UWU Conference Proceedings - UWUCP

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Browsing UWU Conference Proceedings - UWUCP by Author "Abeygunaratne, S.S."

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    Role of microorganisms against hydrocarbon contamination; Bioremediation
    (Uva Wellassa University of Sri Lanka, 2015) Arachchi, S.M.W.; Munasinghe, M.M.E.; Sabaragamukorale, S.T.; Abeygunaratne, S.S.; Rodrigo, W.W.P.; De Silva, D.P.D.C.; Dalpatadu, K.S.L.; Senaratne, S.G.; Gunathilaka, P.A.D.H.N.
    The development of human civilization has changed its path since the industrial revolution. Since then began the use of hydrocarbon sources as the primary energy source of the world. The use of oil as fuel has led to intensive economic development worldwide. Even though these compounds contribute to the global economy on massive scale they in turn have perilous effects on the biotic and abiotic components of the ecosystem. In the stages of oil refinement, transportation, storage and on daily activities, unavoidable oil spills take place in small amounts. However, the accidental large oil spills draw the attention of the public to find remediation solutions. The methods of remediation can be physical, chemical or biological or may be a combination of two or more of these techniques. Hydrocarbon utilizing bacteria, fungi and cyanobacteria have been found in soil, marine and fresh water ecosystems (Okoh, 2002). Although several countries have already used methods including microorganisms for bioremediation of petroleum spills, it has not been previously used in Sri Lanka. Therefore, the objective was to isolate indigenous bacterial strains from hydrocarbons contaminated soils to assess their potential for bioremediation and to develop a bio-product for bioremediation. Methodology Three sites with soil contaminated by different petroleum hydrocarbons were identified in Ceylon Petroleum Corporation, Sapugaskanda, Kelaniya, Sri Lanka. A total of 18 soil samples (6 from each site) were collected randomly by simple soil sampling method (American Society for Testing and Materials, 1998). A weight of 10 g of soil was diluted in 90 ml of 0.1% sterile Sodium pyrophosphate solution containing 30 g of sterile glass beads. After shaking the mixture for 1 hour at 175 rpm, the and were vortexed for 1 minute. A volume of 120 µl of each dilution was spread on Luria Broth (LB) agar medium and was incubated at 28 °C for 7 days. The colonies appeared were inoculated on a Bushnell Haas (BH) liquid and solid mediums supplemented with 50 µl of hydrocarbons followed by an incubation at 28 °C for 7 days. The identified colonies were subjected to genomic DNA extraction using the Phenol-Chloroform method. The extracted genomic DNA samples were sent over to Macrogen, Korea for 16S rRNA sequencing. The overnight grown bacterial cultures were centrifuged at 16000 g for 3 minutes at 4ºC. The pellet was resuspended in 200 µl of TE buffer and was vortexed and centrifuged at 16000 g for 1 minute at 4ºC and a volume of 1.5 µl of Protinase K was added and mixed. To this 20 µl (1/10) of 10% SDS was added, mixed well and incubated for 1 hour at 37 ºC. After the incubation, equal volume of Phenol: Chloroform (1:1) was added and centrifuged at 16000 g for 2 minutes at 4ºC. The aqueous layer was taken out without disturbing the protein layer and transferred into a fresh tube. A volume of 2V of 100% ice cold Ethanol and 0.1V Sodium acetate were added, mixed well and were incubated at 0 ºC for 1 hour. The solution mixture was centrifuged at 16000 g for 5 minutes at 4ºC. The supernatant was discarded and the pellet was dried and dissolved in 40 µl of nuclease-free water by tapping. For the selection of immobilizing agent, 10 g of autoclaved saw dust and rice husk each were mixed with 7.5 ml of Yeast Extract Glucose (YEG) broth separately and was autoclaved. Then the washed, pure bacterial cells were inoculated on to autoclaved rice husk and saw dust at room temperature and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator. The immobilized samples were washed with sterile saline water for 3 times and were inoculated on BH agar plates with diesel. Pure cultures of selected bacterial strains were inoculated with LB agar and were incubated over- night. A single colony of each bacterial strain was inoculated on 5 ml of LB broth. The cultured cells were centrifuged at 2000 g at 4°C for 10 minutes and the pellet was dissolved in 5 mL of phosphate buffer and re-centrifuged under the same conditions. Then the pellet was re-suspended in 5 ml of phosphate buffer. A mass of 14 g of autoclaved rice husk were mixed with 21 ml of YEG broth and was autoclaved. Then 2 ml of washed Bacterial cultures were inoculated on 2 g of autoclaved rice husk at room temperature separately and were incubated at 30 °C at 150 rpm for 5-6 days in a shaking incubator until a heavy culture develops. A volume of 20 ml of water was contaminated with 2 ml of diesel and 0.2 g of immobilized rice husk was added on top of the oil layers under sterile conditions. Turbidity and the time taken for the disruption of oil layer in the water were compared with a control.