Screening for Efficient Hydrocarbon Degradation By Bacterial Isolates From Crude Oil Contaminated Sites of Assam, India

Screening for efficient hydrocarbon degradation by bacterial isolates from crude oil contaminated sites of Assam, India

 

Abhijit Sarma Roy¹, Sounak Bera ², A.K.Ghoshal*^{1, 2}

1Centre for Energy, Indian Institute of Technology Guwahati, Assam-781039, India

² Department of Chemical Engineering, Indian Institute of Technology Guwahati, Assam-781039, India

*Corresponding author (E-mail:aloke@iitg.ernet.in)

India is one of the fastest growing developing nations in the world and consequently the quest for energy reserves is very high. Mineral oil is one of the chief energy resources being utilized in India. A significant portion to the countries oil demand is met from foreign imports and thus crude oil prices has a major say in the economy of the country. India has been steadily improving its crude oil production and thus drilling, storage, transport activities etc. are on a high. Crude oil contamination of land and water bodies in and around drill sites is an emerging problem in different region of the country and Assam being the pioneering state in crude oil drilling activities in India is also facing similar problems. Different crude oil components on contact with soil, alters the soil intrinsic characteristics and renders the soil unfit for plant growth. Beside the microbial flora associated with the soil undergo a drastic change due to crude oil contamination. Besides this different chemical compounds are being increasingly used to aid different activities during drilling to increase the oil yield, many of these chemical compounds are also very much toxic to the soil. Different research groups around the world have been actively participating in research related to remediation of such crude oil contaminated sites, biological remediation using hydrocarbon degrading bacterial specie are being reported by researchers across the globe. Biological remediation of crude oil contaminated sites is very effective because it ensures complete degradation of the toxic compound as well as it is very cost effective. In the present study the main focus was laid towards isolation of efficient bacterial sp. from different crude oil contaminated sites of Assam and accordingly four sites Guwahati Refinery, Guwahati, Digboi Refinery and drill sites, Digboi, Duliajan and Jorajan were selected. 23 bacterial species belonging to different genera were isolated from fifteen soil/ sediment/ sludge and crude oil samples collected from the four contaminated sites. Isolation was done in Minimal salt medium (MSM) by enrichment culture technique. The Physicochemical and hydrocarbon contaminant characterization of all the fifteen samples collected from the four sites were also done. The pH of the samples ranged from 4.96 to 10.3, conductivity of the samples was between 0.6 to 28.6µsimencm^-1. Highest TPH (Total Petroleum Hydrocarbons) was found (494.6g/kg) and lowest was (5.26g/kg). Highest aliphatic compounds was (121011.3mg/kg, 12.1 %) and lowest recorded was (5.26mg/kg). Highest moisture content of the samples was (52.66%) and lowest moisture content was found to be (4.52%). Heavy metals- Arsenic, mercury and chromium were present in trace quantities while. Sodium, potassium, iron (Fe III), chloride, sulfate, nitrate and phosphates were also found to be present in the samples. Among the 23 bacterial isolates screened for degradation ability 7 isolates were found to be gram's negative while 16 isolates were found to be Gram's positive. Few biochemical characters of the isolates were also being done following standard protocols. In gelatin hydrolysis test only 4 isolates showed positive results while in catalase test only one of the isolates showed negative results. In starch hydrolysis test only 5 isolates showed positive results while the rest all of the strains were found to be negative. Emulsification activity is an important criteria for selection of hydrocarbon degrading bacteria, in the E₂₄ assay for assessing the emulsification assay of the bacterial isolates 10 strains showed positive result while the others showed no or insignificant activity. All the strain showed growth in MSM media amended with 100 ppm phenol as the sole source of carbon. One of the strain designated as DJ2 was able to grow at a phenol concentration of 2000 ppm. 16s rRNA sequencing of the bacterial isolates were also completed for accurate identification of the bacterial species and the sequence obtained were deposited in NCBI Genbank   and the accession number KC992288 to KC992310 were generated for the 23 strains. In vitro hydrocarbon degradation ability of the isolates with BTEX (Benzene, Tolune, Ethylbenzene and Xylene) is being continued now. Our preliminary findings suggests that some of the bacterial isolates have good potential of biodegradation of different crude oil fractions and these strains might be useful in developing an efficient consortia for actual field scale bioremediation of crude oil contaminated sites of Assam.

Keywords: Crude oil contamination, BTEX, Bioremediation, TPH