(427i) 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