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Isolation and genetic identification of PAH degrading bacteria from a microbial consortium

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Abstract

Polycyclic aromatic hydrocarbons (PAH; naphthalene, anthracene and phenanthrene) degrading microbial consortium C2PL05 was obtained from a sandy soil chronically exposed to petroleum products, collected from a petrochemical complex in Puertollano (Ciudad Real, Spain). The consortium C2PL05 was highly efficient degrading completely naphthalene, phenanthrene and anthracene in around 18 days of cultivation. The toxicity (Microtox™ method) generated by the PAH and by the intermediate metabolites was reduced to levels close to non-toxic in almost 40 days of cultivation. The identified bacteria from the contaminated soil belonged to γ-proteobacteria and could be include in Enterobacter and Pseudomonas genus. DGGE analysis revealed uncultured Stenotrophomonas ribotypes as a possible PAH degrader in the microbial consortium. The present work shows the potential use of these microorganisms and the total consortium for the bioremediation of PAH polluted areas since the biodegradation of these chemicals takes place along with a significant decrease in toxicity.

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References

  • Aitken MD, Long TC (2004) Biotransformation, biodegradation and bioremediation of PAH. In: Singh A, Ward OP (eds) Soil biology, vol 2. Biodegradation and bioremediation. Springer, Berlin, Germany, pp 84–124

    Google Scholar 

  • Amman RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    Google Scholar 

  • Anzai Y, Kim H, Park JY, Walabayashi H, Oyaizu H (2000) Phylogenetic affiliation of the Pseudomonas based on 16S rRNA sequence. Int J Syst Evol Microbiol 50:1569–1589

    Google Scholar 

  • Bossert I, Kachel MW, Bartha R (1984) Fate of hydrocarbons during oily sludge disosal in soil. Appl Environ Microbiol 47:763–767

    PubMed  CAS  Google Scholar 

  • Bugg T, Foght JM, Pickard MA, Gray MR (2000) Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a. Appl Environ Microbiol 66:5387–5392. doi:10.1128/AEM.66.12.5387-5392.2000

    Article  PubMed  CAS  Google Scholar 

  • Chaineau CH, Rougeux G, Yepremian C, Oudot J (2005) Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biol Biochem 37:1490–1497. doi:10.1016/j.soilbio.2005.01.012

    Article  CAS  Google Scholar 

  • Cui Z, Lai Q, Dong C, Shao Z (2008) Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the middle Atlantic ridge. Environ Microbiol 10:2138–2149. doi:10.1111/j.1462-2920.2008.01637.x

    Article  PubMed  CAS  Google Scholar 

  • Delille D, Delille B, Pelletelier E (2002) Effectiveness of bioremediation of crude oil contaminated subantarctic intertidal sediment: the microbial response. Microb Ecol 44:118–126. doi:10.1007/s00248-001-1047-z

    Article  PubMed  CAS  Google Scholar 

  • Dibble JR, Bartha R (1979) Effect of environmental parameters on the biodegradation of oil sludge. Appl Environ Microbiol 37:729–739

    PubMed  CAS  Google Scholar 

  • Dimitriou-Christidis P, Autenrieth RL, McDonald TJ, Desai AM (2007) Measurement of biodegradability parameters for single unsubstituted and methylated polycyclic aromatic hydrocarbons in liquid bacterial suspensions. Biotechnol Bioeng 97:922–932. doi:10.1002/bit.21268

    Article  PubMed  CAS  Google Scholar 

  • Dowd SE, Sun Y, Secor PR, Rhoads DD, Woldcott BM, James GA, Wolcott RD (2008) Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol 8:43–57. doi:10.1186/1471-2180-8-43

    Article  PubMed  CAS  Google Scholar 

  • Edgehill RU (1999) Bioremediation by inoculation with microorganisms. In: Adriano DC, Bollag JM, Frnakenberger WT, Sims RC (eds) Bioremediation of contaminated soils. American Society of Agronomy, Madison, pp 289–314

    Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evol Int J Org Evol 39:783–791. doi:10.2307/2408678

    Google Scholar 

  • Grant RJ, Muckian LM, Clipson NJW, Doyle EM (2006) Microbial community changes during the bioremediation of creosote-contaminated soil. Lett Appl Microbiol 44:293–300. doi:10.1111/j.1472-765X.2006.02066.x

    Article  CAS  Google Scholar 

  • Grifoll M, Awlidonoc SA, Gatlin CV, Chapman PJ (1995) Actions of a versatile fluorine-degrading bacterial isolate on polycyclic aromatic compounds. Appl Environ Microbiol 61:3711–3723

    PubMed  CAS  Google Scholar 

  • Gupta A, Singh R, Khare SK, Gupta MN (2006) A solvent tolerant isolate of Enterobacter aerogenes. Bioresour Technol 97:99–103. doi:10.1016/j.biortech.2005.02.028

    Article  PubMed  CAS  Google Scholar 

  • Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Hambrick GA, DeLuane RD, Patrick WH (1980) Effect of estuarine sediment pH and oxidation-reducction potential on microbial hydrocarbon degradation. Appl Environ Microbiol 40:365–369

    PubMed  CAS  Google Scholar 

  • Hansen HP, Koroleff F (1999) Determination of nutrients. In: Grassoff K, Ehrdardt M, Kermling M (eds) Methods of seawater analysis. Wiley-VCH, Weinheim, Germany, pp 159–228

    Chapter  Google Scholar 

  • Hesham AE, Wang Z, Zhang Y, Zhang J, Lu W, Yang M (2006) Isolation and identification of a yeast strain capable of degrading four and five ring aromatic hydrocarbons. Ann Microbiol 56:109–112

    Article  CAS  Google Scholar 

  • Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42:182–192

    Google Scholar 

  • International Association of Reseach Cancer (1983) Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Polynuclear aromatic compounds. Part 1. IARC Monogr 32:355–364

    Google Scholar 

  • Jacques RJS, Okeke BC, Bento FM, Teixeria AS, Peralba MCR, Camargo FAO (2008) Microbial consortium bioaugmentation of a polycyclic aromatic hydrocarbons contaminated soil. Bioresour Technol 99:2637–2643. doi:10.1016/j.biortech.2007.04.047

    Article  PubMed  CAS  Google Scholar 

  • Kampfer P, Ruppel S, Remus R (2005) Enterobacter radicincitans sp. nov., a plant growth promoting species of the family Enterobactriaceae. Syst Appl Microbiol 28:213–221. doi:10.1016/j.syapm.2004.12.007

    Article  PubMed  CAS  Google Scholar 

  • Kanaly RA, Harayama S (2000) Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria. J Bacteriol 182:2059–2067. doi:10.1128/JB.182.8.2059-2067.2000

    Article  PubMed  CAS  Google Scholar 

  • Kästner M (2000) Degradation of aromatic and polyaromatic compounds. In: Rehm HJ, Reed RG, Pühler A, Stadler P (eds) Biotechnology, environmental processes II. Soil decontamination. Wiley-VCH, Weinheim, Germany, pp 211–240

    Google Scholar 

  • Lane DJ (1991) 16S/23S sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. Wiley, Chichester, pp 115–175

    Google Scholar 

  • Li JL, Chen BH (2008) Effect of non-ionic surfactants on biodegradation of phenanthrene by a marine bacteria of Neptunomonas naphthovorans. J Hazard Mater 162:66–73. doi:10.1016/j.jhazmat.2008.05.019

    Article  PubMed  CAS  Google Scholar 

  • Ma Y, Wang L, Shao Z (2006) Pseudomonas, the dominant polycyclic aromatic hydrocarbon-degrading bacteria isolated from Antarctic soils and the role of large plasmids in horizontal gene transfer. Environ Microbiol 8:455–465. doi:10.1111/j.1462-2920.2005.00911.x

    Article  PubMed  CAS  Google Scholar 

  • Madden TL, Tatusov RL, Zhang J (1996) Applications of network BLAST sever. Methods Enzymol 266:131–141. doi:10.1016/S0076-6879(96)66011-X

    Article  PubMed  CAS  Google Scholar 

  • Margesin R, Schinner F (2001) Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area. Appl Environ Microbiol 67:3127–3133. doi:10.1128/AEM.67.7.3127-3133.2001

    Article  PubMed  CAS  Google Scholar 

  • Menn FM, Easter JP, Sayler GS (2000) Bacterial activity enhancement and soil decontamination. In: Rehm HJ, Reed GR, Pühler A, Staler P (eds) Biotechnology environmental processes II. Soil decontamination. Wiley-VCH, Weinheim, pp 425–439

    Google Scholar 

  • Microbics Corporation (1992) Microtox manual vol III, condensed protocols. A toxicity testing handbook. Microbics Corporation, Carslbad

    Google Scholar 

  • Muyzer G, Hottentrager S, Teske A, Wawer C (1995) Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA—a new molecular approach to analyse the genetic diversity of mixed microbial communities. In: Akkermans ADL, van Elsas JD, de Bruijn FJ (eds) Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, pp 1–23

    Google Scholar 

  • Pelletelier E, Delille D, Delille B (2004) Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oiled residues. Mar Environ Res 57:311–327. doi:10.1016/j.marenvres.2003.07.001

    Article  CAS  Google Scholar 

  • Sabaté J, Viñas M, Solanas AM (2004) Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils. Int Biodeter Biodegr 54:19–25. doi:10.1016/j.ibiod.2003.12.002

    Article  CAS  Google Scholar 

  • 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. doi:10.1128/AEM.71.3.1202-1209.2005

    Article  PubMed  CAS  Google Scholar 

  • Sun W, Sierra R, Fernandez N, Sanz JL, Amils R, Legatzki A, Maier MR, Field JA (2009) Molecular characterization and in situ quantification of anoxic arsenite oxidizing denitrifying enrichment cultures. FEMS Microbiol Ecol 68:72–85. doi:10.1111/j.1574-6941.2009.00653.x

    Article  PubMed  CAS  Google Scholar 

  • Swofford DL (2003) PAUP*. Phylogenetic analysis using parsimony (*and other methods), version 4.0b 10. Sinauer Associates, Sunderland, MA

    Google Scholar 

  • Toledo FL, Calvo C, Rodelas B, González-López J (2006) Selection and identification of bacteria isolated from waste crude oil with polycyclic aromatic hydrocarbons removal capacities. Syst Appl Microbiol 29:244–252. doi:10.1016/j.syapm.2005.09.003

    Article  PubMed  CAS  Google Scholar 

  • Viñas M, Grifoll M, Sabaté J, Solanas AM (2002) Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities. J Ind Microbiol Biotechnol 28:252–260. doi:10.1038/sj.jim.7000236

    Article  PubMed  Google Scholar 

  • Viñas M, Sabaté J, Guasp C, Lalucat J, Solanas AM (2005) Culture-dependent and independent approaches establish the complexity of a PAH degrading microbial consortium. Can J Microbiol 51:897–909. doi:10.1139/w05-090

    Article  PubMed  Google Scholar 

  • Warmer KH, Peters CA (2005) Polycyclic aromatic hydrocarbon biodegradation rates: a structure-based study. Environ Sci Technol 39:2571–2578. doi:10.1021/es048939y

    Article  CAS  Google Scholar 

  • Wilke BM (2005) Determination of chemical and physical soil properties. In: Margesin R, Schinner F (eds) Manual of soil analysis: monitoring and assessing soil bioremediation. Springer, Berlin, pp 47–97

    Chapter  Google Scholar 

  • Wrenn BA, Venosa AD (1996) Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Can J Microbiol 42:252–258

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work has been funded by the Spanish Ministery of Environment (1.1-373/2005/3-B and 013/2006/2-1.1) and by Fundación Alfonso Martin Escudero. We are deeply indebted to Dr. Marc Viñas for his help and advice during the development of the experiments. We also thank Repsol-YPF for providing the soil samples and to L.S. Jardón and J.A. Villa for helping with the lab work and the anonymous reviewers for helpful comments and improvement of the text.

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Authors and Affiliations

  1. Department of Biology and Geology, ESCET, Universidad Rey Juan Carlos, C/Tulipán s/n, 28933, Móstoles, Madrid, Spain

    M. Carmen Molina, Natalia González & Raquel Simarro

  2. Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/Tulipán s/n, 28933, Móstoles, Madrid, Spain

    L. Fernando Bautista & Raquel Sanz

  3. Department of Molecular Biology, Universidad Autónoma de Madrid, C/Darwin nº 2, 28049, Madrid, Spain

    Irene Sánchez & José L. Sanz

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  1. M. Carmen Molina
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  2. Natalia González
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  3. L. Fernando Bautista
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  4. Raquel Sanz
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  5. Raquel Simarro
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  6. Irene Sánchez
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  7. José L. Sanz
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Correspondence to M. Carmen Molina.

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Molina, M.C., González, N., Bautista, L.F. et al. Isolation and genetic identification of PAH degrading bacteria from a microbial consortium. Biodegradation 20, 789–800 (2009). https://doi.org/10.1007/s10532-009-9267-x

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  • DOI: https://doi.org/10.1007/s10532-009-9267-x

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