Role of microorganisms against hydrocarbon contamination; Bioremediation
No Thumbnail Available
Date
2015
Journal Title
Journal ISSN
Volume Title
Publisher
Uva Wellassa University of Sri Lanka
Abstract
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.
Description
Keywords
Science and Technology, Technology, Biotechnology, Power System, Environmental Science, Industrial