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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 1 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.901.127 Hydrocarbon Degradation by Bacteria Isolated from Cow Dung S. K. Mondal*, D. P. Samantaray and B. B. Mishra Department of Microbiology, Odisha University of Agriculture and Technology Bhubaneswar- 751 003, India *Corresponding author ABSTRACT Keywords CD, Cow dung, Hydrocarbon degradation, Pseudomonas sp. Article Info Accepted: 15 December 2019 Available Online: 20 January 2020 Petrochemical is the fastest growing industry, also manufacturing variety of chemicals. However the waste generated during the manufacturing is a major environmental concern due to its persistence in soil and water bodies. An alternative to chemical treatment is bioremediation which aims to enhance the normal processes of microbial degradation and clear the pollutant from the environment. Cow dung is a nitrogen-rich material and is of economic importance as organic fertilizers and also contributes diverse species of microorganisms which further has capability to degrade pollutants like hydrocarbon. Therefore an attempt was made to evaluate potential of the bacteria isolated from cow dung for hydrocarbon degradation in MSM media. Two bacterial isolates CD-5 Bacillus sp. BBMRH (KF175230) and CD-7 Pseudomonas sp. BBMB (KF965279) were found effective and further utilised for degradation of diesel, petrol, kerosene, and petrol-ether. Introduction Life’s quality on earth is connected to the environment. Releases of recalcitrant and toxic petrochemicals into the environment have a negative impact on biosphere and the environment. Crude oil when released to the environment causes a variety of pollutions. It physically, chemically and biologically degrades the soil and water because of the presence of toxic compounds, such as polycyclic aromatic hydrocarbons, benzene and its substituted cyclo-alkane rings, in relatively high concentrations. These hazardous contaminants find their way into the plants, animals and human beings by direct or indirect pathways in food chain (Adams et al., 2014). Crude petroleum is a complex mixture of aliphatic and aromatic hydrocarbons which includes volatile constituents of gasoline, petrol, kerosene, lubricating oil and solid asphaltene. Serious calamities have been seen in developed and developing countries, due to contamination of 1134 Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 soil and marine environment by crude oil and petroleum products .The main sources of this pollution can originate through oil leaks and human actions and during extraction, refinement, transportation, utilization and storage of petroleum products (Agarry and Ogunleye, 2012). For reduction of hydrocarbon pollution chemical and mechanical methods are used which are very expensive and time consuming. Hydrocarbons including PAHs have been foretold as substrates which supports microbial growth (Bushnell and Haas, 1941; Speight, 1991; Ehrlich, 1995). Bioremediation is a process in which indigenous oil– consuming microflora, called petrophiles, can naturally degrade large hydrocarbons and utilize them as an energy source by using enzymes and can be useful in cleaning up of the contaminated sites (Harder, 2004 and Alexander, 1999). Microbes isolated from cow dung have considerable potential for biodegradation and biotransformation of petroleum product, further contributing to plant production and biogeochemical processes (Adegunloye et al., 2007). There are reports that bioremediation of benzene using cow dung microflora in a bioreactor (Akinde and Obire, 2008) and it was found that Pseudomonas putida isolated from cow dung micro flora was potential benzene degrader at various concentrations. Keeping in view of this, the objective of this study was to investigate the efficiency of cow dung microflora in the bioremediation of diesel, petrol, kerosene and petrol-ether under controlled conditions. Materials and Methods Screening for hydrocarbon utilization The bacteria isolated from fresh cow dung (Mondal et al., 2015, 2016) were inoculated on enrichment Mineral Salt Medium (MSM) which is composed of K2HPO4 (1.8 g/L); NH4Cl (4 g/L); MgSO4.7H2O (0.2 g/L); NaCl (0.1 g/L); Na2SO4.7H2O (0.01 g/L) and distilled water (1L) Jyothi et al., (2012).The medium was supplemented with 1% (v/v) filter sterilized hydrocarbons (petrol, diesel) to serve as the only source of the carbon and energy. The organisms were inoculated and screened on the Mineral Salt Agar medium (MSM+ Agar-20 g/L) with diesel as a sole of carbon source. The plates were incubated at 37°C for 5-7 days. Hydrocarbon degradation assay For the study of bio-degradation of hydrocarbon by bacterial isolates four sources (diesel, petrol, kerosene and petrol-ether) was taken in to consideration. Sterile autoclaved Mineral Salt medium was prepared, dispensed into 100 ml conical flask and inoculated with freshly prepared pre-selected bacterial isolates (1%) with varying percentages of filteredsterilized hydrocarbon source concentration (1%, 2%, 3%, 4%, 5% and etc.) and were incubated in rotary shaker incubator for 24 hours at optimum temperature and pH at 100120 rpm. After 24 hours of incubation these flasks containing varying concentrations of hydrocarbon sources were analysed spectrophotometrically at 595nm against control as standard which contain 24 hours incubated Mineral Salt broth medium and bacterial isolates. Results and Discussion Screening for hydrocarbon utilization During screening of bacterial isolates for the utilisation of hydrocarbon it was found the bacteria coded as CD-5(Bacillus sp. BBMRHaccession number KF175230) Mondal et al., (2015) and CD-7 (Pseudomonas sp. BBMB accession number KF965279)Mondal et al., (2016),utilized Diesel up to 1% in Mineral 1135 Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 salt media. Sukumar and Nirmala, (2016) isolated four bacterial strains from diesel contaminated soil which were Pseudomonas aeruginosa, P. putida, Arthobacter sp. and Bacillus sp., the diesel degrading efficiency of isolated organisms was tested in BH medium supplemented with diesel and DCPIP for 14 days. Graph.1 Growth of Bacillus sp. &Pseudomonas sp. in MSM medium containing diesel as the carbon source 1.2 Bacillus sp. Pseudomonas sp. O.D at 595 nm 1 0.8 0.6 0.4 0.2 0 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% DIESEL % Graph.2 Growth of Bacillus sp. & Pseudomonas sp.in MSM medium containing petrol as the carbon source 1136 Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 Graph.3 Growth of Bacillus sp. &Pseudomonas sp.in MSM medium containing kerosene as the carbon source Graph.4 Growth of Bacillus sp. &Pseudomonas sp.in MSM medium containing petrol ether as the carbon source Hydrocarbon degradation assay The bacteria inoculated in Mineral Salt medium broth were supplemented with varying percentage (v/v) of filter sterilized hydrocarbons (diesel, petrol, kerosene and petrol-ether) as single source of the carbon. The inoculated organisms after incubating at 34°C, pH 7 for Bacillus sp.and 36°C, pH 7 for Pseudomonas sp. in optimised conditions (temperature and pH) for 24hours. Optical density (OD) were measured at 595 nm and found that Bacillus sp. degraded maximum diesel at 12%,petrol at 8%, kerosene at 9% 1137 Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 and had maximum growth at10% on petrolether, whereas Pseudomonas sp. degraded maximum diesel at 8 %, petrol at 8%, kerosene at 8% and had optimal degradation rate at 9% in petrol-ether (graph-1, 2,3and 4). Jyothi et al., (2012) isolated and identified the hydrocarbon degrading bacteria associated with environmental samples collected from soil near petrol, diesel pumps and water samples and chlorine water from swimming pool. These organisms were studied for their biodegrading activities on hydrocarbons (diesel and petrol) using enrichment medium. The microbial growths were determined using calorimeter blanked at 595 nm, which revealed that bacteria Bacillus megaterium, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, Lactobacillus acidophillus, Neisseria flavescence and Corynebacterium xerosis were the potent degraders of hydrocarbons (petrol and diesel). It is evident from the findings that the two isolates viz. Bacillus sp. And Pseudomonas sp. isolated from cow dung has the potential to degrade hydrocarbon and can be employed in hydrocarbon contaminated sites for biodegradation. References Adams, G.O., Tawari-Fufeyin, P. and Ehinomen, I. 2014. Laboratory Scale Bioremediation of Soils from Automobile Mechanic Workshops Using Cow Dung. Journal of Applied & Environmental Microbiology, 2(4):128-134. Adegunloye, D. V., Adetuyi, F.C., Akinyosoye, F.A. and Doyeni, M.O. 2007. Microbial analysis of compost using cow dung as booster. Pakistan Journal of Nutrition, 6(5): 506-510. Agarry, S.E and Oladipupo, O.O. 2012. 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P. and Mishra, B. B. 2015. Optimization of Pigment Production by a Novel Bacillus sp. BBMRH isolated from Cow Dung. J. Pure and Applied Microbiology. 9(3):2321-2326. Mondal, S. K., Samantaray, D. P. and Mishra, B. B. 2016. Standardization of bioparameters for pigment production by Pseudomonas SP. BBMB isolated from cow dung. The Bioscan. 11(3): 15411545. Speight, J.G. 1991. The chemistry and technology of petroleum. Marcel Dekker, New York, N.Y. Sukumar, S. and Nirmala, P. 2016. Screening of diesel oil degrading bacteria from petroleum hydrocarbon contaminated soil. International Journal of Advanced Research in Biological Sciences, 3(8): 1822. 1138 Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1134-1139 How to cite this article: Mondal, S. K., D. P. Samantaray and Mishra, B. B. 2020. Hydrocarbon Degradation by Bacteria Isolated from Cow Dung. Int.J.Curr.Microbiol.App.Sci. 9(01): 1134-1139. doi: https://doi.org/10.20546/ijcmas.2020.901.127 1139