Determination of Location of Phenol Resistant Gene in Phenol Degrading Bacterial Strains Expressing Phenol Hydroxylase Activity

Abstract

Phenols in waste water consist of variety of hydroxybenzene and substituted hydroxybenzene. These phenolic compounds in waste water get mixed up with river, oceans and also contaminate the soil which is very dangerous for flora and fauna even at low concentration. Nowadays degradation of such xenobiotic compounds by microbial activity is considered as the widely accepted process of waste treatment and environment pollution control. This treated waste water can then be used for irrigation purposes in agriculture as this treated waste water will have negligible toxic pollutant and deemed fit as per the norms of CPCB. So, the present study has been done to isolate the phenol degrading bacterial strains expressing Phenol hydroxylase activity on Minimal Salt Media (MSM), Different growth factors were optimized. Three strains NY-1, NS-1 & SP-2 were found to be resistant to phenol concentration up to the range of 5-7 mM and degradation was found to be in the range of 80-90 %. Mode of ring cleavage was found to be ortho-cleavage which is carried out by the enzyme Phenol hydroxylase. Therefore to determine location of phenol resistant gene acridine orange mediated plasmid curing was done taking antibiotic resistant property of the bacterial strain of interest as selectable marker.The concentration of acridine orange at which the plasmid of the bacterial isolates were cured was found to be in the range of 25-40 μg/ml, above which the concentration of acridine orange was found to be lethal to bacterial growth. At 40 μg/ml, plasmids of all the three bacterial isolates got cured completely. The cured bacterial colonies were screened using the replica plate method. The colonies that showed no growth on the replica plate containing appropriate antibiotic salt as compared with the master plate were further screened for their phenol degrading capability. These colonies were inoculated in the Minimal Salt Media and were kept for incubation at 37°C for 24-72 h under shaking condition. Phenol degradation was almost negligible, which indicated that genes encoding for enzyme Phenol hydroxylase was plasmid borne.