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Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons

  • DECAPAGE Project: Hydrocarbon degradation in coastal sediments*
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Abstract

Crude oil is a common environmental pollutant composed of a large number of both aromatic and aliphatic hydrocarbons. Biodegradation is carried out by microbial communities that are important in determining the fate of pollutants in the environment. The intrinsic biodegradability of the hydrocarbons and the distribution in the environment of competent degrading microorganisms are crucial information for the implementation of bioremediation processes. In the present study, the biodegradation capacities of various bacteria toward aliphatic and aromatic hydrocarbons were determined. The purpose of the study was to isolate and characterize hydrocarbon-degrading bacteria from contaminated soil of a refinery in Arzew, Algeria. A collection of 150 bacterial strains was obtained; the bacterial isolates were identified by 16S rRNA gene sequencing and their ability to degrade hydrocarbon compounds characterized. The isolated strains were mainly affiliated to the Gamma-Proteobacteria class. Among them, Pseudomonas spp. had the ability to metabolize high molecular weight hydrocarbon compounds such as pristane (C19) at 35.11 % by strain LGM22 and benzo[a] pyrene (C20) at 33.93 % by strain LGM11. Some strains were able to grow on all the hydrocarbons tested including octadecane, squalene, phenanthrene, and pyrene. Some strains were specialized degrading only few substrates. In contrast, the strain LGM2 designated as Pseudomonas sp. was found able to degrade both linear and branched alkanes as well as low and high poly-aromatic hydrocarbons (PAHs). The alkB gene involved in alkane degradation was detected in LGM2 and other Pseudomonas-related isolates. The capabilities of the isolated bacterial strains to degrade alkanes and PAHs should be of great practical significance in bioremediation of oil-contaminated environments.

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References

  • Atlas RM, Atlas MC (1991) Biodegradation of oil and bioremediation of oil dpills. Curr Opin Biotechnol 2:440–443

    Article  CAS  Google Scholar 

  • Babla MT, Al-Awadhi N, Al-Daher R (1998) Bioremediation of oil-contaminated soil : microbiological methods for feasibility assessment and field evaluation. J Microbiol Methods 32:155–164

    Article  Google Scholar 

  • Baheri H, Meysami P (2001) Feasibility of fungi bioaugmentation in composting a flare pit soil. J Hazard Mater 89:279–286

    Article  Google Scholar 

  • Binazadeh M, Karimi IA, Li Z (2009) Fast biodegradation of long chain n-alkanes and crude oil at high concentration with Rhodococcus sp. Moj-3449. Enzym Microb Technol 45:195–202

    Article  CAS  Google Scholar 

  • Boonchan S, Britz ML, Stanley GA (2000) Degradation and mineralization of highmolecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures. Appl Environ Microbiol 66:1007–1019

    Article  CAS  Google Scholar 

  • Bordenave S, Ms G-U, Vilette C, Blanchard S, Caumette P, Duran R (2008) Diversity of ring-hydroxylating dioxygenases in pristine and oil contaminated microbial mats at genomic and transcriptomic levels. Environ Microbiol 10:3201–3211

    Article  CAS  Google Scholar 

  • Bouaid A, Martínez M, Aracil J (2010) Biorefinery approach for coconut oil valorisation: a statistical study. Bioresour Technol 10:4006–4012

    Article  Google Scholar 

  • Boyd C, Larkin MJ, Reid KA, Sharma ND, Wilson K (1997) Metabolism of naphthalene, 1-naphthol, indene, and indole by Rhodococcus sp. strain NCIMB 12038. Appl Environ Microbiol 63:151–155

    CAS  Google Scholar 

  • Brito EMS, Guyonraud R, Goni-Urriza M, Ranchou-Peyruse A, Verbaere A, Crapez MAC, Wasserman JCA, Duran R (2006) Characterization of hydrocarbonoclastic bacterial communities from mangrove sediments in Guanabara Bay, Brazil. Res Microbiol 157:752–762

    Article  CAS  Google Scholar 

  • Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3:351–368

    Article  CAS  Google Scholar 

  • Churchill SA, Harper JP, Churchill PF (1999) Isolation and characterization of a Mycobacterium species capable of degrading three and four-ring aromatic and aliphatic hydrocarbons. Appl Environ Microbiol 65:549–552

    CAS  Google Scholar 

  • Das K, Mukherjee AK (2007) Differential utilization of pyrene as the sole source of carbon by Bacillus subtilis an Pseudomonas aeroginosa strains: role of biosurfactants in enhancing bioavailability. J Appl Microbiol 102:195–203

    Article  CAS  Google Scholar 

  • Delille D, Coulon F, Pelletier E (2004) Biostimulation of natural microbial assemblages in oil-amended vegetated and desert sub-Antarctic soils. Microb Ecol 47:407–415

    Article  CAS  Google Scholar 

  • Desai C, Madamwar D (2006) Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments. Bioresour Technol 98:761–768

    Article  Google Scholar 

  • Fiorella PD, Spain JC (1997) Transformation of 2, 4, 6-Trinitrotoluene by Pseudomonas pseudoalcaligenes. Appl Environ Microbiol 63:2007–2015

    CAS  Google Scholar 

  • Grötzschel S, Koster J, Abed RM, de Beer D (2002) Degradation of petroleum model compounds immobilized on clay by hypersaline microbial mat. Biodegradation 13:273–283

    Article  Google Scholar 

  • Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235

    Article  CAS  Google Scholar 

  • Hasanshahian M, Giti E (2008) Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Int Biodeterior Biodegrad 62:170–178

    Article  Google Scholar 

  • Hua F, Wang H (2011) Uptake modes of octadecane by Pseudomonas sp. DG17 and synthesis of biosurfactant. J Appl Microbiol 112:25–37

    Article  Google Scholar 

  • Johnsen AR, Bendixen K, Karlson U (2002) Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1. Appl Environ Microbiol 68:2683–2689

    Article  CAS  Google Scholar 

  • Juhasz AI, Naidu R (2000) Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene. Int Biodeterior Biodegrad 45:57–88

    Article  CAS  Google Scholar 

  • Kaczorek E, Olszanowski A (2011) Uptake of hydrocarbon by Pseudomonas fluorescens (P1) and Pseudomonas putida (K1) strains in the presence of surfactants: a cell surface modification. Water Air Soil Pollut 214:451–459

    Article  CAS  Google Scholar 

  • Kanaly R, Harayama S (2000) Biodegradation of high-molecular weight polycyclic aromatic hydrocarbons by bacteria. J Bacteriol 182:2059–2067

    Article  CAS  Google Scholar 

  • Karamalidis AK, Evangelou AC, Karabika E, Koukou AI, Drainas C, Voudrias EA (2010) Laboratory scale bioremediation of petroleum-contaminated soil by indigenous microorganisms and added Pseudomonas aeruginosa strain Step. Bioresour Technol 101:6545–6552

  • Katsivela E, Moore ERB, Kalogerakis N (2002) Biodegradation of aliphatic and aromatic hydrocarbons: specificity among bacteria isolated from refinery waste sludge. Water Air Soil Pollut 3:103–115

    Article  Google Scholar 

  • Kloos K, Munch JC, Schloter M (2006) A new method for the detection of alkanemonooxygenase homologous genes (alkB) in soils based on PCR-hybridization. J Microbiol Methods 66:486–496

    Article  CAS  Google Scholar 

  • Lal B, Khanna S (1996) Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans. J Appl Bacteriol 81:355–362

    CAS  Google Scholar 

  • Langlois E (1998) Theses. Biodegradation of nitroaromatics compounds. Laval University- National library of Canada

  • Leahy JG, Colwell RR (1990) Microbial degradation of hydrocarbons in the environment. Microbiol Rev 54:305–315

    CAS  Google Scholar 

  • Lily MK, Bahuguna A, Dangwal K, Garg V (2010) Optimization of an inducible, chromosomally encoded benzo[a]pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447). Ann Microbiol 60:51–58

    Article  CAS  Google Scholar 

  • Ludwig W, Klenk HP (2001) Overview: a phylogenetic backbone and taxonomic framework for procaryotic systematics. In: BDRaC RW (ed) Bergey's manual of systematic bacteriology. Springer-Verlag, New York, N.Y. edn. pp 49–65

  • Maier T, Foerster HH, Asperger O, Hahn U (2001) Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun 286:652–658

    Article  CAS  Google Scholar 

  • Mannee P, Prayad P, Edward S, Upathan A, Ladda T (1998) Biodegradation of crude oil by soil microorganisms in the tropic. Biodegradation 9:83–90

    Article  Google Scholar 

  • Marquez-Rocha FJ, Olmos-Soto J, Rosano-Hernandez MC, Muriel-Garcia M (2005) Determination of the hydrocarbon-degrading metabolic capabilities of tropical bacterial isolates. Int Biodeterior Biodegrad 55:17–23

    Article  CAS  Google Scholar 

  • Mesarch MB, Nakatsu CH, Nies L (2000) Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR. Appl Environ Microbiol 66:678–683

    Article  CAS  Google Scholar 

  • Morgan P, Watkinson RJ (1989) Hydrocarbon degradation in soils and methods for soil biotreatment. Crit Rev Biotechnol 8:305–333

    Article  CAS  Google Scholar 

  • Obayori OS, Adebusoye SA, Adewale AO, Oyetibo GO, Oluyemi OO, Amokun RA, Ilori MO (2000) Differential degradation of crude oil (Bonny Light) by four Pseudomonas strains. J Environ Sci 21:243–248

    Article  Google Scholar 

  • Overcash MR, Pal D (1979) Design of land treatment system for industrial wastes: theory and practice. Ann Arbor Sci, Ann Arbor, pp 159–219

    Google Scholar 

  • Païssé P, Duran R, Coulon F, Goñi-Urriza M (2011) Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments? Appl Microbiol Biotechnol 92:835–844

    Article  Google Scholar 

  • Parales RE, Bruce CN, Schmid A, Wackett LP (2002) Biodegradation, biotransformation, and biocatalysis (B3). Appl Environ Microbiol 68:4699–4709

    Article  CAS  Google Scholar 

  • Penet S, Marchal R, Sghir A, Monot F (2004) Biodegradation of hydrocarbon cuts used for diesel oil formulation. Appl Microbiol Biotechnol 66:40–47

    Article  CAS  Google Scholar 

  • Penet S, Vendeuvre C, Bertoncini F, Marchal R, Monot F (2006) Characterization of biodegradation capacities of environmental microflorae for diesel oil by comprehensive two-dimensional gas chromatography. Biodegradation 17:577–585

    Article  CAS  Google Scholar 

  • Pérez-Silva RM, Rodriguez AA, Gomez Montes de Oca JM, Moreno DC (2006) Biodegradation of crude oil by Pseudomonas aeroginosa AT18 strain. Tecnologia Quimiqua XXVI, N 1

  • Persson NA, Welander TG (1994) Biotreatment of petroleum hydrocarbons-containing sludges by landfarming. In: Hinchee RA, Alleman BC, Hoeppel RE, Miller RN (eds) Hydrocarbon bioremediation. Lewis Publishers, Boca Raton, pp 335–342

    Google Scholar 

  • Pfennig N, Trüper HG (1992) The family Chromatiaceae. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes. Springer-Verlag, New York, pp 3200–3221

    Chapter  Google Scholar 

  • Rentza JA, Alvarezb N, Schnoor PJJ (2008) Benzo[a]pyrene degradation by Sphingomonas yanoikuyae JAR02. Environ Pollut 151:669–677

    Article  Google Scholar 

  • Rocha-Carlos A, Pedregosa Ana M, Laborda F (2011) Biosurfactant-mediated biodegradation of staight and methyl-branched alaknes by Pseudomonas aeroginosa ATCC 55925. AMB Express 1:9

    Article  CAS  Google Scholar 

  • Rojo F (2009) Degradation of alkanes by bacteria. Environ Microbiol 10:2477–90

  • Salgado-Brito R, Neria MI, Mesta-Howard M, Diaz Cedillo F, Wang ET (2007) Oxidation of solid paraffin (C11-40) by Pseudomonas aeroginosa MGP-1. Ann Microbiol 57:321–328

    Article  CAS  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 12:5463–5467

    Article  Google Scholar 

  • Singer ME, Finnerty WR (1984) Microbial metabolism of straight-chain and branched alkanes. In: Atlas RM (ed) Petroleum microbiol. Macmillan, New York, pp 1–59

    Google Scholar 

  • Smith JJ, McFeters GA (1997) Mechanisms of INT (2-(4-iodophenyl)-3(nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditoolyl tetrazolium chloride) reduction in Escherichia coli K-12. J Microbiol Methods 29:161–175

    Article  CAS  Google Scholar 

  • Smits HM, Balada SB, Witholt B, van Beilen JB (2002) Functional analysis of alkane hydroxylases from gram-negative bacteria. J Bacteriol 184:1733–1742

    Article  CAS  Google Scholar 

  • Solano-Serena F, Marchal R, Lebeault JM, Vandecasteele JP (2000) Distribution in the environment of degradative capacities for gasoline attenuation. Biodegradation 11:29–35

    Article  CAS  Google Scholar 

  • Sotsky JB, Greer CW, Atlas RM (1994) Frequency of genes in aromatic and aliphatic hydrocarbons biodegradation pathways within bacterial populations from Alaskan sediments. Can J Microbiol 40:981–985

    Article  CAS  Google Scholar 

  • Sudip KS, Singh OV, Jain RK (2002) Polycyclic aromatic hydrocarbon: environmental pollution and bioremediation. Trends Biotechnol 20:243–248

    Article  Google Scholar 

  • Thangaraj K, Kapley A, Purohit H (2008) Characterization of diverse Acinetobacter isolates for utilization of multiple aromatic compounds. Bioresour Technol 99:2488–2494

    Article  CAS  Google Scholar 

  • Toledo FL, Calovo C, Rodelas J, Gonzales-Lopez J (2006) Selection and identification of bacteria isolated from waste crude oil with polycyclic aromatic hydrocarbons removal capacities. Syst Appl Microbiol 29:244–252

    Article  CAS  Google Scholar 

  • Van Beilen JB, Funhoff EG (2005) Expanding the alkaneoxygenase toolbox: new enzymes and applications. Curr Opin Biotechnol 16:308–314

    Article  Google Scholar 

  • Van Beilen JB, Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74:13–21

    Article  CAS  Google Scholar 

  • Van Hamme JD, Singh A, Ward OP (2003) Recent advances in petroleum microbiology. Microbiol Mol Biol Rev 67:503–549

    Article  Google Scholar 

  • Vila J, Lopez Z, Sabate J, Minguillon C, Solanas AM, Grifoll M (2001) Identification of a novel metabolite of pyrene by mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons. Appl Environ Microbiol 67:5497–5505

    Article  CAS  Google Scholar 

  • Vinas M, Grifoll M, Sabate 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

    Article  CAS  Google Scholar 

  • Vomberg A, Klinner U (2000) Distribution of alkB genes within n-alkane-degrading bacteria. J Appl Microbiol 89:339–348

    Article  CAS  Google Scholar 

  • Whyte LG, Greer CW, Inniss WE (1996) Assessment of the biodegradation potential of psychrotrophic microorganisms. Can J Microbiol 42:99–106

    Article  CAS  Google Scholar 

  • Whyte LG, Bourbonnière L, Greer CW (1997) Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkanes (alk) and naphthalene (nah) catabolic pathways. Appl Environ Microbiol 63:3719–3723

    CAS  Google Scholar 

  • Whyte LG, Schultz A, van Beilen JB, Luz AP, Pellizari VH, Labbe D, Greer CW (2002) Prevalence of alkane monooxygenase genes in Arctic and Antarctic hydrocarboncontaminated and pristine soils. FEMS Microbiol Ecol 41:141–150

    CAS  Google Scholar 

  • Wong JWC, Zhao Z, Zheng G (2010) Biosurfactants from Acinetobacter calcoaceticus BU03 enhance the bioavailability and biodegradation of polycyclic aromatic Hydrocarbons. Proceeding of the annual international conference on soils, sediments, water and energy: vol. 15, Article 5

  • Xie M, Alonso H, Roujeinikova A (2011) An imporved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1. Appl Biochem Biotechnol 165:823–831

    Article  CAS  Google Scholar 

  • Youssef N, Sheik CS, Krumholz LR, Najar FZ, Roe BA, Elshahed MS (2009) Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys. Appl Environ Microbiol 75:5227–5236

    Article  CAS  Google Scholar 

  • Zhang Y, Miller RM (1994) Effect of a Pseudomonas rhamnolipid biosurfactants on cell hydrophobicity and biodegradation of octadecane. Appl Environ Microbiol 60:2101–2106

    CAS  Google Scholar 

  • Zhang Z, Gai L, Hou Z, Yang C, Ma C, Wangd Z, Sun B, He X, Tang H, Ping Xu P (2010) Characterization and biotechnological potential of petroleum-degrading bacteria isolated from oil-contaminated soils. Bioresour Technol 10:8452–8456

    Article  Google Scholar 

  • Zhang Z, Hou Z, Yang C, Ma C, Tao F, Xu P (2011) Degradation of n-alkanes and polycyclic hydrocarbons in petroleum by a newly isolated Pseudomonas aeroginosa DQ8. Bioresour Technol 102:4111–4116

    Article  CAS  Google Scholar 

  • Zhao HP, Wu QS, Wang L, Zhao XT, Gao HW (2009) Degradation of phananthrene by bacterial strain isolated from soil in oil refinery fields in Shanghai China. J Hazard Mater 164:863–869

    Article  CAS  Google Scholar 

  • Zhuang WQ, Zhuang WQ, Maszenan AM, Tay ST (2002) Bacillus naphthovorans sp. nov. from oil-contaminated tropical marine sediments and its role in naphthalene biodegradation. Appl Microbiol Biotechnol 58:547–553

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the laboratory EEM from Pau University. Authors would like to thank SONATRACH-Algeria for providing soil samples from the refinery of Arzew-Oran. G.A. was supported by Algerian University grant in the «program national exceptional- PNE» during her stay at EEM-Pau University.

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Correspondence to A. Guermouche M’rassi.

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Guermouche M’rassi, A., Bensalah, F., Gury, J. et al. Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons. Environ Sci Pollut Res 22, 15332–15346 (2015). https://doi.org/10.1007/s11356-015-4343-8

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