Isolation and Identification of Hydrocarbon Degrading Bacteria and Transformation of the Gene Responsible into Sensitive Bacteria

Author Name(s): *Nuthan Vikas Bathula, Revanth Reddy Pasham
Author Email: nuthanvikas97@gmail.com

Abstract

Environmental pollution due to petroleum oil spillage is a major problem of the day. Bioremediation is an efficient method in oil spill treatment. Bacteria that are capable of degrading complex hydrocarbons into their simplest forms that can be degradable are used in Bioremediation protocol. In the present study, soil contaminated with petroleum was collected from petrol refilling station, to isolate bacteria capable of hydrocarbon degradation. Bushnell Haas media, with diesel as sole carbon source was used as screening media. The isolated bacteria, capable of hydrocarbon degradation was identified as Corynebacterium xerosis using the biochemical tests performed based on the Bergey’s Manual of Determinative Bacteriology. The key enzyme involved in hydrocarbon degradation by Corynebacterium xerosis was identified as protocatechuate 3,4-dioxygenase using various data mining protocols. The protein sequence for protocatechuate 3,4-dioxygenase was retrieved from NCBI data base. The same sequence was used to design the primers for amplification. Amplicons for the gene that codes for protocatechuate 3,4-dioxygenase in Corynebacteria, were obtained using PCR. Soil sample from ground was collected again to isolate bacteria incapable of hydrocarbon degradation. The developed Amplicons were transformed into isolated non-hydrocarbon degrading bacteria. The same bacteria which were incapable of hydrocarbon degradation were able to degrade hydrocarbons after transformation more efficiently.

Keywords

Hydrocarbons, Bushnell Haas Media, Corynebacteria xerosis, Protocatechuate 3,4-dioxygenase

Introduction

Earth has good reservoirs of oils and gas beneath its surface. Crude oil can be extracted from the earth crust using techniques like Multilateral Drilling, Extended Reach Drilling, Complex Path Drilling, etc. Despite using the revolutionary and modernistic technology we have today, still we could see many oil spill accidents happening time to time. The spilled crude oil damages beaches, marshlands, fragile ecosystems, and also destroys wildlife Habitats and Breeding grounds.

Conclusion

Pollution due to petroleum oil spillage is increasing day by day. All eco systems are being affected by this, especially marine organisms and birds at sea shores are getting more effected. Wildlife habitat and breeding grounds are being destroyed by oil spillage in oceans. This challenge can be addressed to some extent by bioremediation, where the microorganisms are used to degrade complex hydrocarbon molecules into simple carbons. In the present study, bacteria capable of degrading hydrocarbons were isolated from soil contaminated with petroleum oil. The isolated hydrocarbon degrading bacteria were screened using Bushnell Haas media. The isolated bacteria were identified as Corynebacterium xerosis using Bergey’s Manual of Determinative Bacteriology. Bacteria belonging to Corynebacterium species are good hydrocarbon degraders. Protocatechuate 3,4-dioxygenase is one of the enzymes secreted by Corynebacterium for hydrocarbon degradation. Amplicons of the gene coding for protocatechuate 3,4-dioxygenase were obtained using PCR. Non-hydrocarbon degrading bacteria are isolated from soil sample and sub-cultured in nutrient broth. The prepared amplicons are transformed into non-hydrocarbon degrading bacteria. The transformation was successful and the transformed bacteria showed good growth in Bushnell Haas media with diesel as the sole source of carbon. This indicates the success of transformation.

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