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Biodegradation of Crude Oil by Constructed Bacterial

Consortia and the Constituent Single Bacteria Isolated
From Malaysia
a a a a
Ainon Hamzah , Chia-Wei Phan , Nur Faizah Abu Bakar & Kok-Kee Wong
a
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, Selangor Darul Ehsan, Malaysia
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To cite this article: Ainon Hamzah , Chia-Wei Phan , Nur Faizah Abu Bakar & Kok-Kee Wong (2013): Biodegradation of Crude
Oil by Constructed Bacterial Consortia and the Constituent Single Bacteria Isolated From Malaysia, Bioremediation Journal,
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 Bioremediation Journal, 17(1):1–10, 2013
Copyright 
c 2013 Taylor and Francis Group, LLC
ISSN: 1088-9868 print / 1547-6529 online
DOI: 10.1080/10889868.2012.731447

Biodegradation of Crude Oil

by Constructed Bacterial Consortia
and the Constituent Single Bacteria
Isolated From Malaysia
Downloaded by [University of Malaya] at 03:27 03 March 2013

Ainon Hamzah, Chia-Wei
Phan, Nur Faizah Abu Bakar,
and Kok-Kee Wong
School of Biosciences and
Biotechnology, Faculty of
Science and Technology,
Universiti Kebangsaan
Malaysia, Selangor Darul
Ehsan, Malaysia
ABSTRACT Three bacterial isolates identified as Pseudomonas aeruginosa
(UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8
(UKMP-7T) based on biochemical, physiological, and morphological char-
acteristics and on 16S rDNA sequences were isolated from groundwater of a
crude oil refinery plant. From these three isolates, four bacterial consortia were
designed by mixing the single bacterial cultures in the following ratios: (P.
aeruginosa:Rhodococcus sp. M15-2, 1:1), (P. aeruginosa:Rhodococcus sp. ZH8, 1:1),
(Rhodococcus sp. M15-2: Rhodococcus sp. ZH8, 1:1), and (P. aeruginosa: Rhodococ-
cus sp. ZH8:Rhodococcus sp. M15-2, 1:1:1), respectively. Bacterial isolates and
consortia showed differing preferences for nitrogen source (0.01% ammonium
chloride, 0.10% yeast extract, or 0.50% peptone) to reach optimum growth.
When fortified with the preferred nitrogen sources and grown in minimal salt
medium, within 7 days all three single isolates and the four bacterial consortia
biodegraded 97.6-99.9% of Tapis Massa oil without any significant differences.

KEYWORDS bacterial consortia, biodegradation, crude oil, nitrogen sources, Pseu-

domonas aeruginosa, Rhodococcus sp.

Address correspondence to Ainon
Hamzah, School of Biosciences and
Biotechnology, Faculty of Science and
Technology, Universiti Kebangsaan
Malaysia, 43000 Bangi, Selangor Darul
Ehsan, Malaysia. E-mail:
antara@ukm.my
INTRODUCTION
Petroleum hydrocarbon continues to be a major contaminant in soil, ground-
water, and the marine environment as a result of accidental spills and extended
leakage at the oil service stations. The conventional methods for coping with
hydrocarbon contamination that are limited to physicochemical approaches
such as booms, skimmers, dispersant, and in situ incineration are expensive,
and their by-products may cause secondary contamination of soil and water re-
sulting in the need for additional posttreatment. Biodegradation using microbes
has been widely studied because it leads to complete degradation of complex
hydrocarbons to carbon dioxide and water (Liang et al. 2009).
Most of the microbes comprising various strains isolated from petroleum-
contaminated soil or water have been shown to have oil degradation

1
 capabilities. Marquez-Rocha and his coworkers (2005)
have isolated genes encoding alkane hydroxylase and
naphthalene dioxygenase, which are involved in degra-
dation of alkanes and aromatics, respectively, from
Pseudomonas sp. and Bacillus sp. in oil-contaminated
soil. More recently, we have isolated Trichoderma virens
UKMP-1M sampled from an oil refinery oxidation
pond in Melaka, Malaysia, and it was shown capable
of biodegrading heavy Khefji Sour crude oil (Hamzah
et al. 2012). Besides that, two local bacterial isolates,
namely, Pseudomonas aeruginosa UKMP-14T and Bacil-
lus cereus UKMP-6G, were also shown to produce the en-
zyme toluene monooxygenase in biodegrading toluene,
one of the hydrocarbon components in crude oil
(Hamzah, Tavakoli, and Rabu 2011).
A mixed bacterial consortium comprising Micrococ-
cus sp., Corynebacterium sp., Flavobacterium sp., Bacil-
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lus sp., and Pseuudomonas sp. has been shown to de-
grade up to 78% of crude oil compared with using
the respective single isolates, whose degradation rate
ranged from 41% to 66% (Rahman et al. 2002). It is
further evidence of the advantage of using a mixture
of bacteria that after exhaustive growth of one strain
on crude oil, a second and a third strain of bacteria
were able to grow in the residual oil. Recent studies,
then, have focused on using mixed bacteria to enhance
oil degradation. The advantage of bacterial consortia
is that they can metabolize a wider range of hydro-
carbons due to the broader enzymatic range offered by
the various strains within the consortium (Ghazali et al.
2004). Hence, researchers have reported the construc-
tion of artificial bacterial consortia by mixing two or
more bacteria to elucidate the synergistic mechanism
in degradation of oil (Komukai-Nakamura et al. 1996;
Ghazali et al. 2004). However, the degradation capacity
of a constructed microbial consortium is not necessarily
simply the result of totaling the capacities of individual
strains. The idea of utilizing bacterial consortia in biore-
mediation has also been recently applied to tackle other
environmental pollutants other than crude oil, for in-
stance, the removal of 2,4,6-trinitrotoluene (TNT) by a
bacterial consortium of seven different genera (Muter
et al. 2012), and rhizoremediation, whereby chlorpyri-
fos residues from soil was significantly reduced by a
defined rhizosphere bacterial consortium (Dubey and
Fulekar 2012).
The objective of this study was to evaluate the
oil degradation capacity of consortia of bacteria
that potentially may degrade crude oil, especially in
A. Hamzah et al.
tropical conditions. The isolated strains P. aeruginosa
(UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and
Rhodococcus sp. ZH8 (UKMP-7T) were first isolated
from hydrocarbon-contaminated groundwater and
then developed into bacterial consortia. The ability
of the consortia to degrade crude oil was evaluated
against the respective single isolates. Consortia whose
mixed bacterial population offered broad enzymatic
capacities were anticipated to enhance the rate of and
capacity for petroleum biodegradation.
MATERIALS AND METHODS
Isolation and Identification
of Bacterial Cultures
Groundwater samples (pH 6.63 ± 0.12; 30.8◦ C ±
1.4◦ C) were collected from the Petronas crude oil refin-
ery in Terengganu, Malaysia. Enrichment and isolation
of oil-degrading cultures were performed in mineral salt
medium (MSM) supplemented with crude oil as sub-
strate. Each single isolate was obtained by serial dilution
of the culture enriched in MSM and plated on nutrient
agar (Oxoid, Hampshire, UK) and incubated at 40◦ C
to screen for isolates that can adapt well to tropical
condition where the maximum temperature reaches no
higher than 40◦ C. The bacterial isolates were identified
using commercial kit Microbact 24E (Oxoid) according
to the manufactuer’s instruction.
The isolates were further confirmed using poly-
merase chain reaction (PCR) assay targeting 16S rDNA
gene. The bacterial DNA was extracted using QIAamp
DNA Mini Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s instruction. The PCR reactions
were performed in a Biometra T-Gradient thermocycler
(Gottingen, Germany). Amplification was performed
in a 50-µl reaction mixture using 16S rDNAs (universal
F: 5 -AGAGTTTGATCCTGGCTCAG-3 , universal
R: 3 -GGTTACCTTGTTACGACTT-5 ) and 1 U Taq
polymerase (Promega Master Mix; Wisconsin, USA).
The PCR product was purified using a QIAquick
PCR Purification Kit (Qiagen) and both 5 and 3
ends of 16S rDNA clones were sequenced with an
ABI PRISM 377 sequencer and compared against
the National Center for Biotechnology Information
(NCBI) nonredundant protein database using the Basic
Local Alignment Search Tool (BLAST) (Altschul et al.,
1997). Multiple sequence alignment was performed
using ClustalW (Thompson, Higgins, and Gibson
1994) on the European Bioinformatics Institute (EBI)
2
 server (www.ebi.ac.uk/clustalw/index.html) to identify
the isolate species. Phylogenetic inferences were
made with SEQBOOT (for bootstrap analysis) and
NEIGHBOR (for neighbour joining analysis) programs
from MEGA4 version 4.0.2 (Tamura et al. 2007).
Construction of Bacterial Consortia
Bacterial consortia were constructed by using three
single isolates, P. aeruginosa (UKMP-8T), Rhodococcus sp.
M15-2 (UKMP-5T), and Rhodococcus sp. ZH8 (UKMP-
7T), of which the growth parameters (pH 7, 40◦ C, 1.0%
[v/v] crude oil, 150 rpm) had been optimized as re-
ported in Hamzah and Abu Bakar (2007). The nitrogen
sources for optimal growth of P. aeruginosa (UKMP-
8T), Rhodococcus sp. M15-2 (UKMP-5T), and Rhodococ-
cus sp. ZH8 (UKMP-7T) were 0.01% ammonium chlo-
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ride (NH4 Cl), 0.10% yeast extract, and 0.50% peptone,
respectively. The standard inoculum for each single iso-
lates were prepared according to Hamzah et al. (2010)
in fresh nutrient broth and the bacterial cells were har-
vested by centrifugation and suspended in 0.85% (w/v)
sterile normal saline to give a cell concentration of 0.5 at
550 nm wavelength using spectrophotometer (UVmini-
1240; Shimadzu, Kyoto, Japan).
Four bacterial consortia were designed by mixing
equal portions of single bacterial cultures as follows:
Consortium 1 consisted of 5% P. aeruginosa (UKMP-8T)
and 5% Rhodococcus sp. M15-2 (UKMP-5T) (1:1, v/v).
Consortium 2 consisted of 5% P. aeruginosa (UKMP-
8T) and 5% Rhodococcus sp. ZH8 (UKMP-7T) (1:1, v/v).
Consortium 3 consisted of 5% Rhodococcus sp. M15-2
(UKMP-5T) and Rhodococcus sp. ZH8 (UKMP-7T) (1:1,
v/v), and Consortium 4 consisted of 3.33% P. aeruginosa
(UKMP- 8T), 3.33% Rhodococcus sp. M15-2 (UKMP-
5T), and 3.33% Rhodococcus sp. ZH8 (UKMP-7T) (1:1:1,
v/v).
Growth of the consortia was optimized by test-
ing using three different nitrogen sources, namely,
0.01% NH4 Cl, 0.10% yeast extract, and 0.50% pep-
tone, to replace the nitrogen source originally used in
the mineral salt medium (MSM). The composition of
MSM was as follows: K2 HPO4 1.8 g, KH2 PO4 1.2 g,
NH4 Cl 4.0 g, MgSO4 ·7H2 O 0.2 g, NaCl 0.1 g, and
FeSO4 ·7H2 O 0.01 g in 1 L distilled water (Zajic and
Supplisson 1972) supplemented with 0.1% trace el-
ements (MnSO4 ·7H2 O 0.1 g/L, CuCl2 0.025 g/L,
NaB4 O7 ·10H2 O 0.025 g/L, Co(NO3 )2 ·6H2 O 0.025 g/
L, ZnCl 0.025 g/L, NH4 NO3 0.01 g/L, and (NH4 )6
3 Crude Oil Biodegradation by Bacterial Consortia
Mo7 O24 ·H2 O 0.025 g/L) (Bouchez, Banchet, and Van-
dercasteele 1995).
All the consortia were grown in MSM added with
1% (v/v) Tapis Massa crude oil and incubated at op-
timum growth parameters for the isolates that were
used to construct the bacterial consortia (pH 7, tem-
perature 40◦ C, 150 rpm agitation) for 7 days (Hamzah
and Abu Bakar 2007). The nitrogen source that pro-
duced the maximum growth of each bacterial consor-
tium (colony-forming units [CFU]/ml) was chosen for
the subsequent biodegradation experiment. A control
without any nitrogen source was run in parallel.
Biodegradation of Tapis Massa
Crude Oil
A total of 10% (v/v) standard inocula for each in-
dividual single isolate and bacterial consortium were
transferred into a 250-ml Erlenmeyer flask with 50 ml
MSM and 1% Tapis Massa crude oil. The samples
were incubated in an orbital shaker (Infors Multi-
tron, Switzerland) at 150 rpm at 40◦ C for 7 days.
The negative-control flask contained MSM with 1%
Tapis Massa crude oil without bacterial inoculates. Af-
ter 7 days, the number of bacterial colonies was enumer-
ated as CFU/ml and the whole flask was extracted using
chloroform to analyze for residual oil in the culture.
Extraction and Analysis of Residual
Crude Oil
After 7 days of incubation, the residual oil in the
medium (MSM with 1% Tapis Massa crude oil +
10% inoculum) was extracted using chloroform (R&M
Chemicals, Essex, UK) at 1:1 and then filtered using
filter paper (0.45 µm; Millipore, Billerica, MA, USA).
The residual oil in the chloroform was then concen-
trated to 1.0 ml using a rotary evaporator (Eyela model
N-1000; Tokyo, Japan). Analysis of total petroleum
hydrocarbons was done using gas chromatography
with flame ionization detection (GC-FID; Clarus 500;
PerkinElmer Instruments, Bridgeport Avenue, Shelton,
USA) fitted with an HP 3390A integrator (Milton Free-
water, OR, USA), flame ionization detector (300◦ C),
and fused silica capillary column (30 cm × 0.32 mm).
The injector and detector were maintained between
60◦ C and 300◦ C and the oven temperature was pro-
grammed to rise from 60◦ C to 320◦ C at 5.0◦ C per
minute increment and held at 300◦ C for 5 min. A total
 of 1 µl of sample was injected and analyzed for 67 min
with nitrogen gas as carrier.
The percentage of total petroleum hydrocarbon
(TPH) degradation was calculated using the following
formulation:

TPH % = [TPHcontrol − TPHwith inoculation /TPHcontrol ]

× 100 (1)

Statistical Analysis
All data were analyzed statistically by one-way anal-
ysis of variance (ANOVA) with 95% confidence level
using SPSS version 13.0 (SPPS, Chicago, IL, USA). Ex-
periments were carried out independently in triplicates.
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RESULTS
Isolation and Identification
of Bacterial Cultures
The enrichment and isolation procedure resulted
in a total of 150 pure cultures growing in mineral salt
medium with 1% (v/v) crude oil as carbon source.
This suggests that these isolates can solubilize and
degrade crude oil and used the carbon source to
support growth. Of these, three isolates, namely,
UKMP-5T, UKMP-7T, and UKMP-8T, grew in
crude oil agar to reach the optical density of 108
CFU/ml within 24 h of incubation at 40◦ C. Partial
sequencing of the 16S rDNA gene showed maximal
similarity of isolate UKMP-5T with Rhodococcus
rubber, UKMP-7T with Rhodococcus sp. M15-2, and
UKMP-8T with P. aeruginosa based on the phylo-
genetic tree constructed using the following strains:
Rhodococcus ruber (DQ375763.1), Rhodococcus sp.
(AY762055.1), Rhodococcus aetherivorans (NR025208.1),
Rhodococcus gordoniae (AY233202.1), Rhodococcus
pyridinivorans (EU816696.1), Rhodococcus zopfii
(AF191343.1), Rhodococcus phenolicus (AM933579.1),
Bacillus flexus (AM992190.2), Stenotrophomonas mal-
tophilis (AJ131117.1), P. aeroginosa (GU220062.1),
Acinetobacter baumanii (AY847284.1), and Acineto-
bacter calcoaceticus (EU022688.1). Exiguobacterium sp.
(AM398225) was used as outgroup strain (Figure 1).
The isolates UKMP-5T, UKMP-7T, and UKMP-
8T were also analyzed morphologically; their bio-
chemical and physiological properties summarized in
Table 1. UKMP-5T and UKMP-7T share similar phe-
FIGURE 1 Phylogenetic trees showing relationships among the
16s rDNA sequences of isolates UKMP-5T, UKMP-7T, and UKMP-
8T and the reference organisms from genus Rhodococcus and
Pseudomonas, respectively. Bootstrap (5000 resampling) values
are indicated at the nodes.
notypic characters, whereas UKMP-8T had different
phenotypic characters in agreement with their assign-
ment to different bacterial genera. Strain UKMP-8T
being gram positive was further tested using triple
sugar iron and indole-methyl red-Voges-Proskauer-
citrate (IMVIC) tests.
Growth Optimization of Consortia
Using Various Nitrogen Sources
All four consortia were found to grow on MSM
medium using crude oil as single carbon source supple-
mented with various nitrogen sources. Figure 2 shows
the effect of 0.01% ammonium chloride, 0.50% pep-
tone, and 0.10% yeast extract on the growth of Con-
sortia 1, 2, 3, and 4.
Consortia 1, 2, and 3, when supplemented with their
respective optimal nitrogen sources, showed significant
growth compared with control (without nitrogen
source) after 7 days of incubation. Only Consortium

A. Hamzah et al. 4
 TABLE 1 Phenotypic Properties of Bacterial Isolates UKMP-5T, growth of Consortium 3 was increased by 42.2% in the
UKMP-7T, and UKMP-8T
presence of 0.50% peptone compared with control,
Isolate whereas the growth of Consortium 4 was increased by
56.3% when supplemented with 0.01% ammonium
Characteristics UKMP-5T UKMP-7T UKMP-8T
chloride compared with control (Figure 2).
Cell morphology
Gram reaction + + −
Shape Coccus Rod Rod Biodegradation of Tapis Massa
(duplet) (duplet) Crude Oil
Size (µm) 0.1 0.8 2
Motility − − + To evaluate the efficiency of single isolates and
Biochemical test consortia in the degradation of Tapis Massa crude
Citrate − − + oil, the total percentage of hydrocarbon found in
H2 S − − n.d.
the control without any inoculations was compared
Glycerol − − n.d.
L-Arabinose − − −
with samples inoculated with P. aeruginosa (UKMP-
Ribose − − n.d. 8T), Rhodococcus sp. M15-2 (UKMP-5T), Rhodococcus sp.
D-Xylose − − n.d. ZH8 (UKMP-7T), Consortium 1 [P. aeruginosa (UKMP-
Galactose − − n.d. 8T) and Rhodococcus sp. M15-2 (UKMP-5T)], Consor-
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Glucose + − + tium 2 [P. aeruginosa (UKMP-8T) and Rhodococcus sp.

Fructose + + n.d.
ZH8 (UKMP-7T)], Consortium 3 [Rhodococcus sp. M15-
Mannose − − −
Sorbose − − n.d.
2 (UKMP-5T) and Rhodococcus sp. ZH8 (UKMP-7T)],
Rhamnose − − n.d. and Consortium 4 [P. aeruginosa (UKMP- 8T), Rhodococ-
Inositol − − n.d. cus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8
Mannitol − − − (UKMP-7T)]. As the incubation progressed, all cultures
Sorbitol − − n.d. became turbid and it was observed that the crude oil
Esculin + + +
was emulsified into many small oil droplets suspended
Amidone − − n.d.
Gelatin n.d. n.d. +
within the medium, suggesting the secretion of biosur-
Triple sugar iron factant.
Slant n.d. n.d. Alkaline After 7 days, gas chromatographic analysis of the
Butt n.d. n.d. Alkaline hydrocarbon extracted with chloroform as control de-
Gas n.d. n.d. − tected peaks with carbon numbers between C14 to C38,
H2 S n.d. n.d. −
including pristane (Figure 3). All the peaks were re-
IMVIC
Indole formation n.d. n.d. −
duced to undetectable levels after 7 days with the ex-
Methyl Red n.d. n.d. − ception of pristane in samples inoculated with Rhodococ-
formation cus sp. M15-2 (UKMP-5T) and Rhodococcus sp. ZH8
Voges-Proskauer n.d. n.d. − (UKMP-7T). At the end of 7 days, Rhodococcus sp.
reaction M15-2 (UKMP-5T) reduced 94.29% of pristane and
Oxidative- n.d. n.d. Oxidative
Rhodococcus sp. ZH8 (UKMP-7T) reduced 89.35% of
fermentative reaction
MacConkey agar n.d. n.d. Lack lactose
pristane when compared with control. The average per-
fermentor centage of degradation of Tapis Massa crude oil by sin-
Oxidative reaction n.d. n.d. + gle isolates of P. aeruginosa (UKMP-8T), Rhodococcus sp.
Catalase n.d. n.d. + M15-2 (UKMP-5T), and Rhodococcus sp. ZH8 (UKMP-
Note. n.d. = not determined. 7T) was 99.9% ± 0.12%, 99.3% ± 0.25%, and 99.1 ±
1.00%, respectively, when compared with the control
4 supplemented with ammonium chloride, yeast, and (Figure 4).
peptone showed no significant increase in bacterial Inoculation with Consortium 1 reduced all the peaks
population compared with the control. Addition of to undetectable levels except for carbon number C17
0.01% ammonium chloride increased the growth of with 94.70% reduction. All peaks were reduced to unde-
Consortium 1 by 53.1% and of Consortium 2 by tectable levels in samples inoculated with Consortium
43.7% compared with their respective controls. The 2 except C16, C17, and C18 with 99.42%, 86.12%,

5 Crude Oil Biodegradation by Bacterial Consortia

Downloaded by [University of Malaya] at 03:27 03 March 2013
FIGURE 2 Nitrogen sources that supported bacterial growth in Consortia 1, 2, 3, and 4 (AC = ammonium chloride; YE = yeast extract;
P = peptone). The nitrogen source that supported the maximum growth of each consortia.
and 85.49% of reduction observed, respectively. As for
the addition of Consortium 3, C16 to C24 were still
detectable but with percentages of reduction ranging
from 86.60% to 99.98%. Similar results of chromato-
graphic analysis were also obtained for Consortium
4; all peaks were reduced to undetectable levels ex-
cept for C16 to C24, whose reduction values ranged
from 89.12% to 99.97%. Overall, Consortia 1, 2, 3,
and 4 significantly degraded 98.4% ± 0.59%, 98.1% ±
1.50%, 97.6% ± 2.67%, and 98.5% ± 1.74% of the total
petroleum hydrocarbons, respectively, when compared
with control. Inoculum efficiencies in biodegrading the
total petroleum hydrocarbon in the Tapis Massa crude
oil were found to be (in the descending order) as fol-
lows: P. aeruginosa (UKMP-8T) ≥ Rhodococcus sp. M15-
2 (UKMP-5T) ≥ Rhodococcus sp. ZH8 (UKMP-7T) ≥
Consortium 4 ≥ Consortium 1 ≥ Consortium 2 ≥
Consortium 3 (Figure 4).
DISCUSSION
Bacteria isolated from petroleum-contaminated sites
have been known to be well adapted to oil-
contaminated soil or liquid environment (Sugiura et al.
1997; Rahman et al. 2003). In this study, three iso-
lates identified as P. aeruginosa, Rhodococcus sp. M15-2,
and Rhodococcus sp. ZH8 were found to possess oil-
degrading capabilities. The genus Pseudomonas has been
A. Hamzah et al.
reported to carry the alkane hydroxylase gene re-
sponsible for hydroxylation of alkanes, a major com-
ponent of crude oil and naphthalene dioxygenase
responsible for the initial attack on aromatic hydrocar-
bon degradation (Hamzah, Tavakoli, and Rabu 2011).
The genus Rhodococcus also harbor the alkane hydroxy-
lase gene (alkB), which might be involved in medium-
and long-chain alkane oxidation (Hassanshahian, Emti-
azi, and Cappello 2012). Furthermore, Pornsunthawee
et al. (2008) isolated P. aeruginosa SP4 from petroleum-
contaminated soil in Thailand that produced biosur-
factant that was observed to emulsify crude oil.
The second genus, Rhodococcus, obtained in this
study is also increasingly recognized as good oil de-
grader because it has been reported to contain multiple-
degradative-enzyme systems that can degrade a wide
range of hydrocarbons (Pini et al. 2007), is ubiquitous
and robust in the environment (Larkin et al. 1998), and
is known to have high cell surface hydrophobicity
and be able to produce biosurfactant, resulting in bet-
ter physical contact with hydrophobic components of
crude oil (Yakimov et al. 1999). Formation of oil-water
emulsion is important because it was reported that the
contact between hydrophobic hydrocarbons and bacte-
rial membrane-bound degradative enzymes is increased
in emulsified crude oil, thus facilitating the biodegra-
dation process (Zhang and Miller 1992). This further
suggests that P. aeruginosa and Rhodococcus sp. isolated
6
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FIGURE 3 GC chromatogram for Tapis Massa crude oil in (a) control, without inoculation, and inoculated with (b) Pseudomonas
aeruginosa, UKMP-8T; (c ) Rhodococcus sp. M15-2, UKMP-5T; (d ) Rhodococcus sp. ZH8, UMP-7T; (e ) Consortium 1; (f ) Consortium 2; (g )
Consortium 3; and (h) Consortium 4 when incubated at 40◦ C with an agitation rate of 150 rpm for 7 days. (Figure available in color online.)

7 Crude Oil Biodegradation by Bacterial Consortia


FIGURE 4 Degradation of Tapis Massa crude oil by single iso-
lates and consortia incubated at 40◦ C, pH 7, with an agitation rate
of 150 rpm for 7 days. Different letters show significant differences
(p < .05) between the different bacteria and consortia.
in this study are strong candidates suitable to be used
for crude oil degradation.
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All bacteria need a source of nitrogen to support
maximal growth because nitrogen is a key building
block of proteins, enzymes, and nucleic acids. There-
fore, it is important to add nitrogen sources in the
medium for maximum hydrocarbon biodegradation
(Margesin, Zimmerbauer, and Schinner 2000). Shabir
et al. (2007) reported that addition of nitrogen en-
hanced bacterial growth and, hence, increased the
biodegradation rate of crude oil and kerosene. This
suggests that nitrogen is an important macronutrient
for enhancement of the growth of bacteria in build-
ing up enzymes to degrade oils and use them as sub-
strate. Bacteria in Consortia 1, 2, and 4 tended to use
NH4 + ions rather than peptone and yeast extract, which
are structurally more complex. A study involving the
biodegradation of Bombay High crude oil by Nocar-
diopsis sp. concluded that the biodegradation rate of
crude oil supplemented with NH4 Cl was higher (66%
± 1.8%) than either that of yeast extract (47% ± 2.9%)
or of peptone (36% ± 2.6%) (Dixit and Pant 1999).
Results of this study suggest that consortia contain-
ing only Rhodococcus sp. preferred peptone as source
of nitrogen, whereas consortia mixed with P. aerugi-
nosa (UKMP-8T) preferred ammonium chloride. The
nitrogen sources for optimal growth of P. aeruginosa
(UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and
Rhodococcus sp. ZH8 were 0.01% ammonium chloride
(NH4 Cl), 0.10% yeast extract, and 0.50% peptone, re-
spectively (Hamzah et al. 2010). This information can
be used to biostimulate oil-contaminated areas to en-
hance degradation by tailoring the nitrogen sources to
effectively maximize the targeted bacterial strain.
A. Hamzah et al.
In a study by Rahman et al. (2002), the biodegrada-
tion rate of 1% (v/v) Bombay High (BH) crude oil by
bacterial consortia was found to be higher (78%) than
by the individual component bacterial strains: Micrococ-
cus sp. GS2-22 (49%), Corynebacterium sp. GS5-66 (43%),
Flavobacterium sp. DS5-73 (41%), Bacillus sp. DS6-86
(59%), and Pseudomonas sp. DS10-129 (66%). Rahman
et al. (2003) proposed that the type of bacteria com-
prising a bacterial consortium is a key deciding factor
in the extent of biodegradation potential of the bac-
terial consortium mentioned. Elsewhere, a 97-99% of
lube oil biodegradation was successfully achieved by a
defined bacterial consortium consisting of Commaonas
acidovorans Px1, Bacillus sp. Px2, and Pseudomonas sp.
Px3 (Wang et al. 2010). Moreover, lindane biodegrada-
tion by pure and mixed cultures of Streptomyces sp. were
compared, and approximately 12-fold improvement by
consortium consisting not more than five and six strains
was observed (Fuentes et al. 2011). On the other hand,
in a study by Komukai-Nakamura et al. (1996), the
biodegradation percentages of Arabian light crude oil
by the consortia T4+PR4+PB4+AJ1, T4+PR4+PB4,
and T4+PB4 were found to be almost the same (40% ±
7%) as those of single bacterial isolates T4 (Acinetobacter
sp.) and PR4 (Rhodococcus sp.), suggesting that a bacterial
consortium does not necessarily perform better than its
component single bacterial strains.
In this study, all the three isolates and the four
constructed consortia achieved 97.6% to 99.9% of
biodegradation total petroleum degradation after 7 days
of incubation. However, the biodegradation percent-
age did not directly correlate with the bacterial growth.
In other words, although Consortium 1 achieved the
highest growth, the TPH degraded was not the highest.
Intrinsic factors could limit the degradation even
though the number of bacteria was high. One of the
factors that may contribute to this result is the oxy-
gen supply, since insufficient oxygen was reported
to cause incomplete crude oil degradation (Cerniglia
1992). The high bacterial population in Consortium
1 might deplete the concentration of oxygen needed
to degrade the crude oil especially below the oil-water
interface. On the other hand, although the bacterial
growth of Rhodococcus sp. M15-2 (UKMP-5T), Rhodococ-
cus sp. ZH8 (UKMP-7T), as well as Consortia 2, 3,
and 4, which consist of both of the strains, were rel-
atively low, the biodegradation percentage was rather
high (>97%) (Figure 5). This might be due to the effec-
tiveness of Rhodococcus sp. to mineralize the crude oil
8

FIGURE 5 Growth of bacterial cells after 7 days of incubation
with 1% (v /v ) Tapis Massa crude oil in MSM at 40◦ C with an agita-
tion rate of 150 rpm, compared with control.
Downloaded by [University of Malaya] at 03:27 03 March 2013
and produce extracellular biosurfactants that further ac-
celerated the biodegradation process. Nevertheless, no
significant difference was found in biodegradation ef-
ficiency of Tapis Massa crude oil by the constructed
bacterial consortia as compared with single bacterial
strains. Viewed from this aspect, these bacterial con-
sortia in this study were found to be able to degrade
hydrocarbon without the synergistic effect of multi-
ple bacterial strains. The presence of more than one
bacterial strain in a consortium was associated with a
slight decrease in degradation percentage, possibly due
to competition between the bacteria for hydrocarbon
substrates. One bacterial population might have ad-
vantages over another, for example, requiring a shorter
time to adapt to hydrophobic hydrocarbons, which is
known to be poorly accessible to microbial cells. Thus,
this study showed that the net capacity of any microbial
consortium to degrade hydrocarbon is not necessarily
the result of merely adding together the capacities of
the individual bacterial strains forming the association.
The inability of constructed bacterial consortia en-
hancing degradation compared with single isolates can
be due to the different effect of nitrogen sources on the
optimal growth of the different isolates found within
the consortia. In the growth optimization results, it can
be seen that P. aeruginosa (UKMP-8T) grown on NH4 Cl
as nitrogen source showed higher biomass compared
with Rhodococcus sp. M15-2 (UKMP-5T) and Rhodococ-
cus sp. ZH8 (UKMP-7T), which preferred yeast and
peptone, respectively (Figure 5). Although NH4 Cl also
supported maximum biomass in Consortia 1, 2, and 4,
comparison of nitrogen source preferences of the single
9 Crude Oil Biodegradation by Bacterial Consortia
isolates suggests that NH4 Cl preferentially supported P.
aeruginosa (UKMP-8T), possibly increasing the popula-
tion of P. aeruginosa (UKMP-8T) over Rhodococcus sp.
M15-2 (UKMP-5T) and Rhodococcus sp. ZH8 (UKMP-
7T) in the consortia. However, in the case of Consor-
tium 3, peptone might have preferentially supported
the growth of Rhodococcus sp. ZH8 (UKMP-7T) over
P. aeruginosa (UKMP-8T) and Rhodococcus sp. M15-2
(UKMP-5T). Although these consortia may have started
with a mixture of two to three isolates, the nitrogen
source that was added into the MSM medium might
play a decisive role in selectively supporting the growth
of one species to the disadvantage of others during the
incubation period, thus reducing the synergistic effec-
tiveness that is attributed to the presence of bacterial
mixture. Thus, it can be seen that the extent of biodegra-
dation is highly influenced by types of bacteria used as
well as by the nutrients in the medium where the treat-
ment is conducted (Ghazali et al. 2004). Nevertheless,
all three isolates and the consortia grown on different
nitrogen sources managed to reduce the majority of
the peaks, especially those of the heavy-chain hydro-
carbons from C24 to C38, which normally consist of
complex polycyclic aromatic hydrocarbons (PAHs).
CONCLUSION
Three bacterial strains isolated from groundwater of
a petroleum refinery plant were found to be capable
of degrading Tapis Massa crude oil. The three isolates
were identified as P. aeruginosa (UKMP-8T), Rhodococ-
cus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8
(UKMP-7T) on the basis of a series of physiological,
biochemical, and morphological characteristics and fur-
ther confirmed based on sequences amplification of
DNA extracted from the isolates. All the three iso-
lates and four consortia constructed degraded 97.6%
to 99.9% of the crude oil in the medium after 7 days of
incubation. However, the consortia constructed from
these three genus of bacteria did not enhance degra-
dation as expected. It may be that the nitrogen sources
tested (NH4 Cl, peptone, and yeast extract) promote the
growth of one species to the disadvantage of the other
members of the consortium, thus limiting any possible
synergistic effects. Findings from this study showed that
the use of a single bacterial isolate was more effective
than the constructed bacterial consortia in degrading
Tapis Massa crude oil and can be more advantageous,
 as it would significantly reduce the cost, time, and main-
tenance involved while achieving a satisfactory result.
ACKNOWLEDGMENT
We would like to thank PETRONAS Research Sdn.
Bhd. for providing the grant for this research.
REFERENCES
Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new
generation of protein database search programs. Nucleic Acids Res.
25:3389–3402.
Bouchez, M., D. Banchet, and J. P. Vandercasteele. 1995. Degradation of
polycylic aromatic hydrocarbon by pure strains and by defined strain
association: Inhibition phenomena cometabolism. Appl. Microbiol.
Biotechnol. 43:156–164.
Cerniglia, C. E. 1992. Biodegradation of polycyclic aromatic hydrocar-
bons. Biodegradation 3:351–368.
Dixit, V. S., and A. Pant. 2000. Hydrocarbon degradation and protease
Downloaded by [University of Malaya] at 03:27 03 March 2013
Larkin, M. J., De R. Mot, L. A. Kulakov, and I. Nagy. 1998. Applied aspects
of Rhodococcus genetics. Antoine Van Leeuwenhoek 74:133–153.
Liang, Y., X. Zhang, D. Dai, and G. Li. 2009. Porous biocarrier-enhanced
biodegradation of crude oil contaminated soil. Int. Biodeter. Biode-
grad. 63:80–87.
Marquez-Rocha, F. J., J. Olmos-Soto, M. C. Rosano-Hernandez, and M.
Muriel-Garcia. 2005. Determination of the hydrocarbon-degrading
metabolic capabilities of tropical bacterial isolates. Int. Biodeter.
Biodegrad. 55:17–23.
Margesin, R., A. Zimmerbauer, and F. Schinner. 2000. Monitoring of biore-
mediation by soil biological activities. Chemosphere 40:339–346.
Muter, O., K. Potapova, B. Limane, K. Sproge, I. Jakobsone, and G.
Cepurnieks. 2012. The role of nutrients in the biodegradation
of 2,4,6-trinitrotoluene in liquid and soil. J. Environ. Manage. 98:
51–55.
Pini, F., C. Grossi, S. Nereo, L. Michaud, A. L. Giudice, V. Bruni, F. Baldi,
and R. Fani. 2007. Molecular and physiological characterisation of
psychrotrophic hydrocarbon-degrading bacteria isolated from Terra
Nova Bay (Antarctica). Eur. J. Soil Biol. 17:1–12.
Pornsunthorntawee, O., N. Arttaweepon, S. Paisanjit, P. Somboon-
thanate, M. Abe, R. Rujiravanit, and S. Chavadej. 2008. Isola-
tion and comparison of biosurfactants produced by Bacillus subtilis
PT2 and Pseudomonas aeruginosa SP4 for microbial surfactant-
enhanced oil recovery. Biochem. Eng. J. 42:172–179.

production by Nocardiopsis sp. NCIM 5124. Lett. Appl. Microbiol.

Rahman, K. S. M., T. J. Rahman, Y. Kourkoutas, I. Petsas, R. Marchant,
30:67–69.
and I. M. Banat. 2003. Enhanced bioremediation of n-alkane in
Dubey, K. K., and M. H. Fulekar. 2012. Chlorpyrifos bioremedia-
petroleum sludge using bacterial consortium amended with rham-
tion in Pennisetum rhizosphere by a novel potential degrader
nolipid and micronutrients. Bioresour. Technol. 90:159–168.
Stenotrophomonas maltophilia MHF ENV20. World J. Microbiol.
Rahman, K. S. M., J. Thahira-Rahman, P. Lakshmanaperumalsamy, and I.
Biotechnol. 28:1715–1725.
M. Banat. 2002. Towards efficient crude oil degradation by a mixed
Fuentes, M. S., J. M. Sáez, C. S. Benimeli, and M. J. Amoroso. 2011.
bacterial consortium. Bioresour. Technol. 85:257–261.
Lindane biodegradation by defined consortia of indigenous Strep-
Shabir, G., M. Afzal, F. Anwar, R. Tahseen, and Z. M. Khalid. 2007.
tomyces strains. Water Air Soil Pollut. 222:217–231.
Biodegradation of kerosene in soil by a mixed bacterial culture under
Ghazali, F. M., R. N. Z. A. Rahman, A. B. Salleh, and M. Basri. 2004.
different nutrient conditions. Int. Biodeter. Biodegrad. 61:161–166.
Biodegradation of hydrocarbon in soil by microbial consortium. Int.
Sugiura, K., M. Ishihara, T. Shimauchi, and S. Harayama. 1997. Physio-
Biodeter. Biodegrad. 54:61–67.
chemical properties and biodegradability of crude oil. Environ. Sci.
Hamzah, A., and N. F. Abu Bakar. 2007. Optimization of physical and
Technol. 31:45–51.
nutrient parameters in enhancing the degradation of Tapis crude
Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular
oil by Bacillus sp. UKMP-7T. In Proceedings of the 29th Symposium
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
of Malaysian Society for Microbiology, E15:1–6.
Biol. Evol. 24:1596–1599.
Hamzah, A., M. Abu Zarin, A. Abdul Hamid, O. Omar, and S. Senafi.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTALW: Im-
2012. Optimal physical and nutrient parameters for growth of Tri-
proving the sensitivity of progressive multiple sequence alignment
choderma virens UKMP-1M for heavy crude oil degradation. Sains
through sequence weighting, position-specific gap penalties and
Malays 41:71–79.
weight matrix choice. Nucleic Acids Res. 22:4673–4680.
Hamzah, A., A. Rabu, R. F. Hanim Raja Azmy, and N. A. Yussoff. 2010.
Wang, H., R. Xu, F. Li, J. Qiao, and B. Zhang. 2010. Efficient degrada-
Isolation and characterization of bacteria degrading Sumandak and
tion of lube oil by a mixed bacterial consortium. J. Environ. Sci.
South Angsi oils. Sains Malays 39:161–168.
22:381–388.
Hamzah, A., A. Tavakoli, and A. Rabu. 2011. Detection of toluene degra-
Yakimov, M. M., L. Giuliano, V. Bruni, S. Scarfı, and P. N. Golyshin. 1999.
dation in bacteria isolated from oil contaminated soils. Sains Malays
Characterization of Antarctic hydrocarbon-degrading bacteria ca-
40:1231–1235.
pable of producing bioemulsifiers. Microbiologica 22:249–256.
Hassanshahian, M., G. Emtiazi, and S. Cappello. 2012. Isolation and
E. Zajic, and B. Supplisson. 1972. Emulsification and degradation
characterization of crude-oil-degrading bacteria from the Persian
of “Bunker C” fuel oil by microorganisms. Biotechnol. Bioeng.
Gulf and the Caspian Sea. Mar. Pollut. Bull. 64:7–12.
14:31–43.
Komukai-Nakamura, S., K. Sugiura, Y. Yamauchi-Inomata, H. Toki, K.
Zhang, Y. M., and R. M. Miller. 1992. Enhanced octadecane dispersion
Venkateswaran, S. Yamamoto, H. Tanaka, and S. Harayama. 1996.
and biodegradation by a Pseudomonas rhamnolipid surfactant (bio-
Construction of bacterial consortia that degrade Arabian Light
surfactant). Appl. Environ. Microbiol. 58:3276–3828.
crude oil. J. Ferment. Bioeng. 82:570–574.

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