

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Temporal Changes in Microbial Ecology and Geochemistry in Produced Water from Hydraulically Fractured Marcellus Shale Gas Wells Maryam A. Cluff,† Angela Hartsock,‡,§ Jean D. MacRae,∥ Kimberly Carter,‡,⊥ and Paula J. Mouser*,† †

Department of Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, Ohio 43210, United States National Energy Technology Laboratory, U.S. Department of Energy, Pittsburgh, Pennsylvania 15236, United States § Department of Biology, University of Akron-Wayne College, Orrville, Ohio 44667, United States ∥ Department of Civil and Environmental Engineering, University of Maine, Orono, Maine 04469, United States ⊥ Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States ‡

S Supporting Information *

ABSTRACT: 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 (85%) of detected taxa in our samples (Figure 2). The 16S rRNA sequences shifted from microorganisms typical of surface water communities that were present in injected fluids to sequences with high identity to halotolerant and thermophilic microorganisms during the flowback period and production phase. Pseudomonas, Cobetia, Arcobacter, Pseudoalteromonas, and Marinobacterium genera initially dominated in injected fluids. Halanaerobium was also prominent in injected fluids for Well 3, possibly due to fluid recycling. Halolactibacillus and Vibrio genera increased during the first 2 weeks of flowback (Figure 2). Sequences similar to Burkholderia and Arcobacter were also enriched in relative abundance during the initial flowback period while Idiomarina and Marinobacter increased after about 2 weeks. More than a month after the hydraulic fracturing process, Halanaerobium and Halomonas species greatly dominated detected sequences and continued to dominate at 11 months. Halanaerobium and Marinobacter were consistently detected throughout the three sampling phases. Conversely, the bacterial genus Selenihalanaerobacter and archaeal genera Methanohalophilus and Methanolobus were only detected in produced waters more than two months after hydraulic fracturing occurred (Figure 2). Higher richness was observed in injected fluids, while flowback period and produced fluids generally had lower values (Table 1). Shannon diversity averaged 2.15 ± 0.60 for samples collected within the first 5 days postfracturing and 0.82 ± 0.47 after this time (Table 1). The lowest overall richness and diversity estimates were observed at 49 days after hydraulic fracturing. NMDS analysis of Well 1 and Well 2 samples showed a community shift from the injected population across the x-axis (NMDS 1) during the first 4−5 days of flowback (Figure 3). After this time, an ecological shift away from the initial injected community occurred along the y-axis (NMDS 2) and then back across the x-axis (NMDS 1). This almost circular trajectory is not surprising given that the injected fluids contained recycled flowback fluids. Samples produced at 49 days or later had a distinctly different community composition than those at earlier time points and were separated from injected and flowback samples across NMDS 1. By defining our groups as injected fluids (day 0), initial flowback period (days 4−13.3), and later produced waters (days 49−328), we detected significant differences (anosim, p = 0.002) in their community membership with little change in estimates of viable biomass (data not shown). There was a significant difference in our group variability (betadisp, p < 0.001), with initial flowback period fluids having the largest average distance to group centroid and later produced waters having the lowest (Figure S1, Supporting Information). Combining sequences within these compositionally distinct groups (Figures 2 and 3), we generated pie charts showing the average relative abundance of the dominant taxa during each sampling phase. Cobetia within the family Halomonadaceae (45%), Pseudomonas (17%), and other unclassified bacteria (15%) comprised the majority of sequences in injected fluids. After this time, Halolactibacillus (33%), Arcobacter (17%), Vibrio (9.4%), Thermococcus (8.6%), and Marinobacter (7.3%) dominated in relative abundance during the initial flowback period. In later produced waters, sequences closely related to Halanaerobium (86%), Halomonas within the family Halomonadaceae (2.4%), and other Halomonadaceae (5.3%) were detected most often (Figure 3). Community shifts were

approaching an asymptote several months after hydraulic fracturing. A significant inverse trend was observed between chloride and DOC concentrations during the initial flowback period and later production samples (ρ = −0.96, p < 0.0001 Pearson correlation product). Injected DOC primarily reflects seven organic-based additives containing constituents derived from ethyl and ether glycol formulations, hydrocarbon distillates, and a natural formulation of sucrose and dextrose polymers known as guar gum (Table S1, Supporting Information). Organic carbon in the injected fluids may be sorbed, exchanged, or diluted within formation fluids, reacted within the rock-fluid matrix or through microbially mediated mineralization of organic constituents.20,29 Measured DOC at 328 days (37.5 ± 1.5 mg/L) was within the range of levels reported in other Marcellus shale produced fluids14 and similar in concentration to mature fluids produced from shallower Devonian shale (range of 1 to 47 mg/L DOC).28 The DOC in our produced fluids is likely to reflect the hydrophilic fraction of residual fracturing fluid additives, their transformation products, or hydrocarbons released from the formation, such as low-molecular weight alkanes, isoprenoids, and polyaromatic compounds.28 To further define these compounds, we used LC-QTOF and GC-MS to characterize select organic constituents in injected, flowback period, and produced waters. While exact concentrations could not be determined due to the lack of available standards for many compounds, we identified benzene compounds (benzene, dimethylbenzene (C2-benzene), trimethylbenzenes (C3-benzene), and tetramethylbenzenes (C4-benzene)), naphthalene, and toluene with little change in spectra between injected and flowback fluids (Table S2, Supporting Information). Trimethylnaphthalene, undecane (C11H24), and dodecane (C12H26) compounds were identified in flowback fluids but were not detected in injected fluids. Ethoxylated surfactants with larger masses decreased in abundance between injected and flowback fluids, whereas ethoxylated surfactants with smaller masses showed little change across the samples, possibly due to steric effects or other unknown biogeochemical factors. 4-Ethoxybenzoic acid ethyl ester was identified in injected fluids but absent from flowback fluids. Acetic acid, on the other hand, was identified solely in flowback fluids, suggesting production of this compound by fermentation or incomplete oxidation of injected compounds or gas constituents. Aqueous Geochemistry Changes after Hydraulic Fracturing. Total ionic content as measured by conductivity increased by a factor of 3 between injected fluids and waters produced at 11 months after fracturing. Anions (Cl− and Br−), divalent cations (Ba2+, Ca2+, and Mg2+), and monovalent cations (K+ and Na+) increased until 82 days and then remained relatively constant or decreased between day 82 and 328, suggesting that concentrations had reached equilibrium within the formation (Table 1). Total iron generally increased over the sample period, with the highest measured levels at 328 days. We detected elemental phosphorus and sulfur at levels near detection limits, with no apparent trend. Nitrate, nitrite, and sulfate concentrations were all found to be below the detection limit for all samples (100 mg/L due to high sample dilutions). The pH values varied between 5.2 and 6.3, while DIC and DOC values decreased with time after fracturing. TDN levels increased during the flowback period through the first few months after hydraulic fracturing and then decreased between day 82 and 328 (Table 1). 6511

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0.2 1.6 1.9 12.5 4.8 8.5 20.1 18.9 6.7 21.4 22.4 33.6 2.2 26.9 58.4 33.2 73.6 87.3 126

3.2 2.7 3.3 BDL 3.3 8.8 BDL BDL 2.5 BDL 2.1 1.5 BDL 2.6 BDL 2.5 2.6 1.3 2.5

4.0 18.3 3.6 11.7 9.5 9.3 10.2 11.5 8.5 11.5 4.8 9.3 6.9 BDL 1.0 BDL BDL 1.3 6.1

significantly correlated (p < 0.01) to decreased alkalinity, inorganic carbon and organic carbon concentrations (pH, DIC, and DOC) and a significant increase (p < 0.01) in ionic content (conductivity, TN, Br−, total Fe, and K+) (Table 1 and Figure 3). Specifically, higher carbon and pH values were associated with injected fluids and initial flowback period samples (4−5 days) while higher ionic content was associated with later produced waters (Table 1 and Figure 3). Five OTUs were found to be significant (p < 0.025) indicator genera. Among these, Methanohalophilus within Methanosarcinaceae, Selenihalanaerobacter within Halobacteroidaceae, Flexistipes within Deferribacteraceae, and other unclassified Halanaerobiaceae were solely detected in later produced samples, while Halanaerobium within Halanaerobiaceae was detected throughout all fluid phases but greatly dominated sequences in later produced waters. Cond, conductivity. DIC, dissolved inorganic carbon. DOC, dissolved organic carbon. TDN, total dissolved nitrogen. BDL, below detection limit.

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DISCUSSION Studies on microbial communities in fluids used for hydraulic fracturing (freshwater and mixtures of holding ponds and/or recycled produced fluids) show they contain diverse bacteria frequently associated with surface freshwater ecosystems or low-salt estuaries, including the phyla Actinobacteria, Bacteroidetes, and Proteobacteria (Alpha, Beta, and Gamma).23,33 Gammaproteobacteria detected in injected fluids from this study were associated with Gram-negative heterotrophs Cobetia and Pseudomonas. Cobetia spp. including C. marina, C. crustatorum, and C. pacif ica are capable of growth on glucose, glycerol, or sucrose in salty (∼5% mass/vol) aerobic environments49−51 and often used as model organisms to investigate biofilm mechanisms and antifouling agents on seawater surfaces.52,53 OTUs within the genus Pseudomonas and other unclassified Pseudomonadaceae also comprised a sizable portion of sequences detected in injected fluids from this site. Pseudomonas sequences were also abundant in Barnett shale flowback22 and on a metal cartridge deployed to 1800 m in a Haynesville shale well.29 The Gram-negative Pseudomonas is known for its ability to colonize a wide range of niches where aliphatic and aromatic hydrocarbons are present,54−57 including flooded oil reservoirs.58,59 Detected Pseudomonas were most closely related to Gram-negative P. stutzeri (97−98%) and may be involved in the oxidation of simple or complex organic carbon (including alkane or aromatic compounds) present in injected fluids injected at this site and coupled to nitrate reduction.59,60 Although nitrate levels were below detection limits in this study (100 mg/L) due to sample dilution prior to analysis, our TDN concentrations show nitrogen is present in injected and produced fluids, probably as more oxidized species in injected (surface) fluids recently in contact with the atmosphere, as reported in other Marcellus flowback samples.33 During initial flowback, sequences with high similarity to the deep-sea lactic-acid bacteria Halolactibacillus miurensis (98−99% similarity)61 combined with the detection of acetate provide some evidence that fermentation may occur. This is not surprising given that industry selectively controls for acid-producing bacteria in oil and gas wells using biocides.31 Many different Firmicutes are enriched in flowback fluids from hydraulically fractured wells from other shale systems.22,23,32 While Halolactibacillus were not specifically noted in these referenced studies, they or other fermentative Firmicutes may be important transitional community members producing labile substrates from injected organic compounds or formation constituents that support energy metabolism and carbon assimilation for other trophic levels within

a

56 52 56 121 126 155 164 173 257 185 277 249 267 444 516 398 408 343 300 0 0 0 3.5 4 5 6.5 7 7 7.7 7.7 9.0 13.3 49 49 82 82 328 328 1 2 3 3 1 2 1 1 3 2 3 2 1 1 2 1 2 1 2 well well well well well well well well well well well well well well well well well well well

injected injected injected flowback flowback flowback flowback flowback flowback flowback flowback flowback flowback produced produced produced produced produced produced

9904 3397 2014 452 324 274 15 546 15 286 2954 4101 141 12 011 18 109 8732 9279 18 037 15 192 7570 5281

245 89 66 41 30 38 60 52 100 20 11 61 61 18 14 80 78 42 22

453 167 132 54 34 70 320 192 235 42 14 161 143 27 25 178 189 81 50

2.77 1.12 1.94 2.60 2.46 2.01 1.21 0.16 1.41 0.92 1.63 0.50 1.08 0.06 0.39 0.62 0.94 0.77 0.97

0.14 0.63 0.24 0.13 0.16 0.33 0.35 0.95 0.41 0.49 0.23 0.82 0.40 0.99 0.79 0.78 0.48 0.70 0.50

6.18 6.01 6.30 7.10 6.81 6.93 6.08 6.19 6.19 6.31 6.23 6.28 6.17 6.08 5.95 6.28 6.12 5.76 5.20

42.2 41.1 45.0 59.2 61.9 84.9 90.0 93.3 138.2 99.9 141.8 133.2 150.0 166.4 166.5 176.4 174.5 138.9 136.6

76 53 19 85 73 74 77 62 18 64 41 45 44 28 36 48 54 31 35

402 297 542 225 211 179 210 210 122 176 127 139 94 72 65 76 66 36 39

82 81 88 82 76 84 97 95 95 96 102 99 100 132 130 149 145 37 35

15 434 14 742 17 645 23 895 23 991 40 942 37 832 40 522 61 059 42 804 57 581 60 051 61 435 84 812 84 187 92 018 91 769 95 100 88 598

125 132 169 233 256 389 399 429 687 542 577 690 708 887 882 939 934 931 906

289 270 359 435 436 793 710 819 1539 863 1528 1152 1647 1550 1415 3386 3210 1480 1470

549 499 528 1307 1323 1827 3740 4071 6220 4513 7219 5633 7048 11 162 11 010 12 200 11 964 8340 7640

50 44 48 111 119 170 379 415 587 462 677 580 695 1110 1110 1188 1172 862 786

96 96 96 96 101 115 9928 11 412 15 470 12 030 22 961 14 950 16 238 22 971 23 448 30 500 30 325 21 000 17 500

total P mg/L total Fe mg/L Na+ mg/L K+ mg/L Mg2+ mg/L Ca2+ mg/L Ba2+ mg/L Br− mg/L Cl− mg/L TDN mg/L DOC mg/L DIC mg/L cond mS/cm pH simpson evenness shannon diversity chao richness OTU0.03 no. sequences sample type time days sample ID

Table 1. Summary of Geochemical Data, Richness and Diversity Estimates (Classified at the 97% Level) for Well 1, Well 2, and Well 3 Subsamples Sequenced Using 454-Pyrosequencing of the 16S rRNA Genea

total S mg/L

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Figure 2. Heat map showing dominant operational taxonomic units (OTUs), reported at the genus level where possible, that were detected in samples from three hydraulically fractured Marcellus shale wells between June 2012 and May 2013. Reported values represent the relative abundance or fraction (%) of observed sequences divided by the total number of postfiltered sequences in each sample. Colors represent taxa detected at 50−100% (red), 20−50% (dark orange), 10−20% (light orange), 5−10% (light peach), 1−5% (light blue), less than 1% (medium blue), or not detected (white).

produced fluids. As facultative anaerobes, the physiologies of isolated Vibrio species (V. parahemealyticus, V. alginolyticus, V. harveyi, V. natriegens) suggest sugar or other organic carbon may be coupled to nitrate reduction or fermentation activities under the saline conditions noted in our study.63,64 While nitrate was not detected due to the sample dilutions required in these high salt samples, it has been reported at 4−8 mg/L concentrations in other Marcellus injected fluids,33 suggesting it may be present in these fluids, albeit at concentrations well below our detection limit. Sequences with high similarity to autotrophic Arcobacter species were detected within injected fluids and remained prolific throughout the initial flowback period. The Arcobacter species detected here may be involved in the oxidation of sulfide species using injected electron acceptors.65−67 Arcobacter spp. are usual suspects in oil and gas wells, including produced fluids from hydraulically fractured shale.22,32,33,58 Their dominance here verifies their importance in sulfur cycling across a range of hydrocarbon-bearing formations. Sequences with close identity to Marinobacter were detected in injected fluids and initial flowback samples. This genus has been noted in Marcellus shale holding ponds9 and produced fluids in Haynesville shale wells.29 Marinobacter isolates can readily degrade labile carbon (e.g., amino acids, organic acids, alcohols) or more recalcitrant carbon forms (e.g., aliphatic and/or polycyclic aromatic hydrocarbons) across a considerable salinity and redox range,68−72 which may explain the proliferation of similar microbes in this environment. More than a month after hydraulic fracturing, injected and flowback microbe sequences are greatly outnumbered or diluted by strictly anaerobic, halotolerant species within Halanaerobium associated with sulfidogenic and fermentative activities. Sequences are dominated by H. congolense (97−99% similarity), an oil-field isolate that grows optimally at 24%

Figure 3. Nonmetric multidimensional scaling (NMDS) plot of Bray− Curtis dissimilarity distances for Well 1 and Well 2 samples. The NMDS scores for each sample (time in days noted next to well icon) are plotted with significant environmental factors projected as arrows from the origin, with the arrow magnitude and direction representative of environmental gradients. Pie charts for injected fluids (n = 2), initial flowback period (n = 7), and later produced waters (n = 6) show the average relative abundance of the dominant taxa during each sampling phase. Hand drawn lines indicate statistical groupings in order to facilitate the quick identification of samples for the viewer.

the shale.24,62 Conversely, hydraulic fracturing may stimulate fermenters previously inhibited by product build-up through dilution or dispersion within the shale. Sequences classified within the genus Vibrio were detected at low abundance within injected fluids (90%), associated with increasing ionic content and a reduction in carbon. The temporal trajectory described here points to a need to better characterize microbial populations during this highly engineered process, as water management strategies such as fluid recycling are likely to affect the injected community composition and robustness, and its ability to outcompete indigenous communities, presuming some reside in the shale before fracturing. Our results suggest that exogenous microbial members introduced to the shale during the hydraulic fracturing process have the potential to survive within a deep saline environment and alter the indigenous population temporarily or permanently through competition for newly introduced carbon or energy resources. It also suggests that the majority of injected carbon and oxidative species are attenuated in the deep subsurface during the months after hydraulic fracturing via abiotic or biotic processes. Although biocides were applied within these fluids, our results show that they do not completely control injected or indigenous microorganisms, including acid- and sulfide-producers that generate metabolites that can compromise well casing and grout integrity. Persistence of certain microbial members may be partially related to the recycling of flowback and produced fluids, which can act as a selection-enrichment process. There also appears to be a set of community members indicative of produced fluids, which may be useful for identifying the redox phase of the subsurface or for tracking fluids produced from the deep shale. A better understanding of how hydraulic fracturing operations influence microbial populations and their activities would potentially improve the industry’s ability to maximize recovery of hydrocarbons through control or enhancement of specific populations.

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ASSOCIATED CONTENT

S Supporting Information *

Well design details, injected chemical additives, list of detected organic compounds, and results of Betadisp ecological analysis. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 614-247-4429; e-mail: [email protected]. 6514

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded through startup funds provided to P.J.M. through the Department of Civil, Environmental and Geodetic Engineering and the Subsurface Energy Resources Center at Ohio State University along with National Science Foundation CBET Award #1247338. A.H. was supported in part by an appointment to the U.S. Department of Energy (DOE) Postgraduate Research Program at the National Energy Technology Laboratory (NETL) administered by the Oak Ridge Institute for Science and Education. J.D. MacRae was funded through the UMaine ADVANCE Rising Tide Center, NSF Grant #1008498. We thank the National Energy Technology Laboratory and specifically Richard W. Hammack with NETL, Elizabeth L. Rowan with the U.S. Geological Survey, and our industrial partner for coordinating and facilitating sampling for this study. We also thank the thoughtful comments provided by the four anonymous reviewers of this manuscript.

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