Biodegradation of polycyclic aromatic hydrocarbons by Pseudomonas species

Document Type : Original Research Paper


1 Department of Microbiology, Islamic Azad University, Falavarjan Branch, Falavarjan, Isfahan, Iran

2 Department of Basic Medical Sciences,Islamic Azad University, Khorasgan Branch, Isfahan, Iran

3 Department of Basic Sciences, Islamic Azad University, Khorasgan Branch, Isfahan, Iran


Biodegradation of polycyclic aromatic hydrocarbons, toxic compounds widely distributed in the environment by bacteria, is a cheap and safe cleaning up method. The present study attempts to isolate and characterize dioxygenase-producing bacteria which are able to degrade phenanthrene and pyrene from refinery soils. It also aims to assess in vitro biodegradation. To do so, two contaminated soil samples were collected from Isfahan-Iran refinery. The population of phenanthrene and pyrene degrading bacteria were 2.17 × 103 and 1.19 × 103 CFU/g in sample 1 and  21.50 × 103 and 19.40 × 103 CFU/g in sample 2. A sum of 18 phenanthrene and pyrene degrading bacteria were isolated using enrichment culture technique, three of them getting selected which had dioxygenase activity and produced biosurfactant. Identified as Pseudomonas plecoglossicida ATAI18, Pseudomonas aeruginosa ATAI19, and Pseudomonas stutzeri ATAI21, they were submitted to GenBank under the accession number of KF113842, KF113843, and KF113845 respectively. The degradation rate of pyrene (50 mg/L) by strains ATAI18 and ATAI19 was 45.32% and 31.23%, respectively. The strain ATAI21 degraded 39.38% of phenanthrene (50 mg/L) after 9 days. These isolated bacteria can be used to improve microbial population of other hydrocarbon-polluted soils for faster bioremediation of such areas.


Alquati, C., Papacchini, M., Riccardi, C., Spicaglia, S. and Bestetti, G. (2005). Diversity of naphthalene degrading bacteria from petroleum contaminated soil. Ann. Microbiol., 55, 237-242.
Balogun, S.A. and Fagade, O.E. (2008). Screening for surface-active agent producing bacteria from diesel oil polluted tropical soil. World Appl. Sci. J., 3, 930-933.
Bin, M., Huai, H.C., Yan, H. and Jian, M.X. (2010). Isolations and consortia of PAH-degrading bacteria from the rhizosphere of four crops in PAH-contaminated field. In: Proceedings of the 19th World Congress of Soil Science, Soil Solutions for a Changing World. Brisbane, Australia, 63-66.
Boricha, H. and Fulekar, M.H. (2009). Pseudomonas plecoglossicida as a novel organism for the bioremediation of Cypermethrin. Biol. Med., 1, 1-10.
Ceyhan, N. (2012). Biodegradation of pyrene by a newly isolated  Proteus vulgaris. Sci. Res. Essays, 7, 66-77.
Das, K. and Mukherjee, A.K. (2007). Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from North-East India. Bioresour. Technol., 98, 1339–1345.
Das, N. and Chandran, P. (2011). Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnol. Res. Int., 1-13.
Fritsche, W. and Hofrichter, M. (2008). Aerobic Degradation by Microorganisms: Biotechnology. (In H. J. Rehm, and G. Reed, (pp. 145-155). Germany: Wiley-VCH Verlag GmbH.)
Garrity, G.M., Brenner, D.J., Krieg, N.R. and Staley, J.T. (2005). Bergey's Manual of Systematic Bacteriology, New York: Springer.
Guo, C., Dang, Z., Wong, Y. and Tam, N.F. (2010). Biodegradation ability and dioxgenase genes of PAH-degrading Sphingomonas and Mycobacterium strains isolated from mangrove sediments. Int. Biodeterior. Biodegrad., 64, 419-426.
Haritash, A.K. and Kaushik, C.P. (2009). Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review. J. Hazard. Mater., 169, 1-15.
Hertel, R.F., Rosner, G., Kielhorn, J., Menichini, E., Grover, P.L. and Blok, J. (1998). Selected non-heterocyclic polycyclic aromatic hydrocarbon. World Health Organization, Geneva, Environmental Health Criteria 202.
Jain, P.K. and Bajpai, V. (2012). Biotechnology of bioremediation- a review. Int. J. Environ. Sci., 3, 535-549.
Jiang, Y., Yang, X., Liu, B., Zhao, H., Cheng, Q. and Cai, B. (2004). Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization. Biosci. Biotechnol. Biochem., 68, 1798-800.
Kafilzadeh, F. and Hoshyari pour, F. (2012). Degradation of naphthalene, phenanthrene and pyrene by Pseudomonas sp. and Corynebacterium sp. in the landfills. Int. J. Biosci., 2, 77-84.
Khanna, P., Goyal, D. and Khanna, S. (2012). Characterization of pyrene utilizing Bacillus spp. from crude oil contaminated soil. Braz. J. Microbiol., 43, 606-617.
Kuberan, T., Vidhya Pallavi, J., Anburaj, R., Sundaravadivelan, C., Kumar, P. and Prabakaran, V. (2011). Degradation and quantification of polyaromatic hydrocarbon by Pseudomonas sp. Prime J. Microbiol Res., 1, 1-6.
Kumar, A., Munjal, A. and Sawhney, R. (2011). Crude oil constitution, degradation pathway and associated bioremediation microflora: an overview. Int. J. Environ. Sci., 1, 1420-1439.
Kumar, M., Leona, V., Materano, A.S., Ilzinsa, O., Castro, I.G. and Fuenmayor, S.L. (2006). Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1. Z. Naturforsch, 61, 203-212.
Lalucat, J., Bennasar, A., Bosch, R., Garcı´a-Valde´s, E. and Palleroni, N.J. (2006). Biology of Pseudomonas stutzeri. Microbiol. Mol. Biol. Rev., 70, 510-547.
Madueno, L., Coppotelli, B.M., Alvarez, H.M. and Morelli, I.S. (2011). Isolation and characterization of indigenous soil bacteria for bioaugmentation of PAH contaminated soil of semiarid Patagonia, Argentina. Int. Biodeterior. Biodegrad., 65, 345-351.
Mercadal, J.P.R., Isaac, P., Siñeriz, F. and Ferrero, M.A. (2010). Indigo production by Pseudomonas sp. J26, a marine naphthalene-degrading strain. J. Basic Microbiol., 50, 290-293.
Naama, J.H., Khalil, N. and Fattal, A.H. (2010). Study of Degrading Some Petroleum Cuts by Bacteria. J. Chem. Pharm. Res., 2, 772-784.
Niazy, Z., Hassanshahian, M. and Ataei, A. (2016). Isolation and characterization of dieseldegrading Pseudomonas strains from diesel-contaminated soils in Iran (Fars province). Pollut., 2, 67-75.
Nie, M., Yin, X., Ren, C., Wang, Y., Xu, F. and Shen, Q. (2010). Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3. Biotechnol. Adv., 28, 635-43.
Nishiguchi, M.K., Doukakis, P., Egan, M. Kizirian, D., Phillips, A., Prendini, L., Rosenbaum, H.C., Torres, E., Wyner, Y., DeSalle, R. and Giribet, G. (2002). DNA isolation Protocols: Techniques in Molecular Systematics and Evolution. (In R. DeSalle (Eds.), (pp. 243-281). Birkhäuser, Basel, Germany).
Nnamchi, C.I., Obeta, J.A.N. and Ezeogu, L.I. (2006). Isolation and characterization of some polycyclic aromatic hydrocarbon degrading bacteria from Nsukka soils in Nigeria. Int. J. Environ. Sci. Technol., 3, 181-190.
Nwinyi, O.C. (2012). Molecular and physiological studies on bacterial degradation of polynuclear aromatic hydrocarbons. Ph.D thesis University of Ota, Nigeria.
Obayori, O.S., Ilori, M.O., Adebusoye, S.A., Oyetibo, G.O. and Amund, O.O. (2008). Pyrene-degradation potentials of Pseudomonas species isolated from polluted tropical soils. World J. Microbiol. Biotechnol., 24, 2639-2646.
Okoro, C., Agrawal, A. and Callbeck, C. (2012). Simultaneous Biosurfactant Production and Hydrocarbon Biodegradation by the Resident Aerobic Bacterial Flora of Oil Production Skimmer Pit at Elevated Temperature and Saline Conditions. Life Sci. J., 9, 356-364.
Pacwa-Płociniczak, M., Płaza, G.A., Seget, Z.P. and Cameotra, S.S. (2011). Environmental applications of biosurfactants: recent advances.  Int. J. Mol. Sci., 12, 633-654.
Peng, R.H., Xiong, A.S., Xue, Y., Yan, Fu, X. Gao, F., Zhao, W., Tian, Y.S. and Yao, Q.H. (2008). Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol. Lett., 32, 927–955.
Ping, L., Zhang, C., Zhu, Y., Wu, M., Hu, X., Li, Z. and Zhao, H. (2011). Biodegrading of pyrene by a newly isolated Pseudomonas putida PL2. Biotechnol. Bioprocess Eng., 16,1000-1008.
Providenti, M.A., Flemming, C.A., Lee, H. and Trevors, J.T. (1995). Effect of addition of rhamnolipid biosurfactants or rhamnolipid-producing Pseudomonas aeruginosa on phenanthrene mineralization in soil slurries. FEMS Microbiol. Ecol., 17, 15–26.
Puskarova, A., Buckova, M. and Chovanova K. (2013). Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia. Biologia, 68, 587-591.
Rasdy, N.F.A., Sanagi, M.M., Ibrahim, W.A.W., Abu Naim, A. (2008). Determination of polycyclic aromatic hydrocarbons in palm oil mill effluent by soxhlet extraction and gas chromatography-flame ionization detection. Malaysian J. Anal. Sci., 12, 16-21.
Richards, L.A. (1954). Diagnosis and improvement of saline and alkali soils. USDA Agricultural Handbook. United States, Washington.
Roostan, Z., Safahieh, A., Mojodi, F., Zolgharnein, H., Ghanemi, K. and Abiar, H. (2012). Phenanthrene biodegradation by Pseudomonas aeruginosa and Bacillus subtilis isolated from Persian Gulf sediments. Afr. J. Microbiol. Res., 6, 4585-4591.
Saadoun, I., Mohammad, M.J., Hameed, K.M. and Shawaqfah, M.A. (2008). Microbial populations of crude oil spill polluted soils at the Jordan-Iraq desert (the Badia region). Braz. J. Microbiol., 39, 453-456.
Shekhar, S.K., Godheja, J. and Modi, D.R. (2015). Hydrocarbon Bioremediation Efficiency by five Indigenous Bacterial Strains isolated from Contaminated Soils. Int. J. Curr. Microbiol. App. Sci., 4, 892-905.
Shokrollahzadeh, S., Golmohammad, F. and Shokouhi, H. (2012). Study of Sphingopyxis isolates in degradation of polycyclic aromatic hydrocarbons. Chem. Eng. Trans., 27, 55-60.
Stringfellow, W. and Aitken, M.D. (1995). Competitive Metabolism of Naphthalene, Methylnaphthalenes and Fluorene by Phenanthrene-Degrading Pseudomonads. Appl. Environ. Microbiol., 61, 357-362.
Sutton, S. (2010). The most probable number method and Its uses in enumeration,qualification and validation.  J. Validation Technol., 16, 35-38.
Thapa, B., Ajay, K.K.C. and Anish, G. (2012). A review on bioremediation of petroleum hydrocarbon contaminants in soil. Katmandu U. J. Sci. Eng. Technol., 8, 164-170.
Tian, Y., Liu, H.J., Zheng, T.L., Kwon, K.K., Kim, S.J. and Yan, C.L. (2008). PAHs contamination and bacterial communities in mangrove surface sediments of the Jiulong River Estuary, China. Mar. Pollut. Bull., 57, 707–715.
Ward, O.P. (2010). Microbial biosurfactants and biodegradation. Adv. Exp. Med. Biol., 672, 65-74.
Wick, A.F., Haus, N.W., Sukkariyah, B.F., Haering, K. C. and Daniel, W.L. (2011). Remediation of PAH-contaminated soils and sediments: A literature review. CSES Department, Internal Research Document, 102 pp.
Winter, J.P. and Behan-Pelletier, V.M. (2007). Microarthropods. Soil Sampling and Methods of Analysis. (ed. Gregorich, E.G. and Carter, M.R.), CRC Press Taylor & Francis, USA.
Zhao, H.P. Liang, S.H. and Yang, X. (2011). Isolation and characterization of catechol 2,3-dioxygenase genes from phenanthrene degraders Sphingomonas, sp. ZP1 and Pseudomonas sp. ZP2.  Environ. Biotechnol., 32, 1895-1901.
Zhou, W., He, D., Li, X., Zhang, H., Zeng, X. and Cheng, G. (2013). Isolation and characterization of naphthalenedegrading strains, Pseudomonas sp. CZ2 and CZ5. Afr. J. Microbiol. Res., 7, 13-19.
Zhou, Y., Wei, J., Jiang, T., Gao, W., Ma, Y. and Wei, D. (2007). Characterisation of a thermostable catechol-2,3-dioxygenase from phenanthrene-degrading Pseudomonas sp. strain ZJF08. Ann. Microbiol., 57, 503-508.