biodegradation of hydrocarbons

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bioworld
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biodegradation of hydrocarbons

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Hello friends I’ve been trying to isolate and enrich hydrocarbon degrading organisms from soil (taken from places where there is constant spillage of petrol n oil like garage, gas stations). I inoculated 1ml filtered (through muslin cloth) soil sample (mixed in distilled water) in MSM media (25ml) with toluene (1ml) as a sole carbon source. Incubation was donr for 15 days n 1ml toluene was added every alternate day.
I got growth in the media, gram staining showed the presence of gr +ve as well as -ve cocci, gr +ve as well as gr –ve coccobacilli, gr +ve bacilli with spores… it s quite understandable that majority of these organisms are non bio-degrading organisms.. but wen I enriched for the second time innoculating 1ml of culture frm the 1st flask in again MSM media with toluene, I did not get any growth for 15 days..
Problems I faced- since toluene s immiscible in the media . (n if I use other HC source like naphthalene or camphor, in wat state should I use it? I shud crush it n add the powder in the media or add it in the form of large tablets (in a known concentration))
Please let me know where I’m goin wrong.. or suggest me any alternate way to enrich hydrocarbon degrading organisms frm soil.
Please help!!