Isolation, Purification and Characterization of Oil Degrading Bacteria

most life forms. Episodic and chronic pollution of the environment by oil causes

major ecological perturbations. Marine environments are especially vulnerable

since oil spills of coastal regions and the open sea are poorly containable and

mitigation is difficult. In addition to pollution through human activities, about 250

million litres of petroleum enter the marine environment every year from natural

seepages. Despite its toxicity, a considerable fraction of petroleum oil entering

marine systems is eliminated by the hydrocarbon-degrading activities of microbial

communities,inparticularbyaremarkablerecentlydiscovered groupofspecialists,

the so-called hydrocarbonoclastic bacteria (HCB). Alcanivorax borkumensis was

the first HCB to have its genome sequenced. In addition to hydrocarbons, crude oil

often contains various heterocyclic compounds, such as pyridine, which appear to

be degraded by similar, though separate mechanisms than hydrocarbons.

Materials and Methods

Sampling

Three samples were collected for the entire project. Two samples were of mustard

and one of flax seed or linseed was taken. The samples were oil cakes collected

from different places. Sample 1 (mustard cake) was collected from an oil mill near

Mahewa Gate, Naini, Allahabad. Sample 2 (mustard cake) was collected from

an oil mill in Kotha Parcha, Rambagh, Allahabad. Sample 3 (linseed cake) was

collected from an oil mill infront of Gau Ghat petrol pump, Rambagh, Allahabad.

All the three samples were grinded using mortar and pestle. Then the samples

were air dried for 24 hrs.

Isolation of Oil Degrading Bacteria

Oil degrading bacteria were isolated by observing the oil degrading property. 100

ml of nutrient agar was prepared by adding 0.5 gm peptone, beef extract 0.3 gm,

0.5 gm NaCl in 50 ml distilled water. The pH of solution was maintained at 6.8 the

volume was then made upto 100 ml. The solution was then autoclaved and poured

in autoclaved petri plates and left for overnight incubation at room temperature.

Next day the samples were serially diluted and the concentration of 10-6 and 10-7

was used for inoculation. Inoculation was done in laminar air flow and the plates

were then left in incubator at room temperature for growth. After the growth was

observed, a pure culture was prepared for each culture in previously prepared agar

slants. After observing growth in the slant, calculated amount (1 ml) of mustard

oil was poured in each slant. The slants were then kept in incubator for few days to

observe oil degradation. LB broth was prepared and the cultures which degraded

oil, were inoculated in different flasks containing LB.

International Journal of Bioinformatics and Biological Sciences : v.2 n.3&4. Dec. 2014

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