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Prakash and Irfan. J Bioremed Biodegrad 2011, 2:5

egradation
DOI: 10.4172/2155-6199.1000129
Bioremediation & Biodegradation
Jo

ISSN: 2155-6199

Research Article Open

OpenAccess
Access

Pseudomonas aeruginosa is Present in Crude Oil Contaminated Sites of

Barmer Region (India)
Bharti Prakash and Mohammad Irfan*
Department of Zoology, Government College, Ajmer (Rajasthan) India

Abstract
Purpose: Refinery effluent and oil spills are the major sources of oil pollution. Diverse microbial population
including Bacteria, Fungi and Algae can metabolize the hydrocarbons found in crude oil. Among the microorganisms
bacteria are usually the choice. The aims of present study are
• Isolation of Pseudomonas aeruginosa from crude oil contaminated sites of Barmer Region (India)
• Identification of Pseudomonas aeruginosa on the basis of its specific characteristics
Methods: Oil contaminated soil samples were collected randomly from five different sites of Mangala oil field
in Barmer district (India), where huge amount of crude oil has been discovered by Cairns India Energy Company.
Isolation was carried out by serial dilution agar platting method at 37˚C using Bushnell-Haas agar medium+crude oil
as selective medium.
• Results: The Pseudomonas aeruginosa colonies were identified by a combination of information from
primary and secondary identification. Morphological, physiological and biochemical characteristics of pure
isolates revealed that Gram-negative rods isolate were Pseudomonas aeruginosa.
• Conclusions: Previous observations have identified the Pseudomonas genus most efficient among
hydrocarbon degrading microorganisms. Further, the use of surfactants specially, rhamnolipids has been
found to enhance degradation of crude oil. Pseudomonas aeruginosa is a typical strain for rhamnolipid
production and can utilize crude oil as the sole carbon source. Data from this study showed the presence of
Pseudomonas aeruginosa in the oil contaminated soil and lend weight to this suggestion that Pseudomonas
aeruginosa exhibit oil degrading capabilities.

Keywords: Barmer district (India); Crude oil contaminated soil; - Bacteria can be genetically manipulated to improve their
Pseudomonas aeruginosa bioremediation capabilities.

Introduction There are at least 22 genera of bacteria that can metabolize

Petroleum hydrocarbon continues to be used as the principle source
of energy and hence resulted in an important global environmental
pollutant. Refinery effluent and oil spills are the major sources of oil
pollution. In India 20 refineries and 10 different operating companies
are discharging effluents at an alarming rate. These sources play a
major role in polluting the environment and the abutting landscape.
This severely affects ecosystem. With the focus on the protection of
environment and pollution control in India, most of the refineries
with conventional acid clay process are likely to face serious threat in
coming years and need has been felt for a safe, environmental friendly
process which eliminates the use of acid and at the same time suitable
for smaller capacities since collection of large quantities of used oil is a
limitation in our country.
At present, bioremediation (use of microorganisms to remove
pollutants) is often the most suitable method for remediation of
especially petroleum hydrocarbons, because it is cost effective and it
converts the petroleum hydrocarbons into the harmless by-products
such as carbon dioxide and water.
Diverse microbial population including Bacteria, Fungi and Algae
can metabolize the hydrocarbons found in crude oil. Among the
microorganisms bacteria are usually the choice because:
- They have more rapid metabolic rates.
- Numerous metabolic pathways of various organic pollutants
have been determined in bacteria.
petroleum hydrocarbons which include- Pseudomonas, Aeromonas,
Bacillus, Flavobacterium, Corynebacterium, Micrococcus etc. Based on
crude oil degradation capacity Pseudomonas aeruginosa is the most
active hydrocarbon utilizer in crude oil. Previous observations have
identified the Pseudomonas genus most efficient among hydrocarbon
degrading microorganisms [1-3].
Further the use of surfactants has been found to enhance
degradation of crude oil [4,5]. Among various surfactants, rhamnolipids
are considered to be the most in degrading hydrocarbons [6].
Pseudomonas aeruginosa is a typical strain for rhamnolipid
production and can utilize crude oil as the sole carbon source.
Pseudomonas aeruginosa also able to tolerate and grow in high
concentrations (up to 50% v/v) of crude oil and able to utilize
*Corresponding author: Mohammad Irfan, Department of Zoology, Government
College, Ajmer (Rajasthan), India, Tel: +91-98290 69078; E-mail: irfan11_2000@
yahoo.com
Received June 26, 2011; Accepted November 03, 2011; Published November
06, 2011
Citation: Prakash B, Irfan M (2011) Pseudomonas aeruginosa is Present in Crude
Oil Contaminated Sites of Barmer Region (India). J Bioremed Biodegrad 2:129.
doi:10.4172/2155-6199.1000129
Copyright: © 2011 Prakash B, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

J Bioremed Biodegrad
ISSN: 2155-6199 JBRBD, an open access journal Volume 2 • Issue 5 • 1000129
Citation: Prakash B, Irfan M (2011) Pseudomonas aeruginosa is Present in Crude Oil Contaminated Sites of Barmer Region (India). J Bioremed
Biodegrad 2:129. doi:10.4172/2155-6199.1000129
compounds such as aliphatic and monoaromatic hydrocarbons as well
as alcohols as substrates.
Materials and Methods
Sampling
In the present study, oil contaminated soil samples were collected
randomly from five different sites of Mangala oil field in Barmer
district, Rajasthan where huge amount of crude oil has been discovered
by Cairns India Energy Company.
These samples were collected in an autoclaved vial by using simple
sampling technique. Care was taken in handling and sampling to
avoid contamination of the samples and returned to the laboratory for
bacterial extraction as soon as possible. If necessary, the sample vials
were held under refrigeration at 4˚C until extraction, which was no
later than 48 hours after sampling.
Isolation of bacteria
Crude oil was sterilized by autoclaving at 121˚C for 15 minutes in
a sealed glass test tube. The crude oil acted as a source of carbon for the
oil degrading bacteria.
Bushnell-Haas medium was used for the isolation of hydrocarbon
degrading bacteria. Bushnell-Haas medium was prepared by adding 1g
KH2PO4, 1g K2HPO4, 1g NH4NO3, 0.3g Cholesterol, 0.2g MgSO4.7H2O,
0.05g FeCl3, 0.02g CaCl2.2H2O and finally 15g/L of agar were added;
1000ml of water was added to the flask containing Bushnell-Haas
medium. Since the media had a total volume of more than 1000ml
hence 700ml of this mixture was transferred to a separate 1 liter flask to
avoid spill over during autoclaving. The Bushnell-Haas medium was
then autoclaved at 121˚C for 15 minutes.
The sub samples of 1g soil were accurately weighed before being
transferred aseptically to a bottle containing 99ml of sterile dilute
saline solution to attain a dilution of 10-2. The mixture was then shaken
vigorously and was allowed for the soil to settle at the bottom of the
bottle and 0.1ml of the water was transferred to a test tube containing
9ml of sterile dilute saline solution to attain a dilution of 10-3. A
serial dilution technique subsequently yielded 4 additional test tubes
with dilutions of 10-4, 10-5, 10-6. Using a pipette, 1ml of the solution
from each of the test tubes was inoculated in Bushnell-Haas medium
containing Petri dishes by platting and spreading method. 0.5ml of the
crude oil was then transferred to the Petri dishes. This process selects
for organisms that exhibit hydrocarbon degrading capabilities. The
Petri dishes were incubated at 37˚C for 24-48 hours.
Identification of bacteria
The Pseudomonas aeruginosa colonies were identified by a
combination of information from primary and secondary identification.
Morphological, physiological and biochemical characteristics of
pure isolates were examined according to the Bergey’s Manual of
Determinative Bacteriology.
Primary identification was done on the basis of colony and
cell morphology and Gram staining. Representative colonies of
Pseudomonas aeruginosa appeared on plates were checked for purity
through the microscopy and pure isolates were streaked on slants of
Bushnell-Haas medium on which they developed during isolation and
stored at 4˚C for further investigation.
J Bioremed Biodegrad
ISSN: 2155-6199 JBRBD, an open access journal
Page 2 of 2
After pure cultures had been obtained, four biochemical tests:
Casein, Citrate, Urease and MacConkey were performed for secondary
identification. Tests were conducted as described by Cappuccino and
Sherman [7]. After identification the isolates were deposited in the
culture collection for long-term preservation.
Result
Pseudomonas aeruginosa was successfully isolated from crude oil
contaminated soil samples using serial dilution agar plating method.
This culturing technique based upon the principle that when material-
containing microorganism is cultured each viable microorganism will
develop into a colony [8].
It is known from literature that Pseudomonas aeruginosa colonies
are large, opaque, shiny colonies with serrated edges. On the basis of
Gram staining and microscopic appearance these colonies had a cell
type characteristic of gram-negative rods.
All four of the biochemical test results supported a positive
Pseudomonas aeruginosa identification after 48 hours incubation.
The casein test appeared to show clearing through the medium,
characteristic of a positive casein test. The citrate medium had a color
change from green to dark blue, indicative of a positive result for citrate
test. The urease agar had a color change from yellow to a bright pink,
a positive characteristic for the presence of the enzyme urease. The
MacConkey test showed growth, but the colonies were colorless, not
red, characteristic of no lactose fermentation, or a negative test result.
Discussion
Data from this study showed the presence of Pseudomonas
aeruginosa in the oil contaminated soil and lend weight to this suggestion
that Pseudomonas aeruginosa exhibit oil degrading capabilities which
can be used for the bioremediation of crude oil polluted sites.
Acknowledgment
The authors are grateful to Department of Zoology, Government College,
Ajmer (India) for all facilities provided during the course of the study.
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