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Microbial Degradation of Petroleum Hydrocarbon

Forkan Ahamed
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh.

M Hasibullah
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh.

Jannatul Ferdouse
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

M Nural Anwar
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

Abstract/ Executive Summary:

The present research was undertaken to isolate and characterize petroleum hydrocarbon degrading microbes from ship-breaking yards at Vatiary and Kumira coast in Chittagong. Twenty different petroleum hydrocarbon contaminated composite samples were collected and the total bacterial count was found to vary between 2.7×10³ cfu/ gm and 1.77×10⁷ cfu/ gm. Ten isolates were finally selected through secondary screening by Bushnell-Hass mineral salt medium using kerosene, diesel and engine oil as carbon source. They were provisionally identified and found closely related to the species Listeria monocytogenes, Staphylococcus aureus, Pseudomonas alcaligenes, Listeria grayi, Bacillus pasteurii, Bacillus badius, Bacillus cirroflagellosus, Bacillus circulans, Bacillus brevis and Citrobacter freundii. Greasy spot test was used as the primary indicator of microbial degradation of kerosene, diesel and engine oil. A more specific experiment was carried out to estimate the rate of degradation by the isolates. The highest (96.8%) degradation of diesel was shown by B. brevis, followed by 92%, 88.8% and 84.8% of diesel degradation by the strains of P. alcaligenes, B. cirroflagellosus and C. freundii, respectively. The highest degradation of kerosene (78.26%) and engine oil (43.97%) by S. aureus and L. monocytogenes respectively, were also observed.

Keywords: Microbes, Petroleum hydrocarbon, Degradation

Location: Department of Microbiology, University of Chittagong

Start Date:

End Date:

Program Area: Knowledge Management

Non-commodity: Performance

Objectives:

To isolate and characterize petroleum hydrocarbon degrading microbes from ship-breaking yards at Vatiary and Kumira coast in Chittagong.

Methodology:

Sampling site and collection of samples - Twenty different petroleum hydrocarbon contaminated composite samples (soil, water and combinations of soil and water) were aseptically collected from these ship scrapping areas. The pH, temperature and colour of the samples were recorded. Sterile glass bottle, conical flasks, polythene bags, cottons, forceps and alcohol were used in collecting the samples and after that these were transported and stored at 4^oC. Enumeration of microorganisms - Total bacterial count of the samples was determined by serial dilution followed by pour plate and spread method using nutrient agar as a medium. Selection of the isolates - From a total of 20 samples, 35 colonies were isolated and finally 10 isolates were selected on the basis of their growth in Bushnell- Haas mineral salt medium14 (0.2 gm of MgSO₄, 0.02 gm of CaCl₂, 1.0 gm of KH₂PO₄, 1.0 gm of K₂HPO₄, 1.0 gm of NH₄NO₃, 2 drops of conc. Sol. FeCl₃ in 1000 ml of distilled water) supplemented with petroleum hydrocarbon such as kerosene, diesel and engine oil. Characterization and identification of the selected isolates The isolates were characterized and identified on the basis of their morphological, cultural, physiological and biochemical characteristics. Determination of petroleum hydrocarbon degradation using greasy spot test - To perform this test, a brown paper bag or normal paper was cut into some 40×40 cm square sheets. Then, with a ruler and pencil, the large square was divided into 4×4 cm squares. Each square was designated by the code of the isolates and one was for control. Three drops from each treatment were deposited on the corresponding marked square. Then the circumference of each greasy spot was circled with a pencil and the diameter of each spot was measured and compared with the control. Estimation of petroleum hydrocarbon degradation - Seven different isolates which showed comparatively good degradation capacity in greasy spot test were cultured in three sets of 150 ml conical flask containing 25 ml of mineral salt medium15 (0.5 gm/L of NH₄Cl, 0.5 gm/L of K₂HPO₄, 0.5 gm/L of NaH₂PO₄, 750 ml of natural sea water, 250 ml of distilled water, pH 7.5). The first and second sets of conical flasks were supplemented with 0.2 % of kerosene and diesel, respectively, while the third set was supplemented with 0.16 % of engine oil. Each flask was covered by aluminum foil. The cultures were maintained in shaker at 150 rpm for 7 days at 30^oC. After 7 days of incubation cell free culture broth was extracted with three volumes (75 ml) of toluene and extract was made up to 10 ml and the OD (optical density) was measured at 420 nm. The percentage of degradation was calculated from standard value.

Source of Information: Bangladesh J Microbiol, Volume 27, Number 1, June 2010, pp 10-13

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Prior the screening of hydrocarbon degrading microorganisms, the bacterial populations were estimated in each original sample. Appreciable number of bacteria up to 10⁷cfu/gm has been found to exist in the samples. Of the 10 bacterial isolates, 5 were found to be the member of the genus Bacillus sp. The discovery of P. alcaligenes was not surprising based on their frequency in the soil as well as their frequent biodegrading ability.

Knowledge Management: Journal