Isolation and Characterization of Engine Oil Degrading Indigenous Microorganisms in Kwazulu-Natal, South Africa

Abstract

Uncontrolled releases of petroleum compounds that are carcinogenic, mutagenic and are potent immunotoxicants into soil and groundwater poses a serious threat to human and animal health. Biodegradation of hydrocarbon-contaminated soils has been established as an efficient, economic, versatile and environmentally sound treatment. Ten indigenous microorganisms were isolated from contaminated soils using the enrichment technique. Five isolates with the highest degradation potentials under standard degradation conditions were identified as Acinetobacter calcoaceticus (LT 1 and ETS 2), Acinetobacter sp. (LT 1 A), Citrobacter freundii (MRC 3) and Bacillus pumilus (JL B). B. pumilus achieved 86.94% of diesel degradation in 2 weeks. Additional degradation assay was carried out in liquid media using 3 local commercial fertilizers as nutrient supplements in comparison with the Bushnell-Haas (BH) media. The results show that the addition of fertilizer F1 stimulated diesel degradation by all isolates especially B. pumilus while the addition of fertilizer F3 seemed to strongly inhibit the bacterial ability of diesel degradation. The inoculation with the consortia did not show a higher degradation potential than the individual isolate. The results strongly indicate that environmental conditions of the contaminated sites play a crucial role in the degradation even though additional diesel-degrader has been introduced into the contaminated site.

Full text

African Journal of Biotechnology Vol. 7 (12), pp. 1927-1932, 17 June, 2008
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2008 Academic Journals
Full Length Research Paper
Isolation and characterization of diesel oil degrading
indigenous microrganisms in Kwazulu-Natal, South
Africa
C. Singh and J. Lin*
School of Biochemistry, Genetics, Microbiology and Plant Pathology, University of KwaZulu-Natal (Westville), Private
Bag X 54001, Durban, Republic of South Africa.
Accepted 5 December, 2007
Uncontrolled releases of petroleum compounds that are carcinogenic, mutagenic and are potent
immunotoxicants into soil and groundwater poses a serious threat to human and animal health.
Biodegradation of hydrocarbon-contaminated soils has been established as an efficient, economic,
versatile and environmentally sound treatment. Ten indigenous microorganisms were isolated from
contaminated soils using the enrichment technique. Five isolates with the highest degradation
potentials under standard degradation conditions were identified as Acinetobacter calcoaceticus (LT1
and ETS2), Acinetobacter sp. (LT1A), Citrobacter freundii (MRC3) and Bacillus pumilus (JLB). B. pumilus
achieved 86.94% of diesel degradation in 2 weeks. Additional degradation assay was carried out in
liquid media using 3 local commercial fertilizers as nutrient supplements in comparison with the
Bushnell-Haas (BH) media. The results show that the addition of fertilizer F1 stimulated diesel
degradation by all isolates especially B. pumilus while the addition of fertilizer F3 seemed to strongly
inhibit the bacterial ability of diesel degradation. The inoculation with the consortia did not show a
higher degradation potential than the individual isolate. The results strongly indicate that environmental
conditions of the contaminated sites play a crucial role in the degradation even though additional
diesel-degrader has been introduced into the contaminated site.
Key words: Diesel bioremediation, Bacillus sp, Acinetobacter calcoaceticus sp., Citrobacter freundii.
INTRODUCTION
Petroleum continues to be used as the principle source of
energy; however, despite its important usage, petroleum
hydrocarbons also pose as a globally environmental
pollutant (Plohl et al., 2002). South Africa is especially
vulnerable to oil spills due to the high volume of oil
transported around the coasts. Protective and preventive
measures need to be taken into account to avoid spillage
into the environment. Spillages of oil have become a
common occurrence in recent history. In 1983, a major
spill of 250,000 tons of oil occurred in South Africa by the
tanker known as Castillo De Belluer. In 2000, two large
oil spillages near and in Cape Town threatened the South
*Corresponding author. E-mail: linj@ukzn.ac.za. Tel: +27-31-
2607407. Fax: +27-31-2607809.
African penguin population and damaged major tourist
attraction sites such as Robben Island (Avian Demogra-
phy Unit, 2000). In 2002, another oil-spillage occurred
near 12 km South of the St. Lucia estuary. The cargo was
still being removed in 2004 (Department of Environmental
Affairs and Tourism, 2004) and the estuary house was
only reopened in 2006 to avoid ecological disaster
demonstrating how time-consuming a clean-up operation
can be. Oil spills in coastal areas cause immediate and
obvious problems to animals and plants. There are also
long-term effects on ecosystems related to the release of
toxic components over a prolonged period as the oil
breaks up and the concentration of toxicants in
organisms towards the top of the food chain increases
(Samanta et al., 2002).
Petroleum contamination also results from leakage
above ground and underground storage tanks, spillage

1928 Afr. J. Biotechnol.
during transport of petroleum products, abandoned
manufactured gasoline sites, other unplanned releases
and current industrial processes (Mishra et al., 2001;
Sarkar et al., 2005). Uncontrolled releases of these
compounds into soil and ground water are frequent as a
result of accidents or poor control practices and attract
public interest (Mishra et al., 2001; Roling et al., 2002;
Atlas and Philp, 2005). Petroleum compounds are
considered to be recalcitrant to microbial degradation and
persist in ecosystems because of their hydrophobic
nature and low volatility and thus they pose a significant
threat to the environment (Abed et al., 2002). The
constituents of these contaminants such as diesel oil, are
carcinogenic, mutagenic and are potent immunotoxi-
cants, thus posing a serious threat to human and animal
health (Boonchan et al., 2000; Samanta et al., 2002). Oil
spills, especially in soil contamination have prompted
research on cost-effective, environmentally benign clean-
up strategies (Margesin and Schinner, 2001).
Biodegradation of hydrocarbon-contaminated soils,
which exploits the ability of microorganisms to degrade
and/or detoxify organic contaminants, has been esta-
blished as an efficient, economic, versatile and environ-
mentally sound treatment (Mehrashi et al., 2003). Since
hydrocarbons are natural products, it is not surprising to
find organisms that are able to degrade these energy-rich
substrates (Delille et al., 2002). The ability of microbes to
degrade organic contaminants into harmless constituents
has been explored as a means to biologically treat
contaminated environments. It is the subject of many
research investigations and real-world applications and it
is the basis for the emergent field of bioremediation (Atlas
and Philp, 2005).
This report deals with the isolation of diesel-degrading
bacteria and determination of the biodegradation poten-
tials of the pure cultures of selected bacterial isolates as
well as consortia.
MATERIALS AND METHODS
Collections of diesel contaminated soil samples
Eight diesel-contaminated soil samples were collected from six
different transport companies in and around Durban, South Africa.
100 g of each sample was placed into 500 ml Schott bottles and
stored at 4˚C until further study. The diesel was purchased from a
local garage and stored in the dark at ambient temperature
throughout the study.
Isolation of bacterial diesel degraders
Bushnell – Haas (BH) medium (Atlas, 1994) was used as the
enrichment media with 10% (v/v) diesel as the sole carbon source
to isolate diesel–degrading bacteria. 10 g of the contaminated soil
was added and incubated at 30oC at 170 rpm. After 2 weeks, 1 ml
of enriched media was transferred into freshly prepared enrichment
media and incubated at the same conditions as described above.
Serial dilutions (1/10) from the third enrichment process were plated
out onto BH agar plates, which were covered with 100 µl of diesel
oil and incubated at 30oC. The single colonies were streaked onto
nutrient agar plates, incubated at 30oC overnight, and stored at 4oC
until further use.
The oil-degrading isolates were identified by gram stain, bioche-
mical tests (Balows et al., 1992) and confirmed by 16S rDNA
sequencing (Marchesi et al., 1998). For long–term preservation, the
bacterial isolates were stored in 40% glycerol at -70oC.
Characterization of the degradation potential
The preliminary biodegradation assays were performed as describ-
ed by Mandri and Lin (2007) with modifications. A single colony of
the isolate was inoculated into 10 ml nutrient broth (Merck) at 30oC
overnight. The overnight culture was centrifuged for 15 min at 3500
rpm. The cell pellet was washed twice and was re-suspended with
BH medium until OD600 was equivalent to 1.2.
One ml of bacterial inoculum (1.2 OD600 equivalent) was
transferred into 100 ml BH medium with 5 ml (5%) diesel and was
incubated at 30oC at 170 rpm for two weeks. A control devoid of
the bacterial isolate was prepared for each set of experiments. All
experiments were performed in duplicate.
The biodegradation assays were also performed using selected
isolates and using local commercial fertilizers as nutrient supple-
ments instead of BH media under the same conditions.
The growth patterns were obtained by measuring the optical
density at 600 nm and total viable counts (cfu/ml) of the isolates
were determined by the spread plate technique after the incubation
of the nutrient agar plates at 30oC for 24 h.
Determination of diesel degradation
The level of diesel oil degradation was determined using the gravi-
metric analysis (Chang, 1998; Marquez– Rocha et al., 2001). The
percentage of diesel remaining was calculated compared to the
control.
RESULTS
The preliminary biodegradation assay was carried out to
determine the diesel degradation capabilities of the 10
indigenous microbial cultures that were isolated from
contaminated environments. Preliminary investigation of
these cultures in BH media together with agitation and
aeration for 2 weeks allowed microbial degradation of the
diesel oil as shown in (Figure 1). The isolate JLB accele-
rated a high degradation of 86.94%, whereas isolate SC1
showed the least degradation of 1.484%. The 5 best
diesel-degraders could also degrade diesel up to 60.3%
under stationary conditions (Data not shown). These 5
isolates were identified as A. calcoaceticus (LT1 and
ETS2), Acinetobacter sp. (LT1A), C. freundii (MRC3) and
Bacillus pumilus (JLB). A. calcoaceticus (LT1), Acineto-
bacter sp. (ETS2) and B. pumilus (JLB) with highest
degradation potentials were used for the following study.
Additional degradation assay was carried out in liquid
media using 3 local commercial fertilizers as nutrient
supplements in comparison with the BH media. The
results in Figure 2 demonstrate the degradation rates of
different combinations of bacterial isolates and the
nutrients under the same conditions as described above

Singh and Lin 1929
Figure 1. Percentage of diesel degradation by bacterial isolates under the standard degradation
conditions. LT1 and ETS2: Acinetobacter calcoaceticus; LT1A: Acinetobacter sp.; MRC3: Citrobacter
freundii; and JLB: Bacillus pumilus. The remaining five isolates were unidentified Gram-
negative bacteria.
after 2 weeks. The combination of A. calcoaceticus
(ETS2) and fertilizer F1 exhibited a highest degradation of
90.28%. The addition of fertilizer F1 stimulated the diesel
degradation of the isolates especially B. pumilus (from
53- 81%), while the addition of fertilizer F3 seemed to
strongly inhibit the bacterial ability of diesel degradation.
The fertilizer F2 was the less enhancer than F1 (Data not
shown). The results in Figure 2 also show that the
bacterial consortia of all three isolates did not improve the
level of diesel degradation than the individual isolate
under the same conditions.
DISCUSSION
Microorganisms are extremely diverse and can adapt to
survive in inhospitable environments. Microbes are
capable of breaking down many complex molecules by
adaptation of their degradative enzyme system (Sohal
and Srivastava, 1994). Microorganisms play important
roles in the natural environment; they contribute to the
geological cycle of elements and transformation of
natural chemicals (Watanabe, 2002). Contaminated sites
often harbour a vast array of microbial flora that is
capable of utilizing the contaminant as an energy and
carbon source (Watanabe, 2002; Das and Mukherjee,
2006).
The contaminated soil samples were enriched and
subsequently 10 bacterial isolates were subcultured. The
preliminary assay showed that eight of the ten isolates
possess the diesel degradation potentials. B. pumilus
isolate (JLB) seemed to have the best degradation of
86.94%. Several Acinetobacter isolates were also found
with the diesel degradation potentials. All isolates in this
study have been commonly reported as hydrocarbon–
degraders in various environments (da Cunha et al.,
2006; Ilori et al., 2006; Mihial et al., 2006, Kim and
Crowley, 2007).
Few studies (Annweiller et al., 2000; Ijah and Antai,
2003; Sorkhoh et al., 1993) have reported on the roles of
Bacillus spp. in hydrocarbon bioremediation; although
there are several reports of bioremediation of pollutants
by the action of Bacillus spp. occurring in extreme
environments. Ijah and Antai (2003) reported Bacillus
spp. as being the predominant isolate of all the crude oil
utilizing bacteria characterized from highly polluted soil
samples (30 and 40% crude oil). It was postulated that
Bacillus spp. are more tolerant to high levels of hydro-
carbons in soil due to their resistant endospores. There is
growing evidence that isolates belonging to the Bacillus
sp. could be effective in clearing oil spills (Ghazali et al.,
2004).
Acinetobacter species was the most frequent isolate in
this study as reported in the literature as the most
common bacterial hydrocarbon–degraders (Rusansky et
al., 1987; Kiyohara et al., 1992; Johnson et al., 1996;
Barathi and Vasudevan, 2001; Bhattacharya et al., 2002;
Pokethitiyook et al., 2003; Van Hamme et al., 2003).
Acinetobacter spp. are widespread in nature and can
remove or degrade a wide range of organic and inorganic

1930 Afr. J. Biotechnol.
Figure 2. Percentage of diesel degradation by bacterial isolates using commercial fertilizers (F1 and
F3) as nutrient comparison with BH media under the same degradation conditions. JLB: Bacillus
pumilus isolate; LT1: Acinetobacter calcoaceticus isolate; ETS2: Acinetobacter sp.
compounds (Auling et al., 1991; Wagner et al., 1994;
Boswell et al., 2001; Briganti et al., 1997; Zilli et al., 2001;
Abdel-El-Haleem et al., 2002). Acinetobacter species
have shown the potentials in both environmental and
biotechnological applications (Abdel-El-Haleem 2003).
Another A. calcoaceticus isolate was also obtained in this
laboratory and was found to degrade used engine oil
(Mandri and Lin, 2007).
Biodegradation of complex hydrocarbons in nature
usually requires the cooperation of more than a single
species. Microbial populations that consist of strains that
belong to various genera have been detected in
petroleum-contaminated soil (da Cunha et al., 2006; Ilori
et al., 2006; Mihial et al., 2006, Kim and Crowley, 2007).
This strongly suggests that each strain or genera have
their roles in the hydrocarbon transformation processes
(Ghazali et al., 2004; Cunliffe and Kertesz, 2006).
Individual microorganisms can metabolize only a limited
range of hydrocarbon substrates, so assemblages of
mixed populations with overall broad enzymatic capaci-
ties are required to bring the rate and extent of petroleum
biodegradation further. However, in this study, inoculation
with the consortia did not show a higher degradation
potential (Figure 2). All isolates obtained in this study
were isolated through the same enrichment process;
therefore the best diesel degraders obtained might
possess the similar abilities and occupy the same niches.
The inoculated isolates might compete for the same
carbon source and the consortia might not be able to
perform better than the individual isolate. In addition, the
differences in the cell number, that the cell mass of the
inoculants with the pure isolate was higher than that of
the same isolate in the consortia, probably also contribute
to the observation. Inoculation has been shown to be
efficient when the contaminants belong to a single type of
recalcitrant compound (Thomassin-Lacroix et al., 2002).
In order to explore the potential of in situ bioreme-
diation of indigenous isolates, the local commercial
fertilizers were randomly selected as alternative nutrient
sources instead of BH media. The addition of different
commercial fertilizers caused a significant difference in
the ability of diesel degraders to break down diesel. The
fertilizer F1 stimulated the diesel degradation abilities of
all isolates. B. pumilus (JLB) was found to be the best
diesel-degrader in the presence of fertilizer F1. However,
the addition of fertilizer F3 inhibited dramatically the
degradation abilities of same isolates. Walworth et al.
(1997) reported the enhancement and inhibition of soil

petroleum biodegradation through the use of fertilizer
nitrogen.
Some researchers have reported that inoculation had
positive, marginal or no effects on oil biodegradation
rates (Margesin and Schinner, 1997; Venosa et al.,
1992). Microorganisms that possess the degradative
ability of organic pollutants in cultures may fail to function
when inoculated into the natural environment. Using
different fertilizers as the nutrient sources had a signifi-
cant impact on diesel degradation of the same isolate.
The results strongly indicate that the environmental
conditions including physical and chemical conditions of
the contaminated sites play a crucial role in the degra-
dation even though additional diesel-degrader has been
introduced into the contaminated site. It might be feasible
to harbour microorganisms from a contaminated site,
because the microbes have adapted to a contaminated
environment and utilizes the contaminant as a carbon
and energy source (Sohal and Srivastava, 1994;
Watanabe, 2002; Ghazali et al., 2004; Das and
Mukherjee, 2006).
Bioremediation has been widely received by the public.
However, a number of factors must be taken into consi-
deration before in situ bioremediation can be applied.
These include (i) type and concentration of oil conta-
minated; (ii) prevalent climatic conditions; (iii) type of
environment that has been contaminated; and (iv)
nutrient content as well as pH of the contaminated site
(Rosenberg, 1992). Further research will be directed
towards understanding the roles of individual isolate in
influencing the effectiveness of a microbial association as
well as the optimal degradation conditions in situ.
ACKNOWLEDGEMENTS
This project was financially supported by the National
Research Foundation (GUN 2069344) and the University
Competitive Research Grant, University of KwaZulu-
Natal, South Africa.
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