International Journal of Environmental Bioremediation & Biodegradation
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International Journal of Environmental Bioremediation & Biodegradation. 2017, 5(1), 8-17
DOI: 10.12691/ijebb-5-1-2
Open AccessArticle

Isolation of Indigenous Hydrocarbon Transforming Bacteria from Oil Contaminated Soils in Libya: Selection for Use as Potential Inocula for Soil Bioremediation

Althalb Hakima1, and Singleton Ian2

1Industrial and Production Chemistry Department, Libyan Petroleum Institute, Tripoli, Libya

2School of Applied Sciences, Edinburgh Napier University, Edinburgh, United Kingdom

Pub. Date: March 03, 2017

Cite this paper:
Althalb Hakima and Singleton Ian. Isolation of Indigenous Hydrocarbon Transforming Bacteria from Oil Contaminated Soils in Libya: Selection for Use as Potential Inocula for Soil Bioremediation. International Journal of Environmental Bioremediation & Biodegradation. 2017; 5(1):8-17. doi: 10.12691/ijebb-5-1-2


The Libyan oil industry has left a significant legacy of contamination and methods are required to remediate oil-contaminated soils in the area. In this work hydrocarbon utilizing microorganisms were isolated and identified from contaminated soil samples obtained from an oil Refinery (Zawia, Libya). After initial screening of eleven isolates capable of growth on hexadecane, the five most promising hydrocarbon–utilizing bacteria were isolated and tested for biosurfactant production and emulsification activity. They were identified (using 16S rRNA sequence analysis) as Pseudomonas putida, Pseudomonas species, Betaproteobacterium, Actinomyces species, and Bacillus species. Among the five species tested, Pseudomonas putida showed superior performance in terms of growth on hydrocarbons (1.0×1010 CFU (ml)), E24 emulsifying activity (86%) and ability to transform hydrocarbons in pure culture. Interestingly, gas chromatographic analysis of crude oil treated with P. putida showed a decrease in heavy hydrocarbon fractions demonstrating a clear potential for this microbe to be used as a soil inoculant in bioremediation.

Biosurfactant Emulsifying Activity hydrocarbon–utilizing bacteria Pseudomonas putida Betaproteo bacterium Actinomyces species Bacillus species

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[1]  Al-Asadi, M.K.K, Levels of oil residues in some coastal lagoons, East of Libya. Mesopot. J. Mar. Sci, 27 (1). 67-77. 2012.
[2]  Althalb, H. Potential use of nutrients and biodiesel to bioremediate diesel contaminated sub-soil. Degree of Master of Science. University of Newcastle, United Kingdom, 2007.
[3]  Anon, The analysis of agricultural material- ministry of agriculture fisheries and food. Reference Book 427. Stationary office. London, 1986.
[4]  Banat, I.M, Biosurfactants production and use in microbial enhanced oil recovery and Pollution remediation: a review. Bioresource Technology, 51. 1-12. 1995a.
[5]  Banat, I.M, Makkar, R.S, and Cameotra, S.S, Potential commercia applications of microbial surfactants. Applied Microbiology and Biotechnology, 53. 495-508. 2000a.
[6]  Bento, F.M, Camargo F.A.O, Okeke BC and Jr WTF, Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresource Technology, 96. 1049-1055. 2005a.
[7]  Bento F.M, Camargo F.A.O, Okeke BC and Jr WTF, Diversity of Biosurfactant producing microorganisms isolated from soils contaminated with diesel oil. Microbiological Research, 160. 249-255. 2005b.
[8]  Bossert, I., and Bartha, R., The fate of petroleum in soil ecosystem. In Petroleum Microbiology, Atlas Macmillan Publishers, New York, 1984, 435-473.
[9]  Boopathy, R., Factors limiting bioremediation technologies, Bioresource Technology, 74. 63-67. 2000.
[10]  Bundy, G.J., Paton, G.I., and Campbell, C.D., Combind microbial community level and single species biosensor responses to monitor recovery of oil polluted soil, Soil Biology and Biochemistry, 36. 1149-1159. February. 2004.
[11]  Cooper, D.G., and Goldenberg, Biosurfactants and enhanced oil recovery, in Microbial Enhanced Oil Recovery, Afton, Ok, Doe Conf-85051, 40. 112-114. 1982.
[12]  Farnum, R. and Harkness, M., Laboratory Evaluation of the Biodegradation of TCE at the WWTP Area. GE Global Research Publishers, 2003.
[13]  Ghazali, F., Mohamad, Rahman, R.N.Z., Salleh, A.B. and Basri, M., Biodegradation of hydrocarbons in soil by microbial consortium, International Biodeterioration Biodegradation, 54. 61-67. 2004.
[14]  Gogoi, B.K., Dutta, N.N., Goswarni, P. and Mohan, T.R.K., A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site, Advances in Environmental Research,7. 767-782. 2003.
[15]  Howage, H.M., Khalaf, M.K. and Khattali, A.O., Preliminary study on the oil pollution in the Gulf of Sirte. Journal of Basic and applied sciences, 8. 11-22. 2000.
[16]  Huang, X.D., EL-Alawi, Y., Gurska, J., Glick, B.R. and Greenberg, B.M., Amulti-process Phytoremediation system for persistent total petroleum hydrocarbons (TPH) from soil. Journal microchemical, 81. 139-147. 2005.
[17]  Jùrgensen, K.S., Puustinen, J. Suortti, A.M., Bioremediation of petroleum hydrocarbon contaminated soil by composting in biopiles. Environmental Pollution, 107. 245-254. 2000.
[18]  Keeney, D.R. and Bremner, J.M., Determination and isotope-ratio analysis of different forms of nitrogen in soils. 4. Exchangeable ammonium, nitrate, and nitrite by direct distillation methods. Soil Science Society of America Proceedings, 30. 583-58. 1966.
[19]  Kumar, A., Bisht, B.S., Joshi, V.D., and Dhewa, T., Review on Bioremediation of Polluted Environment: A Management Tool, International Journal of Environmental Sciences, 1 (6). 1079-1093. 2011.
[20]  Lai, C.C., Huang, Y.C., Wei, Y.H., Chang, J.S., Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil, Journal of Hazardous Materials, 167. 609-614. 2009.
[21]  Liebeg, E.W. and Cutright, T., The investigation of enhanced bioremediation through the addition of macro and micro nutrients in a PAH contaminated soil, International Biodeterioration and Biodegradation, 55-64. 1999.
[22]  Liu, B., Banks, M., Schwab, P., Effects of soil water content on biodegradation of phenanthrene in a mixture of organic contaminants, Soil Sediment Contam, 10. 633-658. 2001.
[23]  Lucas, J.A., Wibberley, L.J., Kennedy, E.M., and Dlugogorski, B. Z., Hydrocarbon contaminated soil and the development of an innovative thermal process, ANZ Geo. Environment, 28-30. 2001.
[24]  Lynch, J.M. and Moffat, A.J., Bioremediation-prospects for the future application of innovative applied biological research, Annals of Applied Biology, 146. 217-221. 2005.
[25]  Madigan, M.T., Martinko, J.M. and Parker, J., Biology of Microorganisms, 9th edn. Prentic Hall, Inc. New York. 2000.
[26]  Maila, M.P., Microbial ecology and bio-monitoring of total petroleum contaminated soil environment. Thesis for PhD. university of Pretoria, South African (2005).
[27]  Mcmillen, S., Bioremediation Overview – ChevronTexaco. Presentation at DOE/PERF Bioremediation Works. 1994.
[28]  Muyzer, G., Waal, E.C.D., Uitterlinden, A.G.,. Profiling of complex Microbial populations by denaturing Gradient gel electrophoresis analysis of polymerase chain reaction- amplified genes coding for 16S rRNA. Applied and Environmental Microbiology. 59 (3). 695-700. Mar. 1993.
[29]  Nayak, A.S., Vijaykumar, M.H., Karegoudar, T.B., Characterization of biosurfactant produced by Pseudoxanthomonas sp. PNK-04 and its application in bioremediation, International Biodeterioration & Biodegradation, 63. 73-79. 2009.
[30]  Nikolopoulou, M. and Kalogerakis, N., Enhanced bioremediation of crude oil utilizing lipophilic fertilizers combined with biosurfactants and molasses, Marine Pollution Bulletin, 56. 1855-1861. 2008.
[31]  Odokuma, L.O. and Akponah, E., Effect of nutrient supplementation onbiodegradation and metal uptake by three bacteria in crude oil impacted fresh and brackish waters of the Niger Delta, Cell and Animal Biology, 4. 001-018. 2010.
[32]  Okoh, A.I., Biodegradation alternative in the cleanup of petroleum hydrocarbon pollutants, Biotechnology and Molecular Biology, 1. 38-50. 2006.
[33]  Olsen, S.R., Cole, V.C., Watanabe, F.S. and Dean, L.A., Estimation of available phosphorus in soils by extraction with sodium bicarbonate, Departmentof Agriculture Circular, U.S. 1954.
[34]  Perfumo, A., Banat, I.M., Canganella, F. and Marchant, R., Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1, Applied Microbiology and Biotechnology, 72. 132-138. 2006.
[35]  Peltola, R., Bioavailability aspects of hydrophobic contaminant degradation in soils, Thesis for phD. University of Helsink. 2008.
[36]  Plaza, G.A., Lukasik, K., Wypych, J., Nalecz-Jawecki, G., Berry, C. and Brigmon, R.., Biodegradation of Crude Oil and Distillation Products by Biosurfactant-Producing Bacteria, Polish Journal of Environmental Studies,17. 87-94. 2008a.
[37]  Radwan, S.S., Microbiology of Oil- Contaminated Desert Soils and Coastal Areas in the Arabian Gulf Region, Microbiology of Extreme Soils. Soil Biology ed. by Dion and Nautiyal CS, Berlin, 13. 275-298. 2008.
[38]  Rahman, K.S., Banat, I.M., Thahira, J., Thayumanavan, T. and Lakshmanaperumalsamy, P., Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant, BioresourTechnol, 81. 25-32. 2002.
[39]  Raghavan, P.U.M. and Vivekanandan, M., Bioremediation of oil-spilled sites throughseeding of naturally adapted Pseudomonas putida, International Biodeterioration and Biodegradation, 44. 29-32. 1999.
[40]  Reddy, M.S., Basha, S., Joshi, H.V., Ramachandraiah, G., Seasonal distribution and contamination level of total PAHs and heavy metals in coastal waters of the Alang-Sosiya ship scrapping yard, Gulf of Cambay, India, Chemosphere, 61. 1587-1593. 2005.
[41]  Rowland, A.P., Lindley, D.K., Hall, G.H., Rossal, l.M.J., Wilson, D.R., Benham, D.G., Harrison, A.F. and Daniels, R.E., Effects of beach sand properties, temperature and rainfall on the degradation rates of oil in buried oil/beach sand mixtures, Environmental Pollution,109. 109-118. 2000.
[42]  Schneiker, S., Santos, V.M., Bartels, D., Bekel, T., Brecht, M., Buhrmester, J., Chernikova, T.N., Denaro, R., Ferrer, M., Gertler, C., Goesmann, A., Golyshina, O.V., Kaminski, F., Khachane, A.N., Lang, S., Linke, B., McHardy, A.C., Meyer, F., Nechitaylo, T., Pühler, A., Regenhardt, D., Rupp, O., Sabirova, J.S., Selbitschka, W., Yakimov, M.M., Timmis, K.N., Vorhölter, F.J., Weidner, S., Kaiser, O., Golyshin, P.N., Genome sequence of the ubiquitous hydrocarbon degradingmarine bacterium Alcanivoraxborkumensis, 24. 997-1004. 2006.
[43]  Shukor, M.Y., Dahalan, F.A., Jusoh, A.Z., Muse, R., Shamaan, N.A. and Syed, M.A., Characterization of a diesel-degrading strain isolated from a hydrocarbon-contaminated site, Journal of Environmental Biology, 30. 145-150. 2009.
[44]  Singh, C., Lin, J., Isolation and characterization of diesel oil degrading indigenous microrganisms in Kwazulu-Natal, South Africa, African Journal of Biotechnology. 7. 1927-1932. 2008.
[45]  Sokhn, J., De Leij, F.A.A.M., Hart, T. D and Lynch, J.M., Effect of copper on the degradation of phenanthrene by soil microorganisms, Letters in Applied Microbiology, 33. 164-168. 2001.
[46]  Sulivan, K.M. and Skladany, G.J., Decay theory biological treatment for low level organic contaminated ground-water and industrial waste. In superfund 87: proc, 8th National conference, silver spring, MD 1987.
[47]  Supaphol, S., Panichsakpatana, S., Trakulnaleamsai, S., Tungkananuruk, N., Roughjanajirapa, P. and O’Donell, A.G., The selection of mixed microbial inocula in environmental biotechnology: example using petroleum contaminated tropical soils, Journal of Microbiological Methods, 65. 432-441. 2006.
[48]  Thavasi, R., Jayalakshmi, S. and Banat, I.M., Biosurfactants from marine bacterial isolates, Applied microbiology and microbial biotechnology, 1367-1373. 2010.
[49]  Ueno, A., Ito, Y., Yamamoto, Y., Yumoto, I. and Okuyama, H., Bacterial community changes in diesel-oil-contaminated soil microcosms biostimulated with LURIA BERTANI medium orbioaugmented with a petroleum-degrading bacterium, Pseudomonas eruginosa strain WatG, Basic Microbiol, 46. 310-317. 2006.
[50]  Ward, O., Singh, A. and Hamme, J.V., Accelerated biodegradation of petroleum hydrocarbon waste, Journal of Industrial Microbiology and Biotechnology, 30. 260-270. 2003.
[51]  Widada, J., Nojiri, H., Kasuga, K., Yoshida, T., Habe, H. and Omori, T., Moleculardetection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites, Applied Microbiology and Biotechnolology, 58. 202-209. 2002.