Screening of biosurfactant producing and diesel oil degrading bacteria from petroleum hydrocarbon contaminated surface waters

Onur, Gözde
Hydrocarbon contamination may happen in various ways such as accidents during fuel transportation by trucks and ships, leakage of oil from underground storage tanks, or during extraction and processing of oil. These contaminations can be treated by several methods including physical, chemical and biological treatment. During biological cleaning up, hydrocarbon-degrading bacteria emulsifying hydrocarbons by producing biosurfactants are used. Therefore, isolation and identification of biosurfactant producing and hydrocarbon degrading bacteria are pivotal for effective bioremediation of hydrocarbon contaminated surface waters. Hence, the aim of this study is to isolate and identify efficient biosurfactant producing and diesel oil degrading bacteria to remove spilled diesel oil from surface waters. For this reason, bacteria isolated from the petroleum hydrocarbon-contaminated river water in close vicinity to petrol refinery were screened for their potential to produce biosurfactant and degrade diesel oil. Primary selection of diesel oil degraders was carried out by using conventional enrichment culture technique which was followed by drop-collapse test, oil displacement test and emulsification activity measurement. Primary determination of diesel oil degradation was done by using the gravimetric analysis. Secondary determination was only carried out with potential isolates by using the gas choromatographic (GC) analysis. The results of GC analysis pointed out two isolates, designated as Zn01 and Fe10, effective in diesel oil degradation with 92 and 61% respectively. The isolates Zn01 and Fe10were identified by using 16S rRNA sequencing as Acinetobacter haemolyticus and Acinetobacter calcoaceticus, respectively. Both isolates were further characterized for the presence of two novel catabolic genes (alkB and C23O), responsible for diesel oil degradation, the key enzymes (alkane monooxygenase and catechol 2,3 dioxygenase), encoded by these novel genes, and emulsifying ability of the biosurfactants produced by these two isolates through the use of several methods including DNA extraction, agarose gel electrophoresis, polymerase chain reaction (PCR), protein extraction, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), surface tension measurement, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and zeta potential measurement. The population dynamics of alkB and C23O harboring bacteria in the polluted river water were also monitored by using DNA probes through the fluorescein in situ hybridization (FISH). The study elucidated that Acinetobacter species harboring alkB and C23O seem to have high potential for diesel oil remediation with high emulsifying indices. FISH results also revealed that alkB and C23O harboring bacteria populate in the polluted surface waters successfully.
Citation Formats
G. Onur, “Screening of biosurfactant producing and diesel oil degrading bacteria from petroleum hydrocarbon contaminated surface waters,” M.S. - Master of Science, Middle East Technical University, 2015.