Microbial Ecology

, Volume 72, Issue 3, pp 571–581 | Cite as

Metatranscriptome analysis of active microbial communities in produced water samples from the Marcellus Shale

  • Amit Vikram
  • Daniel Lipus
  • Kyle BibbyEmail author
Environmental Microbiology


Controlling microbial activity is a primary concern during the management of the large volumes of wastewater (produced water) generated during high-volume hydraulic fracturing. In this study we analyzed the transcriptional activity (metatranscriptomes) of three produced water samples from the Marcellus Shale. The goal of this study was to describe active metabolic pathways of industrial concern for produced water management and reuse, and to improve understanding of produced water microbial activity. Metatranscriptome analysis revealed active biofilm formation, sulfide production, and stress management mechanisms of the produced water microbial communities. Biofilm-formation and sulfate-reduction pathways were identified in all samples. Genes related to a diverse array of stress response mechanisms were also identified with implications for biocide efficacy. Additionally, active expression of a methanogenesis pathway was identified in a sample of produced water collected prior to holding pond storage. The active microbial community identified by metatranscriptome analysis was markedly different than the community composition as identified by 16S rRNA sequencing, highlighting the value of evaluating the active microbial fraction during assessments of produced water biofouling potential and evaluation of biocide application strategies. These results indicate biofouling and corrosive microbial processes are active in produced water and should be taken into consideration while designing produced water reuse strategies.


Hydraulic fracturing Metatranscriptome RNA-seq Produced water Wastewater Sulfate reduction Stress response Biofouling Methanogenesis Biofilm Alginate 



This technical effort was performed under the RES contract RES1000027/183U as part of the National Energy Technology Laboratory’s Regional University Alliance (NETL-RUA), a collaborative initiative of NETL.

Supplementary material

248_2016_811_MOESM1_ESM.docx (631 kb)
ESM 1 (DOCX 631 kb)


  1. 1.
    Kargbo DM, Wilhelm RG, Campbell DJ (2010) Natural gas plays in the Marcellus Shale: challenges and potential opportunities. Environ Sci Technol 44:5679–5684. doi: 10.1021/es903811p CrossRefPubMedGoogle Scholar
  2. 2.
    Engelder T (August 2009) Marcellus 2008: report card on the breakout year for gas production in the Appalachian Basin. Fort Worth Basin Oil and Gas Magazine, pp 18–22Google Scholar
  3. 3.
    Bibby KJ, Brantley SL, Reible DD, Linden KG, Mouser PJ, Gregory KB, Ellis BR, Vidic RD (2013) Suggested reporting parameters for investigations of wastewater from unconventional shale gas extraction. Environ Sci Technol 47:13220–13221CrossRefPubMedGoogle Scholar
  4. 4.
    Gregory KB, Vidic RD, Dzombak DA (2011) Water management challenges associated with the production of shale gas by hydraulic fracturing. Elements 7:181–186. doi: 10.2113/gselements.7.3.181 CrossRefGoogle Scholar
  5. 5.
    Keranen KM, Weingarten M, Abers GA, Bekins BA, Ge S (2014) Sharp increase in central Oklahoma seismicity since 2008 induced by massive wastewater injection. Science 345:448–451. doi: 10.1126/science.1255802 CrossRefPubMedGoogle Scholar
  6. 6.
    Maloney KO, Yoxtheimer DA (2012) Production and disposal of waste materials from gas and oil extraction from the Marcellus Shale play in Pennsylvania. Environ Pract 14:278–287. doi: 10.1017/S146604661200035X CrossRefGoogle Scholar
  7. 7.
    Vidic RD, Brantley SL, Vandenbossche JM, Yoxtheimer D, Abad JD (2013) Impact of shale gas development on regional water quality. Science 340:1235009. doi: 10.1126/science.1235009 CrossRefPubMedGoogle Scholar
  8. 8.
    Mohan AM, Hartsock A, Hammack RW, Vidic RD, Gregory KB (2013) Microbial communities in flowback water impoundments from hydraulic fracturing for recovery of shale gas. FEMS Microbiol Ecol 86:567–580. doi: 10.1111/1574-6941.12183 CrossRefGoogle Scholar
  9. 9.
    Cluff MA, Hartsock A, MacRae JD, Carter K, Mouser PJ (2014) Temporal changes in microbial ecology and geochemistry in produced water from hydraulically fractured Marcellus Shale gas wells. Environ Sci Technol 48:6508–6517. doi: 10.1021/es501173p CrossRefPubMedGoogle Scholar
  10. 10.
    Struchtemeyer CG, Elshahed MS (2012) Bacterial communities associated with hydraulic fracturing fluids in thermogenic natural gas wells in North Central Texas, USA. FEMS Microbiol Ecol 81:13–25. doi: 10.1111/j.1574-6941.2011.01196.x CrossRefPubMedGoogle Scholar
  11. 11.
    Bottero S, Enzien M, van Loosdrecht MCM, Bruining J, Heimovaara T (2010) Formation damage and impact on gas flow caused by biofilms growing within proppant packing used in hydraulic fracturing SPE 128066; SPE International Symposium and Exhibition on Formation Damage Control, Lafayette, LA,, Richardson, TX, February 10–12, 2010Google Scholar
  12. 12.
    Murali Mohan A, Hartsock A, Bibby KJ, Hammack RW, Vidic RD, Gregory KB (2013) Microbial community changes in hydraulic fracturing fluids and produced water from shale gas extraction. Environ Sci Technol 47:13141–13150. doi: 10.1021/es402928b CrossRefPubMedGoogle Scholar
  13. 13.
    Mohan AM, Bibby KJ, Lipus D, Hammack RW, Gregory KB (2014) The functional potential of microbial communities in hydraulic fracturing source water and produced water from natural gas extraction characterized by metagenomic sequencing. PLoS ONE 9:e107682. doi: 10.1371/journal.pone.0107682 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cray JA, Bell AN, Bhaganna P, Mswaka AY, Timson DJ, Hallsworth JE (2013) The biology of habitat dominance; can microbes behave as weeds? Microb Biotechnol 6:453–492CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Martini AM, Walter LM, Budai JM, Ku TCW, Kaiser CJ, Schoell M (1998) Genetic and temporal relations between formation waters and biogenic methane: upper Devonian Antrim Shale, Michigan Basin, USA. Geochim Cosmochim Acta 62:1699–1720. doi: 10.1016/S0016-7037(98)00090-8 CrossRefGoogle Scholar
  16. 16.
    Clark ID, Ilin D, Jackson RE, Jensen M, Kennell L, Mohammadzadeh H, Poulain A, Xing YP, Raven KG (2015) Paleozoic-aged microbial methane in an Ordovician shale and carbonate aquiclude of the Michigan Basin, southwestern Ontario. Org Geochem 83:118–126. doi: 10.1016/j.orggeochem.2015.03.006 CrossRefGoogle Scholar
  17. 17.
    Sharma S, Mulder ML, Sack A, Schroeder K, Hammack R (2014) Isotope approach to assess hydrologic connections during Marcellus Shale drilling. Groundwater 52:424–433. doi: 10.1111/gwat.12083 CrossRefGoogle Scholar
  18. 18.
    Tucker YT, Kotcon J, Mroz T (2015) Methanogenic Archaea in Marcellus Shale: a possible mechanism for enhanced gas recovery in unconventional shale resources. Environ Sci Technol 49:7048–7055. doi: 10.1021/acs.est.5b00765 CrossRefPubMedGoogle Scholar
  19. 19.
    Vikram A, Lipus D, Bibby K (2014) Produced water exposure alters bacterial response to biocides. Environ Sci Technol 48:13001–13009. doi: 10.1021/es5036915 CrossRefPubMedGoogle Scholar
  20. 20.
    Moore SL, Cripps CM (2012) Bacterial survival in fractured shale-gas wells of the Horn River Basin. J Can Pet Technol 51:283CrossRefGoogle Scholar
  21. 21.
    Vikram A, Bomberger JM, Bibby KJ (2015) Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 59:3433–3440. doi: 10.1128/aac.05152-14 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kahrilas GA, Blotevogel J, Stewart PS, Borch T (2015) Biocides in hydraulic fracturing fluids: a critical review of their usage, mobility, degradation, and toxicity. Environ Sci Technol 49:16–32. doi: 10.1021/es503724k CrossRefPubMedGoogle Scholar
  23. 23.
    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: 1621–1624. doi:
  24. 24.
    Clifford RJ, Milillo M, Prestwood J, Quintero R, Zurawski DV, Kwak YI, Waterman PE, Lesho EP, Mc Gann P (2012) Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR. PLoS ONE 7:e48558. doi: 10.1371/journal.pone.0048558 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Akyon B, Stachler E, Wei N, Bibby K (2015) Microbial mats as a biological treatment approach for saline wastewaters: the case of produced water from hydraulic fracturing. Environ Sci Technol 49:6172–6180. doi: 10.1021/es505142t CrossRefPubMedGoogle Scholar
  26. 26.
    Baker BJ, Sheik CS, Taylor CA, Jain S, Bhasi A, Cavalcoli JD, Dick GJ (2013) Community transcriptomic assembly reveals microbes that contribute to deep-sea carbon and nitrogen cycling. ISME J 7:1962–1973. doi: 10.1038/ismej.2013.85 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829. doi: 10.1101/gr.074492.107 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Meyer F, Paarmann D, D’Souza M, Olson R, Glass E, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J, Edwards R (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinf 9:386CrossRefGoogle Scholar
  29. 29.
    Abubucker S, Segata N, Goll J, Schubert AM, Izard J, Cantarel BL, Rodriguez-Mueller B, Zucker J, Thiagarajan M, Henrissat B, White O, Kelley ST, Methé B, Schloss PD, Gevers D, Mitreva M, Huttenhower C (2012) Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput Biol 8:e1002358. doi: 10.1371/journal.pcbi.1002358 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Aziz R, Bartels D, Best A, DeJongh M, Disz T, Edwards R, Formsma K, Gerdes S, Glass E, Kubal M, Meyer F, Olsen G, Olson R, Osterman A, Overbeek R, McNeil L, Paarmann D, Paczian T, Parrello B, Pusch G, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2 CrossRefPubMedGoogle Scholar
  32. 32.
    Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30. doi: 10.1093/nar/28.1.27 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mitra S, Rupek P, Richter D, Urich T, Gilbert J, Meyer F, Wilke A, Huson D (2011) Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG. BMC Bioinf 12:S21CrossRefGoogle Scholar
  34. 34.
    Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JLM, D’Auria G, De Lima AF, La Cono V, Genovese M, McKew BA, Hayes SL, Harris G, Giuliano L, Timmis KN, McGenity TJ (2007) Limits of life in MgCl2-containing environments: chaotropicity defines the window. Environ Microbiol 9:801–813. doi: 10.1111/j.1462-2920.2006.01212.x CrossRefPubMedGoogle Scholar
  35. 35.
    Yakimov MM, La Cono V, Spada GL, Bortoluzzi G, Messina E, Smedile F, Arcadi E, Borghini M, Ferrer M, Schmitt-Kopplin P, Hertkorn N, Cray JA, Hallsworth JE, Golyshin PN, Giuliano L (2015) Microbial community of the deep-sea brine Lake Kryos seawater–brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA. Environ Microbiol 17:364–382. doi: 10.1111/1462-2920.12587 CrossRefPubMedGoogle Scholar
  36. 36.
    Enning D, Venzlaff H, Garrelfs J, Dinh HT, Meyer V, Mayrhofer K, Hassel AW, Stratmann M, Widdel F (2012) Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust. Environ Microbiol 14:1772–1787. doi: 10.1111/j.1462-2920.2012.02778.x CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Cray JA, Stevenson A, Ball P, Bankar SB, Eleutherio EC, Ezeji TC, Singhal RS, Thevelein JM, Timson DJ, Hallsworth JE (2015) Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms. Curr Opin Biotechnol 33:228–259CrossRefPubMedGoogle Scholar
  38. 38.
    Youssef NH, Savage-Ashlock KN, McCully AL, Luedtke B, Shaw EI, Hoff WD, Elshahed MS (2014) Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales. ISME J 8:636–649. doi: 10.1038/ismej.2013.165 CrossRefPubMedGoogle Scholar
  39. 39.
    Johnson EF, Mukhopadhyay B (2005) A new type of sulfite reductase, a novel coenzyme F420-dependent enzyme, from the Methanarchaeon Methanocaldococcus jannaschii. J Biol Chem 280:38776–38786. doi: 10.1074/jbc.M503492200 CrossRefPubMedGoogle Scholar
  40. 40.
    Pires RH, Venceslau SS, Morais F, Teixeira M, Xavier AV, Pereira IAC (2005) Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP ComplexA membrane-bound redox complex involved in the sulfate respiratory pathway. Biochemistry 45:249–262. doi: 10.1021/bi0515265
  41. 41.
    Thauer RK, Kaster A-K, Seedorf H, Buckel W, Hedderich R (2008) Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Microbiol 6:579–591CrossRefPubMedGoogle Scholar
  42. 42.
    Zhou A, Baidoo E, He Z, Mukhopadhyay A, Baumohl JK, Benke P, Joachimiak MP, Xie M, Song R, Arkin AP, Hazen TC, Keasling JD, Wall JD, Stahl DA, Zhou J (2013) Characterization of NaCl tolerance in Desulfovibrio vulgaris Hildenborough through experimental evolution. ISME J 7:1790–1802. doi: 10.1038/ismej.2013.60 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Gifford SM, Sharma S, Rinta-Kanto JM, Moran MA (2011) Quantitative analysis of a deeply sequenced marine microbial metatranscriptome. ISME J 5: 461–472. doi:
  44. 44.
    Goltsman DSA, Comolli LR, Thomas BC, Banfield JF (2015) Community transcriptomics reveals unexpected high microbial diversity in acidophilic biofilm communities. ISME J 9:1014–1023. doi: 10.1038/ismej.2014.200 CrossRefGoogle Scholar
  45. 45.
    Urich T, Lanzén A, Qi J, Huson DH, Schleper C, Schuster SC (2008) Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome. PLoS ONE 3:e2527. doi: 10.1371/journal.pone.0002527 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Moore LE, Ledder RG, Gilbert P, McBain AJ (2008) In vitro study of the effect of cationic biocides on bacterial population dynamics and susceptibility. Appl Environ Microbiol 74:4825–4834. doi: 10.1128/aem.00573-08 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kümmerer K (2004) Resistance in the environment. J Antimicrob Chemother 54:311–320. doi: 10.1093/jac/dkh325 CrossRefPubMedGoogle Scholar
  48. 48.
    Baron JL, Vikram A, Duda S, Stout JE, Bibby K (2014) Shift in the microbial ecology of a hospital hot water system following the introduction of an on-site monochloramine disinfection system. PLoS ONE 9:e102679. doi: 10.1371/journal.pone.0102679 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Oren A, Hallsworth JE (2014) Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei. FEMS Microbiol Lett 359:134–142CrossRefPubMedGoogle Scholar
  50. 50.
    Raivio TL, Silhavy TJ (2001) Periplasmic stress and ECF sigma factors. Annu Rev Microbiol 55:591–624. doi: 10.1146/annurev.micro.55.1.591 CrossRefPubMedGoogle Scholar
  51. 51.
    Zhou J, He Q, Hemme CL, Mukhopadhyay A, Hillesland K, Zhou A, He Z, Van Nostrand JD, Hazen TC, Stahl DA, Wall JD, Arkin AP (2011) How sulphate-reducing microorganisms cope with stress: lessons from systems biology. Nat Rev Micro 9: 452–466. doi:
  52. 52.
    Landfald B, Strøm AR (1986) Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol 165:849–855PubMedPubMedCentralGoogle Scholar
  53. 53.
    Yim HH, Villarejo M (1992) osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli. J Bacteriol 174:3637–3644Google Scholar
  54. 54.
    Zheng X, Ji Y, Weng X, Huang X (2015) RpoS-dependent expression of OsmY in Salmonella enterica serovar Typhi: activation under stationary phase and SPI-2-inducing conditions. Curr Microbiol 70:877–882. doi: 10.1007/s00284-015-0802-1 CrossRefPubMedGoogle Scholar
  55. 55.
    Wang Y (2002) The function of OmpA in Escherichia coli. Biochem Biophys Res Commun 292:396–401. doi: 10.1006/bbrc.2002.6657 CrossRefPubMedGoogle Scholar
  56. 56.
    Hu P, Brodie EL, Suzuki Y, McAdams HH, Andersen GL (2005) Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. J Bacteriol 187:8437–8449. doi: 10.1128/jb.187.24.8437-8449.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Brioukhanov AL, Netrusov AI (2004) Catalase and superoxide dismutase: distribution, properties, and physiological role in cells of strict anaerobes. Biochem Mosc 69:949–962. doi: 10.1023/B:BIRY.0000043537.04115.d9 CrossRefGoogle Scholar
  58. 58.
    Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Syst 4:1CrossRefGoogle Scholar
  59. 59.
    Gomez-Alvarez V, Revetta R, Domingo JW (2012) Metagenome analyses of corroded concrete wastewater pipe biofilms reveal a complex microbial system. BMC Microbiol 12:122CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Heaney CD, Wing S, Campbell RL, Caldwell D, Hopkins B, Richardson D, Yeatts K (2011) Relation between malodor, ambient hydrogen sulfide, and health in a community bordering a landfill. Environ Res 111:847–852. doi: 10.1016/j.envres.2011.05.021 CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Wuchter C, Banning E, Mincer T, Drenzek NJ, Coolen MJ (2013) Microbial diversity and methanogenic activity of Antrim Shale formation waters from recently fractured wells. Frontiers Microbiol 4. doi: 10.3389/fmicb.2013.00367
  62. 62.
    Blauch ME (2010) Developing effective and environmentally suitable fracturing fluids using hydraulic fracturing flowback waters. Unconventional Gas Conference, vol. 131784. Society of Petroleum Engineers, Pittsburgh, PAGoogle Scholar
  63. 63.
    Guss AM, Mukhopadhyay B, Zhang JK, Metcalf WW (2005) Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H2 metabolism between closely related species. Mol Microbiol 55:1671–1680. doi: 10.1111/j.1365-2958.2005.04514.x
  64. 64.
    Buan NR, Metcalf WW (2010) Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase. Mol Microbiol 75:843–853. doi: 10.1111/j.1365-2958.2009.06990.x CrossRefPubMedGoogle Scholar
  65. 65.
    Crable BR, Plugge CM, McInerney MJ, Stams AJM (2011) Formate formation and formate conversion in biological fuels production. Enzym Res 2011:8. doi: 10.4061/2011/532536 CrossRefGoogle Scholar
  66. 66.
    Zengler K, Richnow HH, Rossello-Mora R, Michaelis W, Widdel F (1999) Methane formation from long-chain alkanes by anaerobic microorganisms. Nature 401:266–269CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringUniversity of PittsburghPittsburghUSA
  2. 2.Department of Computational and Systems BiologyUniversity of PittsburghPittsburghUSA

Personalised recommendations