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Isolation and characterization of biosurfactant producing Bacillus sp. from diesel fuel-contaminated site

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Abstract

Among hydrocarbon pollutants, diesel oil is a complex mixture of alkanes and aromatic compounds which are often encountered as soil contaminants leaking from storage tanks and pipelines or as result of accidental spillage. One of the best ecofriendly approaches is to restore contaminated soil by using microorganisms able to degrade those toxic compounds in a bioremediation process. In the present study, nineteen bacteria were isolated by enrichment culture technique from diesel spilled soil collected from electric generator shed of NBAIM, Mau. All the isolates were subjected to screening for lipase production and twelve isolates were found to be positive for lipase. When the isolates were screened for biosurfactant production using CTAB-methylene blue agar plates, only one isolate viz. 2NBDSH3 was found positive which was found to be phylogenetically closely related with Bacillus flexus. Despite having low emulsification index, the bacterium could degrade 88.6% of diesel oil in soil. Biosurfactant from the isolate was extracted and characterized through infra-red spectroscopy which indicated its possible lipopeptide nature which was further supported by strong absorption in UV range in the UV-Vis spectrum. The results of the present study indicated that the isolate either does not produce any bioemulsifier or produces very low amount of emulsifier rather it produces a lipopeptide biosurfactant which helps in degradation of diesel oil by lowering the surface tension. The bacterium thus isolated and characterized can serve as a promising solution for ecofriendly remediation of bacterium diesel contaminated soils.

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References

  • Adeyemo, I.S., Agbolade, J.O., and Olufunmilola, O., Microflora associated with diesel powered generators contaminated soil arena of Obafemi Awolowo and Oduduwa Universities in ILE–IFE, Osun State, Nigeria, Global J. Biol., Agric. Health Sci., 2013, vol. 2, no. 4, pp. 65–73.

    Google Scholar 

  • Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, New York: Greene Pub. Associates, 232, 1992.

    Google Scholar 

  • Bai, G., Brusseau, M.L., and Miller, R.M., Biosurfactantenhanced removal of residual hydrocarbon from soil, J. Contam. Hydrol., 1997, vol. 25, pp. 157–170.

    Article  CAS  Google Scholar 

  • Bento, F.M., Camargo, F.A., Okeke, B.C., and Frankenberger, W.T., Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation, Bioresour. Technol., 2005, vol. 96, pp. 1049–1055.

    Article  CAS  PubMed  Google Scholar 

  • Cooper, D.G. and Goldenberg, B.G., Surface-active agents from two Bacillus species, Appl. Environ. Microbiol., 1987, vol. 53, pp. 224–229.

    PubMed Central  CAS  PubMed  Google Scholar 

  • Denger, K. and Schink, B. New halo- and thermotolerant fermenting bacteria producing surface-active compounds, Appl. Microbiol. Biotechnol., 1995, vol. 44, pp. 161–166.

    Article  CAS  Google Scholar 

  • Desai, J.D. and Banat, I.M., Microbial production of surfactants and their commercial potential, Microbiol. Mol. Biol. Rev., 1997, vol. 61, pp. 47–64.

    PubMed Central  CAS  PubMed  Google Scholar 

  • Edwards, U., Rogall, T., Blöcker, H., Emde, M., and Böttger, E.C., Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA, Nucleic Acids Res., 1989, vol. 17, pp. 7843–7853.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hazra, C., Kundu, D., and Chaudhari, A., Lipopeptide biosurfactant from Bacillus clausii BS02 using sunflower oil soapstock: evaluation of high throughput screening methods, production, purification, characterization and its insecticidal activity, RSC Adv., 2015, vol. 5, pp. 2974–2982.

    Article  CAS  Google Scholar 

  • Ilori, M.O., Adebusoye, S.A. and Ojo, A.C., Isolation and characterization of hydrocarbon-degrading and biosurfactant- producing yeast strains obtained from a polluted lagoon water, World J. Microbiol. Biotechnol., 2008, vol. 24, pp. 2539–2545.

    Article  CAS  Google Scholar 

  • Jirasripongpun, K., The characterization of oil-degrading microorganisms from lubricating oil contaminated (scale) soil, Lett. Appl. Microbiol., 2002, vol. 35, pp. 296–300.

    Article  CAS  PubMed  Google Scholar 

  • Kanaly, R.A. and Harayama, S., Biodegradation of highmolecular- weight polycyclic aromatic hydrocarbons by bacteria, J. Bacteriol., 2000, vol. 182, pp. 2059–2067.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kayode-Isola, T., Eniola, K., Olayemi, A., and Igunnugbemi, O., Response of resident bacteria of a crude oilpolluted river to diesel oil, Amer.-Euras. J. Agro., 2008, vol. 1, pp. 06–09.

    Google Scholar 

  • Kebria, D.Y., Khodadadi, A., Ganjidoust, H., Badkoubi, A., and Amoozegar, M., Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel, Int. J. Environ. Sci. Technol., 2009, vol. 6, pp. 435–442.

    Article  CAS  Google Scholar 

  • Khanna, P., Goyal, D., and Khanna, S., Characterization of pyrene utilizing Bacillus spp. from crude oil contaminated soil, Brazil. J. Microbiol., 2012, vol. 43, pp. 606–617.

    Article  CAS  Google Scholar 

  • Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y. S., Lee, J.-H., and Yi, H., Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species, Int. J. Syst. Evol. Microbiol., 2012, vol. 62, pp. 716–721.

    Article  CAS  PubMed  Google Scholar 

  • Kosaric, N. and Sukan, F. V., Biosurfactants: Production: Properties: Applications, New York: CRC, 1993.

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Langworthy, D., Stapleton, R., Sayler, G., and Findlay, R., Lipid analysis of the response of a sedimentary microbial community to polycyclic aromatic hydrocarbons, Microb. Ecol., 2002, vol. 43, pp. 189–198.

    Article  CAS  PubMed  Google Scholar 

  • Makkar, R. and Cameotra, S., Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis, J. Ind. Microbiol. Biotechnol., 1998, vol. 20, 48–52.

    Article  CAS  Google Scholar 

  • Marquez-Rocha, F.J., Hernández-Rodrí, V., and Lamela, M.T., Biodegradation of diesel oil in soil by a microbial consortium, Water, Air, Soil Pollut., 2001, vol. 128, pp. 313–320.

    Article  CAS  Google Scholar 

  • Morikawa, M., Ito, M., and Imanaka, T., Isolation of a new surfactin producer Bacillus pumilus A-1, and cloning and nucleotide sequence of the regulator gene, psf-1, J. Ferment. Bioeng., 1992, vol. 74, pp. 255–261.

    Article  CAS  Google Scholar 

  • Mukherjee, S., Das, P., and Sen, R., Rapid quantification of a microbial surfactant by a simple turbidometric method, J. Microbiol. Methods, 2009, vol. 76, pp. 38–42.

    Article  CAS  PubMed  Google Scholar 

  • Pekdemir, T., Ishigami, Y., and Uchiyama, H., Characterization of aescin as a biosurfactant for environmental remediation, J. Surfactants Deterg., 1999, vol. 2, pp. 337–341.

    Article  CAS  Google Scholar 

  • Rahman, K., Rahman, T. J., Kourkoutas, Y., Petsas, I., Marchant, R., and Banat, I., Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients, Bioresour. Technol., 2003, vol. 90, pp. 159–168.

    Article  CAS  PubMed  Google Scholar 

  • Rahman, K., Thahira-Rahman, J., Lakshmanaperumalsamy, P., and Banat, I., Towards efficient crude oil degradation by a mixed bacterial consortium, Bioresour. Technol., 2002, vol. 85, pp. 257–261.

    Article  CAS  PubMed  Google Scholar 

  • Samad, M.Y.A., Razak, C.N.A., Salleh, A.B., Yunus, W.Z.W., Ampon, K., and Basri, M., A plate assay for primary screening of lipase activity, J. Microbiol. Methods, 1989, vol. 9, pp. 51–56.

    Article  CAS  Google Scholar 

  • Satpute, S., Bhawsar, B., Dhakephalkar, P., and Chopade, B., Assessment of different screening methods for selecting biosurfactant producing marine bacteria, Indian J. Mar. Sci., 2008, vol. 37, p. 243.

    CAS  Google Scholar 

  • Singh, A., Kumar, K., Pandey, A.K., Sharma, A., Singh, S.B., Kumar, K., Arora, A., and Nain, L., Pyrene degradation by biosurfactant producing bacterium Stenotrophomonas maltophilia, Agric. Res., 2015, vol. 4, pp. 1–6.

    Article  Google Scholar 

  • Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S., MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol., 2011, vol. 28, pp. 2731–2739.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • USEPA, SW-846, Test Methods for Evaluating Solid Waste (SW-846). Methods 8015 and 3540A. United States Environmental Protection Agency, 1986.

  • Viramontes-Ramos, S., Portillo-Ruiz, M.C., Ballinas-Casarrubias, M.d.L., Torres-Muñoz, J.V., Rivera-Chavira, B.E., and Nevárez-Moorillón, G.V., Selection of biosurfactan/bioemulsifier-producing bacteria from hydrocarbon- contaminated soil, Brazil. J. Microbiol., 2010, vol. 41, pp. 668–675.

    Article  CAS  Google Scholar 

  • Willumsen, P.A. and Karlson, U., Screening of bacteria, isolated from PAH-contaminated soils, for production of biosurfactants and bioemulsifiers, Biodegradation, 1996, vol. 7, pp. 415–423.

    Article  CAS  Google Scholar 

  • Wu, T., Xie, W., Yi, Y., Li, X., Yang, H., and Wang, J., Surface activity of salt-tolerant Serratia spp. and crude oil biodegradation in saline soil, Plant Soil Environ., 2012, vol. 58, no. 9, pp. 412–416.

    CAS  Google Scholar 

  • Yakimov, M.M., Timmis, K.N., Wray, V., and Fredrickson, H.L., Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50, Appl. Environ. Microbiol., 1995, vol. 61, pp. 1706–1713.

    PubMed Central  CAS  PubMed  Google Scholar 

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Correspondence to Hillol Chakdar.

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Yadav, A.K., Manna, S., Pandiyan, K. et al. Isolation and characterization of biosurfactant producing Bacillus sp. from diesel fuel-contaminated site. Microbiology 85, 56–62 (2016). https://doi.org/10.1134/S0026261716010161

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