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OCCURRENCE OF CRUDE OIL DEGRADING BACTERIA IN GASOLINE
AND DIESEL STATION SOILS
Pattanathu K.S.M. Rahman
*1
, Thahira-Rahman
1
, Perumal Lakshmanaperumalsamy
2
,
and Ibrahim M. Banat
1
1. Biotechnology Group, School of Biological and Environmental Sciences,
University of Ulster, Coleraine, County Londonderry, Northern Ireland - BT52 1SA.
2. Department of Environmental Sciences, Bharathiar University,
Coimbatore – 641 046, Tamilnadu, India.
* Corresponding Author Present address:
Dr Pattanathu K.S.M. Rahman
Chemical and Bioprocess Engineering Group
School of Science and Engineering
Teesside University, Middlesbrough - TS1 3BA
Teesvalley, United Kingdom.
Tel: +44-1642-384669
Email: p.rahman@tees.ac.uk
Running title: Biodegradation of petroleum hydrocarbons
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SUMMARY
Microbial enumeration and identification were carried out on several oil contaminated
soil samples collected from gasoline and diesel stations. Bacteria were the most dominant
microbiota and were therefore classified to generic level. Eleven main genera were
detected and Corynebacterium was the predominant genus in all the samples.
Biochemical characterisation and substrate utilisation showed high percentage of lipolytic
ability combined with high inorganic nitrogen utilisers. The ability of these cultures to
degrade crude oil was tested individually and in mixed bacterial consortium at different
temperatures and pH values. Maximum crude oil biodegradation of 78% was achieved
using a bacterial consortium containing five cultures (Micrococcus sp. GS2-22,
Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and
Pseudomonas sp. DS10-129) with 1% crude oil at 30
o
C and pH 7.5. Such a consortium
may be useful for bioaugmentation of oil contaminated environments.
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INTRODUCTION
Petroleum continues to serve as the principle source of energy. Wide scale production,
transport, use and disposal of petroleum globally has made it a lead contaminant in both
prevalence and quantity in the environment. As a result of the increase in automobiles, the
number of gasoline/diesel stations and automobile service stations is ever increasing. In
gasoline, diesel stations and service stations, oil is spilled during transfer and servicing
operations. During accidental spills, action will be taken to remove or remediate or recover
the contaminant immediately, whereas in the gasoline and diesel stations the spills due to
leakage may be small but continuous and prolonged. Because of its persistence, the chance
for groundwater contamination is greater.
The addition of hydrocarbons to an ecosystem may result in a selective increase in
microorganisms capable of utilising the hydrocarbons and those that are capable of
utilising metabolites produced by the hydrocarbon-utilisers (Venkateswaran et al. 1995;
Ferrari et al. 1996). The enhancement or reduction will depend upon the chemical
composition of the contaminating hydrocarbons and on the species of microorganisms
present within the microbial community of the particular ecosystem (Atlas 1995).
The Gulf War and an increasing trend in tanker accidents have focussed attention on the
problem of hydrocarbon contamination in the environment. The traditional methods to cope
with oil spills are mainly through physical containment and collection using floating
booms, applications of adsorbents such as straw, shredded polyurethane or porous volcanic
glasses and/or dispersal by detergents (Ollis 1992). Biological methods have an edge over
the physico-chemical treatment regimes in removing spills as they offer in situ bio-
degradation of oil fractions by the microorganisms (Ferrari et al.1996)
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The fate of petroleum hydrocarbons in the environment, including biodegradation, is
largely determined by abiotic factors, which influence the extent of weathering. This paper
describes the microbial population and microbial diversity of samples from gasoline and
diesel stations and explores the possibility of using selected bacterial cultures and bacterial
consortium to degrade Bombay high crude oil at various environmental conditions.
MATERIALS AND METHODS
Screening of samples
The soil samples (100g each) were collected from ten Gasoline spilled (GS) and diesel
spilled (DS) stations for the isolation of oil degrading microorganisms. The samples were
collected in pre-sterilised glass bottles and transported to the laboratory for analysis.
Enumeration and isolation of heterotrophic bacteria, actinomycetes and fungi was carried
out through serial dilution-agar plating technique using Nutrient agar, Kuster’s agar and
Rose Bengal agar medium (HIMEDIA) respectively.
Characterisation of bacteria
The isolates were grouped to various genera as per Bergey's Manual of Determinative
Bacteriology (Holt et al., 1994).
These cultures were characterised depending on their
morphology, gram staining, spore staining, motility, oxidase, catalase, oxidation
fermentation, gas production, ammonia formation, nitrate and nitrite reduction, indole
production test, methyl-red and Voges-Proskauer test, citrate and mannitol utilisation test,
hydrolysis of casein, gelatin, starch, urea and lipid (Aaranson 1970).
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Growth of bacteria on crude oil
The bacterial cultures isolated from oil spilled environment were inoculated in Mineral
salts medium (Kennedy et al., 1975) with 1% Bombay High crude oil as carbon source. It
was kept in the shaker at 200 rpm at 30°C for a period of seven days. The growth was
recorded and categorized spectrophotometrically as low growth with optical density (OD)
in the range 0.21-0.4, moderate growth (OD 0.41-0.6), high growth (OD 0.61-0.8) and
excellent growth (OD 0.81-1.0) all measured at 620nm.
Preparation of inoculum
The bacterial isolates that exhibited excellent growth on crude oil, Micrococcus sp. GS2-
22, Bacillus sp. DS6-86, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73 and
Pseudomonas sp. DS10-129, were selected. A loopful of culture was inoculated into 100ml
sterile nutrient broth. The flasks were incubated and shaken at 200 rpm, for 12h at 30°C.
One ml volumes of the culture broth from each of the above five isolates were mixed to
prepare mixed bacterial consortium.
Degradation of crude oil
Individual bacterial cultures and bacterial consortium (1.0 %) were transferred to 250 ml
conical flasks, each containing 100 ml of sterile defined mineral salts medium with 1%
Bombay High (BH) crude oil. An uninoculated control was also studied concurrently. The
flasks were incubated at 30°C and shaken at 200 rpm for 20 days. At 2-day intervals, a set of
flasks was used for the enumeration of microbial population and estimation of crude oil content
