International Journal of Emerging trends in Engineering and Development ISSN 2249-6149

Available online on http://www.rspublication.com/ijeted/ijeted_index.htm Issue 2, Vol.5 (July 2012)

ISOLATION AND IDENTIFICATION OF

HYDROCARBON DEGRADING MICRO-ORGANISMS
FROM CRUDE OIL CONTAMINATED SOILS
Ibrahim Abdulhaqq Ameen#1, Baba Yahaya Danjuma#2 and Olabode Yusuf Raji #3
Chemical Engineering Programme, School of Engineering and Engineering Technology,
Abubakar Tafawa Balewa University Bauchi-Nigeria,
#1 +2348038181200,
#2 +2347032666006,
#3 +2348036380099,

ABSTRACT

This research work is aimed at Isolating and Identifying Hydrocarbon degrading bacteria
from Crude oil contaminated soils. A soil sample was contaminated with two different crude oils
(Escravos and Urals) so as to isolate and identify the bacteria capable to acclimatize and degrade
the crude oil contaminants present. The bacteria strains in a serial diluted solution were
inoculated on nutrient agar plate incubated at 370C. The isolates were characterized for Gram
reaction, cell morphology and various biochemical/enzymatic analysis such as Triple sugar iron,
Citrate, Oxidase, Indole, and Urease tests. The result revealed the presence of Pseudomonas
aeruginosa and Proteus vulgaris which are highly adapted bacteria with great potential to
biodegrade total petroleum hydrocarbons (TPH) from crude oil contaminated soil.

Key words: Isolation and Identification, Inoculation, Hydrocarbon degrading bacteria,

contaminated soil
Correspondence Author: Ibrahim A.A.

INTRODUCTION
Pollution is the introduction of contaminants into a natural environment that causes
instability, disorder, harm or discomfort to the ecosystem i.e. physical systems or living
organisms [6]. Pollution can take the form of chemical substances or energy, such as noise, heat,
or light. Pollutants, the elements of pollution, can be foreign substances or energies, or naturally
occurring; when naturally occurring, they are considered contaminants when they exceed natural
levels. The major sources of land pollution by oil include oil spillage during oil exploration and
exploitation, accidental breakage of pipelines, oil tanker collision and discharge of oily water
from industries. Nigeria being an oil producing country is seriously facing the problem of land
pollution through oil spillage.

Bioremediation is a modern method in which the natural ability of micro-organisms is

employed for the reduction of the concentration and/or toxicity of various chemical substances,
such as petroleum derivatives, aliphatic and aromatic hydrocarbons, industrial solvents,
pesticides and metals [5]. Some microorganisms can decompose or transform the chemical
substances present in petroleum and petroleum derivatives. Hydrocarbons from crude oil
represent substrates for microorganisms, hence, when an accidental oil spill occurs, the number
of hydrocarbon degrading microorganisms in the ecosystem increases [2]. A single

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International Journal of Emerging trends in Engineering and Development ISSN 2249-6149
Available online on http://www.rspublication.com/ijeted/ijeted_index.htm Issue 2, Vol.5 (July 2012)

microorganism can degrade only certain types of petroleum compounds, but a mixed population
– microbial community enables a higher level of degradation. Petroleum hydrocarbons can be
degraded by microorganisms such as bacteria, fungi, yeast and microalgae [9]. However, bacteria
play the central role in hydrocarbon degradation. The driving force for petroleum biodegradation
is the ability of microorganisms to utilize hydrocarbons to satisfy their cell growth and energy
needs.

MATERIALS AND METHODS

Materials
In this study the following materials were used:
(i) Crude oil (Escravos and Urals)
(ii) Soil
(iii) Distilled water
The equipment used in this study were:
(i) Digital weighing balance**
(ii) Petri dishes*
(iii) Spatula*
(iv) Measuring cylinder* (1000 ml)
(v) Cotton wool
(vi) Beaker* (250 ml)
(vii) Test tube*
(viii) Wire loop**
(ix) Straight wire**
**M/s. Contech Instruments Limited (http://www.contechbalance.com/)
*Pyrex laboratory glassware Limited (http://www.pyrexware.com/)

Methods
Soil sample from surface soil (0-15 cm depth) was randomly collected from Abubakar
Tafawa Balewa University, Yelwa Campus, Bauchi Nigeria. The soil sample was sieved using 2
mm mesh to remove debris and large particles. 19 kg of the soil sample was weighed into two
different containers labeled samples A and B and then contaminated with 1.045 kg of Escravos
and Urals crude oil respectively. The contaminated soil samples were left for three weeks to
allow the acclimatization of microorganisms.

Serial Dilution and Inoculums Preparation

Ten sterile test tubes were arranged in a rack, 1 ml of sterile peptone water was added to the
first test tube and 4.5 ml to each of the remaining test tubes. Approximately 1 g of the
contaminated soil sample was added to the first test tube and homogenized; 0.5 ml of the mixture
was transferred from the first test tube to the second test tube and mixed. 0.5 ml was transferred
again from the second test tube to the third test tube and mixed. This was done continuously until
the tenth test tube where 0.5 ml was taken from it and then discarded (Serial dilution).

Each of the dilution was sub-cultured unto nutrient agar plates and incubated overnight, at
37 C and the dilutions were also incubated overnight at 37 oC for motility test. The
o

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microorganisms were identified using the standard Bacteriological procedures after the
incubation time-out, as described by [3].

Fig 1: Serial dilution setup

A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor
at each step is constant, resulting in a geometric progression of the concentration in a logarithmic
fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M, 0.0001 M, 0.00001 M,
0.000001 M, 0.0000001 M, 0.00000001 M, 0.000000001 M, and 0.0000000001 M.

Fig 2: Dilutions Sub-cultured onto Nutrient agar plates.

Serial dilutions are used to accurately create highly diluted solutions as well as solutions for
experiments resulting in concentration curves with a logarithmic scale [1]. Each of the dilution
was sub-cultured unto nutrient agar plates as presented in figure 11 below, where Control, A, B,
C…I correspond to the dilution factors of sub cultured solutions. The dilution factors are 10 0, 10-
1
, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, and 10-9.

The two contaminated soil samples were subjected to series of tests according to the standard
procedures [3]. Bacteria isolates were characterized on the basis of their morphology and
biochemical reactions as shown in Table 1, 2 & 3 below. These properties were compared with
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International Journal of Emerging trends in Engineering and Development ISSN 2249-6149
Available online on http://www.rspublication.com/ijeted/ijeted_index.htm Issue 2, Vol.5 (July 2012)

the standards presented in the Difco manual (Differentiation of Enterobacteriaceae by

Biochemical tests) for bacteria identification.

RESULTS AND DISCUSSION OF RESULTS

Table 1: Gram staining of sub-cultured dilutions
S/N Dilution Sample A Shape Colour Sample B Shape Colour
(Escravos) (Urals)
1 Control GNR1* Cylindrica Pink GNR/GPR Cylindrica Pink/
l l purple
2 10-1 GNR/GPR Cylindrica Pink/ purple GNR/GPR Cylindrica Pink/
l l purple
-2 2 5
3 10 GNR * Cylindrica Pink GNR * Cylindrica Pink
l l
4 10-3 GNR3* Cylindrica Pink GPR Cylindrica purple
l l
5 10-4 GPR Cylindrica purple GPR Cylindrica purple
l l
-5 4
6 10 GNR * Cylindrica Pink GPR Cylindrica purple
l l
7 10-6 GPR Cylindrica purple GPR Cylindrica purple
l l
8 10-7 GPR Cylindrica purple GPR Cylindrica purple
l l
-8
9 10 GPR Cylindrica purple GPR Cylindrica purple
l l
10 10-9 GPR Cylindrica purple GPR Cylindrica purple
l l
Note: Biochemical tests were only carried out on the GNR labeled *1-5
Key:
GPR = Gram Positive Rods
GNR = Gram Negative Rods

The type of the biochemical test carried out on a microorganism isolate depends on its gram
reaction;

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Available online on http://www.rspublication.com/ijeted/ijeted_index.htm Issue 2, Vol.5 (July 2012)

Biochemical test

Gram positive Gram negative

(Catalase, Coagulate test) (Sugar, Citrase, Indole, Urase test)

Fig 3: Biochemical test for gram positive/gram negative

Table 2: Biochemical test result

S/N GRAMING MOT TSI URASE CITRATE INDOLE OXIDASE ISOLATE
1. GNR + + (±)V V + + Proteus vulgaris

2. GNR + + Pseudomonas
Aeruginosa

3. GNR + + (±)V V + + Proteus vulgaris

4. GNR + + Pseudomonas
Aeruginosa

5. GNR + + Pseudomonas
Aeruginosa

Key:
MOT = Motility
TSI = Tripple sugar iron

4.2 DISCUSSION OF RESULTS

The stained slide revealed rod-like shape of bacteria and appeared pinkish when viewed
under the microscope. This indicates Gram negative rod bacteria. All the isolates appear motile
when viewed under the Microscope at 100 times objective with emulsion oil and showed positive
reaction to the Triple Sugar Iron (TSI) test. With these two biochemical tests, isolate 2, 4, and 5
were able to be identified as Pseudomonas aeruginosa. Isolates 1 and 3 were further subjected to
Urease, Citrate, Indole and Oxidase tests where they revealed positive reactions as described in
the procedures above. These indicate the presence of Proteus vulgaris.

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The results of the morphological and biochemical properties of bacteria isolates from soil
contaminated crude oil are presented in the Table 2. From the results obtained, bacteria species
of Pseudomonas aeruginosa and Proteus vulgaris were identified using the Difco biochemical
test manual chart. The identification of Pseudomonas aeruginosa and Proteus vulgaris were in
accordance with the work of Fulekar, M.H. [4] and Okoh, A.I. [7] who stress the hydrocarbon
degrading potentials of the identified bacteria. The ability to isolate and identify certain oil
microorganisms from oil-contaminated environment is commonly taken as evidence that these
microorganisms are the active degraders of the contaminant [8].

It can also be deduced from the result that Pseudomonas aeruginosa has a greater ability to
acclimatize with contaminants from Escravos and Urals since it was isolated and identified from
the two samples of contaminated soils where Proteus vulgaris was only identified in the
Escravos crude oil contaminated soil. This indicated its universality as attested by most literature
[8].

CONCLUSION AND RECOMMENDATION

Conclusion
From the result obtained in this study, the following conclusions can be drawn:
 Two bacteria species were identified from Escravos crude oil contaminated soil namely;
Pseudomonas aeruginosa and Proteus vulgaris.
 One bacteria specie was identified from Urals crude oil contaminated soil namely;
Proteus vulgaris.

Recommendation
The study showed that Pseudomonas aeruginosa and Proteus vulgaris are highly adapted
bacteria with great potential to biodegrade hydrocarbons from crude oil contaminated soil,
therefore, it is recommended that further studies should be carried out on them in order to
maximize the potential and utilize the ability as regards bioremediation of crude oil contaminated
soil.

REFERENCES

[1] Aneja K. R. (2005): “Experiments in Microbiology, Plant Pathology and Biotechnology”.

New Age Publishers, p. 69. ISBN 812241494X

[2] Atlas, R.M., (1995): Petroleum Biodegradation and Oil Spill Bioremediation. Marine
Pollution Bulletin 31, 178-182.

[3] Cheesborugh M., (2000): “District Laboratory Practice in Tropical Countries”; Cambridge
University press UK; p.70.

[4] Fulekar, M.H. (2005). “Bioremediation of Fenvalerate by Pseudomonas aeruginosa in a

Scale up Bioreactor” Biosciences Biotechnology Research Asia. Bucharest University,
Romania. Vol. 14, No. 6, 2009, pp. 4900-4905

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[5] Korda A., Santas P., Tenente A., Santas R., (1997): „Appl. Microbial. Biotechnol’, 48:
677.New York.

[6] " Merriam-Webster Online Dictionary – Definition of Pollution ". Merriam-webster.com.

2010-08-13. http://www.merriam-webster.com/dictionary/pollution. Retrieved 2010-08-26.

[7] Okoh, A.I., (2003). “Biodegradation of bonny light crude oil in soil microcosm by some
bacterial strains isolated from crude oil flow stations saver pits in Nigeria”. Afr. J.
Biotechnol., Mc millan publishers. vol. 2: Pp 104-108.
http://www.academicjournals.org/AJB/abstracts/abstracts2003/Mayabstracts2003/Okoh.htm

[8] Olajide P. O. and Ogbeifun L.B. (2010): “Hydrocarbon biodegrading potentials of a Proteus
vulgaris strain isolated from fish samples” Gale, Cengage Learning Science Publications; Pp
80.

[9] Riser-Roberts E., (1992): „Bioremediation of Petroleum Contaminated Sites‟. Boca Raton
(Florida): CRC Press Inc; Pp78-79.

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