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Hydrocarbon and Lipid Microbiology Protocols pp 181–197Cite as

DNA- and RNA-Based Stable Isotope Probing of Hydrocarbon Degraders

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Abstract

The microbial degradation of hydrocarbons in contaminated environments can be driven by distinct aerobic and anaerobic populations. While the physiology and biochemistry of selected degraders isolated in pure culture have been intensively studied in recent decades, research has now started to take the generated knowledge back to the field, in order to identify microbes truly responsible for degradation in situ. Partially, this has been facilitated by stable isotope probing (SIP) of nucleic acids. This chapter discusses the concepts and important methodological foundations of SIP and provides a detailed workflow for the application of DNA- and rRNA-based SIP to degraders of petroleum hydrocarbons in aerobic and anaerobic systems. SIP is capable of providing direct knowledge on intrinsic hydrocarbon degrader populations in diverse environmental and technical systems, which is an important step toward more integrated concepts in contaminated site monitoring and bioremediation.

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References

  1. Neufeld JD, Wagner M, Murrell JC (2007) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1:103–110

    Article  CAS  PubMed  Google Scholar 

  2. Gutierrez-Zamora ML, Manefield M (2010) An appraisal of methods for linking environmental processes to specific microbial taxa. Rev Environ Sci Bio-Technol 9:153–185

    Article  Google Scholar 

  3. Jeon CO, Madsen EL (2013) In situ microbial metabolism of aromatic-hydrocarbon environmental pollutants. Curr Opin Biotechnol 24:474–481

    Article  CAS  PubMed  Google Scholar 

  4. Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649

    Article  CAS  PubMed  Google Scholar 

  5. Manefield M, Whiteley AS, Griffiths RI, Bailey MJ (2002) RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl Environ Microbiol 68:5367–5373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Madsen EL (2006) The use of stable isotope probing techniques in bioreactor and field studies on bioremediation. Curr Opin Biotechnol 17:92–97

    Article  CAS  PubMed  Google Scholar 

  7. Cupples AM (2011) The use of nucleic acid based stable isotope probing to identify the microorganisms responsible for anaerobic benzene and toluene biodegradation. J Microbiol Meth 85:83–91

    Article  CAS  Google Scholar 

  8. Kleinsteuber S, Schleinitz K, Vogt C (2012) Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers. Appl Microbiol Biotechnol 94:851–873

    Article  CAS  PubMed  Google Scholar 

  9. DeRito CM, Pumphrey GM, Madsen EL (2005) Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl Environ Microbiol 71:7858–7865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liou JSC, DeRito CM, Madsen EL (2008) Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment. Environ Microbiol 10:1964–1977

    Article  CAS  PubMed  Google Scholar 

  11. Gutierrez T, Singleton DR, Berry D, Yang T, Aitken MD, Teske A (2013) Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP. ISME J 7:2091–2104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jones MD, Crandell DW, Singleton DR, Aitken MD (2011) Stable-isotope probing of the polycyclic aromatic hydrocarbon-degrading bacterial guild in a contaminated soil. Environ Microbiol 13:2623–2632

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pilloni G, von Netzer F, Engel M, Lueders T (2011) Electron acceptor-dependent identification of key anaerobic toluene degraders at a tar-oil-contaminated aquifer by Pyro-SIP. FEMS Microbiol Ecol 78:165–175

    Article  CAS  PubMed  Google Scholar 

  14. Sun W, Cupples AM (2012) Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing. Appl Environ Microbiol 78:972–980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bastida F, Jechalke S, Bombach P, Franchini AG, Seifert J, von Bergen M, Vogt C, Richnow HH (2011) Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies. FEMS Microbiol Ecol 77:357–369

    Article  CAS  PubMed  Google Scholar 

  16. Oka AR, Phelps CD, McGuinness LM, Mumford A, Young LY, Kerkhof LJ (2008) Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing. Appl Environ Microbiol 74:6476–6480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fowler SJ, Gutierrez-Zamora M-L, Manefield M, Gieg LM (2014) Identification of toluene degraders in a methanogenic enrichment culture. FEMS Microbiol Ecol 89:625–636

    Article  CAS  PubMed  Google Scholar 

  18. Manefield M, Whiteley AS, Bailey MJ (2004) What can stable isotope probing do for bioremediation? Int Biodeter Biodegr 54:163–166

    Article  CAS  Google Scholar 

  19. Uhlik O, Leewis MC, Strejcek M, Musilova L, Mackova M, Leigh MB, Macek T (2013) Stable isotope probing in the metagenomics era: a bridge towards improved bioremediation. Biotechnol Adv 31:154–165

    Article  CAS  PubMed  Google Scholar 

  20. Wang Y, Chen Y, Zhou Q, Huang S, Ning K, Xu J, Kalin RM, Rolfe S, Huang WE (2012) A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community. PLoS ONE 7:e47530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jeon CO, Park W, Padmanabhan P, DeRito C, Snape JR, Madsen EL (2003) Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc Natl Acad Sci USA 100:13591–13596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Leigh MB, Pellizari VH, Uhlik O, Sutka R, Rodrigues J, Ostrom NE, Zhou J, Tiedje JM (2007) Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs). ISME J 1:134–148

    Article  CAS  PubMed  Google Scholar 

  23. Singleton DR, Hu J, Aitken MD (2012) Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading Betaproteobacterium. Appl Environ Microbiol 78:3552–3559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kazy S, Monier A, Alvarez P (2010) Assessing the correlation between anaerobic toluene degradation activity and bssA concentrations in hydrocarbon-contaminated aquifer material. Biodegradation 21:793–800

    Article  CAS  PubMed  Google Scholar 

  25. Yagi JM, Madsen EL (2009) Diversity, abundance, and consistency of microbial oxygenase expression and biodegradation in a shallow contaminated aquifer. Appl Environ Microbiol 75:6478–6487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Baldwin BR, Biernacki A, Blair J, Purchase MP, Baker JM, Sublette K, Davis G, Ogles D (2010) Monitoring gene expression to evaluate oxygen infusion at a gasoline-contaminated site. Environ Sci Technol 44:6829–6834

    Article  CAS  PubMed  Google Scholar 

  27. Manefield M, Griffiths RI, Leigh MB, Fisher R, Whiteley AS (2005) Functional and compositional comparison of two activated sludge communities remediating coking effluent. Environ Microbiol 7:715–722

    Article  CAS  PubMed  Google Scholar 

  28. Kasai Y, Takahata Y, Manefield M, Watanabe K (2006) RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. Appl Environ Microbiol 72:3586–3592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kasai Y, Kodama Y, Takahata Y, Hoaki T, Watanabe K (2007) Degradative Capacities and Bioaugmentation Potential of an Anaerobic Benzene-degrading Bacterium Strain DN11. Environ Sci Technol 41:6222–6227

    Article  CAS  PubMed  Google Scholar 

  30. Jechalke S, Franchini AG, Bastida F, Bombach P, Rosell M, Seifert J, von Bergen M, Vogt C, Richnow HH (2013) Analysis of structure, function, and activity of a benzene-degrading microbial community. FEMS Microbiol Ecol 85:14–26

    Article  PubMed  Google Scholar 

  31. Singleton DR, Jones MD, Richardson SD, Aitken MD (2013) Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil. Appl Microbiol Biotechnol 97:8381–8391

    Article  CAS  PubMed  Google Scholar 

  32. Paliwal V, Puranik S, Purohit H (2012) Integrated perspective for effective bioremediation. Appl Biochem Biotechnol 166:903–924

    Article  CAS  PubMed  Google Scholar 

  33. Farhadian M, Vachelard C, Duchez D, Larroche C (2008) In situ bioremediation of monoaromatic pollutants in groundwater: a review. Bioresour Technol 99:5296–5308

    Article  CAS  PubMed  Google Scholar 

  34. Whiteley AS, Thomson B, Lueders T, Manefield M (2007) RNA stable-isotope probing. Nat Protocols 2:838–844

    Article  CAS  PubMed  Google Scholar 

  35. Neufeld JD, Vohra J, Dumont MG, Lueders T, Manefield M, Friedrich MW, Murrell JC (2007) DNA stable-isotope probing. Nat Protocols 2:860–866

    Article  CAS  PubMed  Google Scholar 

  36. Neufeld J, Dumont M, Vohra J, Murrell J (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53:435–442

    Article  CAS  PubMed  Google Scholar 

  37. Rickwood D (1992) Centrifugal methods for characterizing macromolecules and their interactions. In: Rickwood D (ed) Preparative centrifugation: a practical approach. Oxford University Press: Oxford, pp 143–186

    Google Scholar 

  38. Lueders T, Manefield M, Friedrich MW (2004) Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ Microbiol 6:73–78

    Article  CAS  PubMed  Google Scholar 

  39. Buckley DH, Huangyutitham V, Hsu S-F, Nelson TA (2007) Stable isotope probing with 15N achieved by disentangling the effects of genome G + C content and isotope enrichment on DNA density. Appl Environ Microbiol 73:3189–3195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Cupples AM, Shaffer EA, Chee-Sanford JC, Sims GK (2007) DNA buoyant density shifts during 15N-DNA stable isotope probing. Microbiol Res 162:328–334

    Article  CAS  PubMed  Google Scholar 

  41. Meselson M, Stahl FW (1958) The replication of DNA in Escherichia coli. Proc Natl Acad Sci USA 44:671–682

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lueders T, Wagner B, Claus P, Friedrich MW (2004) Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ Microbiol 6:60–72

    Article  CAS  PubMed  Google Scholar 

  43. Whiteley AS, Manefield M, Lueders T (2006) Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies. Curr Opin Biotechnol 17:67–71

    Article  CAS  PubMed  Google Scholar 

  44. Zemb O, Lee M, Gutierrez-Zamora ML, Hamelin J, Coupland K, Hazrin-Chong NH, Taleb I, Manefield M (2012) Improvement of RNA-SIP by pyrosequencing to identify putative 4-n-nonylphenol degraders in activated sludge. Water Res 46:601–610

    Article  CAS  PubMed  Google Scholar 

  45. Bell TH, Yergeau E, Martineau C, Juck D, Whyte LG, Greer CW (2011) Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using 15N-DNA-based stable isotope probing and pyrosequencing. Appl Environ Microbiol 77:4163–4171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kim S-J, Park S-J, Jung M-Y, Kim J-G, Madsen EL, Rhee S-K (2014) An uncultivated nitrate-reducing member of the genus Herminiimonas degrades toluene. Appl Environ Microbiol 80:3233–3243

    Article  PubMed  PubMed Central  Google Scholar 

  47. Uhlik O, Wald J, Strejcek M, Musilova L, Ridl J, Hroudova M, Vlcek C, Cardenas E, Mackova M, Macek T (2012) Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil. PLoS One 7:e40653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Verastegui Y, Cheng J, Engel K, Kolczynski D, Mortimer S, Lavigne J, Montalibet J, Romantsov T, Hall M, McConkey BJ et al. (2014) Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities. mBio 5:e01157–01114

    Google Scholar 

  49. Kleindienst S, Herbst F-A, Stagars M, von Netzer F, von Bergen M, Seifert J, Peplies J, Amann R, Musat F, Lueders T et al (2014) Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps. ISME J 8:2029–2044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Neufeld JD, Chen Y, Dumont MG, Murrell JC (2008) Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environ Microbiol 10:1526–1535

    Article  CAS  PubMed  Google Scholar 

  51. Sul WJ, Park J, Quensen JF III, Rodrigues JLM, Seliger L, Tsoi TV, Zylstra GJ, Tiedje JM (2009) DNA-Stable isotope probing integrated with metagenomics for retrieval of biphenyl dioxygenase genes from polychlorinated biphenyl-contaminated river sediment. Appl Environ Microbiol 75:5501–5506

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zumsteg A, Schmutz S, Frey B (2013) Identification of biomass utilizing bacteria in a carbon-depleted glacier forefield soil by the use of 13C DNA stable isotope probing. Environ Microbiol Rep 5:424–437

    Article  CAS  PubMed  Google Scholar 

  53. Kunapuli U, Lueders T, Meckenstock RU (2007) The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation. ISME J 1:643–653

    Article  CAS  PubMed  Google Scholar 

  54. Bombach P, Chatzinotas A, Neu TR, Kästner M, Lueders T, Vogt C (2010) Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques. FEMS Microbiol Ecol 71:237–246

    Article  CAS  PubMed  Google Scholar 

  55. Cheng L, Ding C, Li Q, He Q, Dai L-r, and Zhang H (2013) DNA-SIP reveals that Syntrophaceae play an important role in methanogenic hexadecane degradation. PLoS ONE 8:e66784

    Google Scholar 

  56. Graue J, Kleindienst S, Lueders T, Cypionka H, Engelen B (2012) Identifying fermenting bacteria in anoxic tidal-flat sediments by a combination of microcalorimetry and ribosome-based stable-isotope probing. FEMS Microbiol Ecol 81:78–87

    Article  CAS  PubMed  Google Scholar 

  57. Herrmann S, Kleinsteuber S, Chatzinotas A, Kuppardt S, Lueders T, Richnow H-H, Vogt C (2010) Functional characterization of an anaerobic benzene-degrading enrichment culture by DNA stable isotope probing. Environ Microbiol 12:401–411

    Article  CAS  PubMed  Google Scholar 

  58. Winderl C, Penning H, von Netzer F, Meckenstock RU, Lueders T (2010) DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment. ISME J 4:1314–1325

    Article  PubMed  Google Scholar 

  59. Heider J, Fuchs G (1997) Anaerobic metabolism of aromatic compounds. Eur J Biochem 243:577–596

    Article  CAS  PubMed  Google Scholar 

  60. Singleton DR, Hunt M, Powell SN, Frontera-Suau R, Aitken MD (2007) Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria. J Microbiol Meth 69:180–187

    Article  CAS  Google Scholar 

  61. Lueders T, Pommerenke B, Friedrich MW (2004) Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil. Appl Environ Microbiol 70:5778–5786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Singleton DR, Powell SN, Sangaiah R, Gold A, Ball LM, Aitken MD (2005) Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil. Appl Environ Microbiol 71:1202–1209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Mahmood S, Paton GI, Prosser JI (2005) Cultivation-independent in situ molecular analysis of bacteria involved in degradation of pentachlorophenol in soil. Environ Microbiol 7:1349–1360

    Article  CAS  PubMed  Google Scholar 

  64. Gallagher E, McGuinness L, Phelps C, Young LY, Kerkhof LJ (2005) 13C-carrier DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing. Appl Environ Microbiol 71:5192–5196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Cupples AM, Sims GK (2007) Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing. Soil Biol Biochem 39:232–238

    Article  CAS  Google Scholar 

  66. Grob C, Taubert M, Howat AM, Burns OJ, Chen Y, Murrell JC (2015) Generating enriched metagenomes from active microorganisms with DNA stable isotope probing. Hydrocarb Lipid Microbiol Protocols. doi:10.1007/8623_2015_81

  67. Pilloni G, Granitsiotis MS, Engel M, Lueders T (2012) Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes. PLoS One 7:e40467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Bartram AK, Poon C, Neufeld JD (2009) Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant. Biotechniques 47:1016–1019

    Article  Google Scholar 

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Acknowledgments

The author acknowledges the Helmholtz Society and the Deutsche Forschungsgemeinschaft (DFG, SPP-1319) for the support during the preparation of this manuscript.

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Authors and Affiliations

  1. Institute of Groundwater Ecology, Helmholtz Zentrum München – German Research Centre for Environmental Health, Neuherberg, Germany

    Tillmann Lueders

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  1. Tillmann Lueders
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Correspondence to Tillmann Lueders .

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Editors and Affiliations

  1. Dept. Biological Sciences, University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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Lueders, T. (2015). DNA- and RNA-Based Stable Isotope Probing of Hydrocarbon Degraders. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_74

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  • DOI: https://doi.org/10.1007/8623_2015_74

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