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The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
Microbial Degradation of Petroleum Hydrocarbons in a Polluted
Tropical Stream
Sunday A. Adebusoye 1*, Matthew O. Ilori 1, Olukayode O. Amund 1, Olakunle D. Teniola 2, S. O. Olatope 2
1 Department of Botany and Microbiology, University of Lagos, Lagos, Nigeria
2 Biotechnology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria,
Email: olakunleteniola@yahoo.com, Telephone: (234)-8023533428
Abstract: Enrichments of water samples from a polluted stream with crude oil resulted in the isolation of nine
bacteria belonging to the following genera: Acinetobacter, Alcaligenes, Bacillus, Corynebacterium, Flavobacterium,
Micrococcus and Pseudomonas. A mixed culture was developed from the assemblage of the nine bacterial species.
The defined microbial consortium utilized a wide range of pure HCs including cycloalkane and aromatic HCs.
Utilization of crude oil and petroleum cuts i.e., kerosene and diesel resulted in increase in total viable count (till day
10) contaminant with drops in pH and residual oil concentration. Crude oil, diesel and kerosene were degraded by
88%, 85% and 78% respectively in 14 days. Substrate uptake studies with axenic cultures showed that growth was
not sustainable on either cyclohexane or aromatics while degradation of the petroleum fraction fell below 67% in
spite of extended incubation period (20 day). From the GC analysis of recovered oil, while reductions in peaks of n-
alkane fractions and in biomarkers namely n-C17/pristane and n-C18/phytane ratios were observed in culture fluids
of pure strains, complete removal of all the HC components of kerosene, diesel and crude oil including the
isoprenoids was obtained with the consortium under 14 days. Study shows that assemblage of microbial cultures
offer a more extensive degradation than pure cultures. [The Journal of American Science. 2006;2(3):48-57].
Key Words: Mixed culture; Hydrocarbons; Degradation; Bacteria; Phytane; Pristane; Microbial strains; Residual oil
Introduction The present-day methods of ridding the
environment of spilled oil most especially in Nigeria
include mechanical collection, use of sorbent materials,
sinking, burning, dispersion, etc., all of which have
undesirable ecological consequences (Ekundayo and
Obire, 1987). Microbial degradation is the major and
ultimate natural mechanism by which one can clean-up
the PHC pollutants from the environment (Atlas, 1992;
Amund and Nwokoye, 1993; Lal and Khanna, 1996).
However each individual strain is usually characterized
by an ability to utilize only a few kinds of hydrocarbons
(HCs) Yeasts, for example, can oxidize only the
aliphatic HCs (West et al., 1984; Okpokwasili and Ibe,
1987). Such bacterial genera as Acinetobacter,
Arthrobacter, Bacillus, Corynebacterium,
Flavobacterium, Vibrio and Pseudomonas contain
species that together can degrade most constituents of
crude oil, including the aliphatic, alicyclic, aromatic,
and polycyclic HCs (Atlas, 1992; Ko and Lebeault,
1999). It has been observed that pure cultures of the
individuals species have only limited substrate ranges
and are of little help in consuming the complex HC
mixtures found in crude oil (Colwell and Walker, 1977;
Continual crude oil spills in the Niger Delta area of
Nigeria due to pipeline bursting and due to oil tanker
accident and similar occurrences elsewhere have drawn
attention to the problem of petroleum hydrocarbons
(PHCs) contamination in the environment. Whatever the
origin of contamination, some petroleum or
decomposition products may reach groundwater
reserves, lakes or water courses providing water for
domestic and industrial use. Apart from possible
hazards to health such as liver damage and skin
problems (Okonkwo, 1984), such contamination is
objectionable because of the very low concentration at
which PHCs and associated materials can be detected by
their smell and taste (Anyaegbu, 1987; Nwankwo and
Irrechukwu, 1987). This problem is most serious in
areas which rely on groundwater and rivers as major
sources of drinking water; as constantly experienced in
Lagos and Niger Delta areas of Nigeria. The pollutant
may also inhibit some microbial communities that are
important in some biogeochemical cycles of that
ecosystem and this affects the productivity of such
ecosystems (Rhodes and Hendricks, 1990).
48
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
Okpokwasili and Ibe, 1987; Adams and Jackson, 1996).
Since the HC mixtures differ markedly in volatility,
solubility and susceptibility to degradation, it is
therefore evident that the necessary enzymes needed
cannot be found in a single organism. Therefore, a
mixed culture of microbial community is required for
complete biodegradation of oil pollutants.
A great deal has been learned about the
microbiology of PHCs by pure cultures under laboratory
conditions (Amanchukwu et al., 1989; Amund and
Adebiyi, 1991; Dixit and Pant, 2000). But to understand
the fate of petroleum in soil and aquatic environments,
natural assemblages of organisms must be examined.
Use of natural populations as in ocular will enable
individual species in the consortium to consume
different HC components of the oil and also permit
some of the interactions that occur in nature to occur in
the laboratory: competition among organisms,
commensalism, and possible sequential co-metabolic
events (Lal and Khanna, 1996). The use of consortia of
known microbial composition has gained recent
attention owing to its effectiveness over natural mixed
populations of unknown species. Lal and Khanna (1996)
showed that a combination of Alcaligenes calcoaceticus
and Alcaligenes odorans effected higher degradation
rates than that shown by consortia of unknown
microbial populations. In earlier study, Okpokwasili and
James (1995) observed better utilization of kerosene by
a pure culture than a mixed culture. One of the possible
reasons given by the authors was antagonistic properties
of individual organisms in the consortium.
Oil pollution is a continuous phenomenon most
especially in oil producing countries. Thus oil pollution
despite the progress of recent years will remain a
considerable problem. The microbes’ scavenging
versatility need to be harnessed to a greater extent than
at present. In this communication, the petroleum
degrading potentials of axenic cultures as well as
assemblage of pure bacterial strains from a polluted
stream was examined with the hope of isolating and
stocking useful organisms with high crude oil degrading
potentials as candidate organisms for clean-up of
petroleum contaminated systems.
Materials and Methods
Water samples
Water Samples were collected during dry season
from three locations along the course of a polluted
stream in Lagos, Nigeria. These were Shomolu, Abule-
Oja and Iwaya tagged I, II, and III respectively. These
stations were about few kilometres from one another.
Two replicate samples were collected from each site and
were transported immediately to the laboratory for
further work which commenced upon arrival. The
stream had the following characteristics: pH, 6.2 – 6.7;
conductivity, 117.0 – 624.0 µs/cm; total dissolved
solids, 363 – 719 mg/L; total suspended solids, 442 –
719 mg/L; total acidity, 45 – 79 g/L; nitrate, 44 – 79
mg/L; sulphate, 8 – 13 mg/L; salinity, 50 – 715 mg/L.
All heavy metals were below the detection limit. The
water was pale-brown in colour and had an offensive
odour particularly at station III.
Chemicals and crude oil
Higher purity n-alkane, cycloalkane, and aromatic
HCs were obtained from Farmex Nigeria Limited,
Sango-Otta. Escravos high crude oil and petroleum cuts
were obtained from Chevron Nigeria Limited.
Utilization of crude oil by microorganisms in the
polluted stream
The water samples were aseptically dispensed into
two sets of conical flasks in two replicates. Sterile
Escravos light crude oil at 1.0% (v/v) was added to one
set while the other set served as control. All the flasks
were incubated at ambient temperature (29.0 ± 1.0 oC)
in a gyratory shaker incubator operated at 120 rpm for
14 days. The total viable counts (TVCs) in each flask
were monitored at intervals. TVCs were obtained after
serial dilutions in sterile distilled water, spread-plating
of appropriate aliquots in tryptone soy agar (TSA) and
incubation of plates for 24 – 36 h at room temperature.
Assessment of bacterial populations
Total viable heterotrophic bacterial count (TVC)
was determined by spread-plating each water sample
(after appropriate dilution in sterile distilled water) onto
TSA. The spread plates were incubated at 30 oC and
examined for bacterial growth at 24 h. Enumeration of
HC-degrading bacteria was performed by spread
inoculation on mineral salts medium (MSM) formulated
according to Mills et al. (1978). The medium contained
the following in g/L of distilled water; NaCl, 10.0; KCl,
0.29; MgSO4.7H2O, 0.42; KH2PO4, 0.83; NaNO3, 0.42;
Na2HPO4, 1.25. The medium was solidified with
purified agar (20.0 g). The pH was adjusted to 7.2 prior
autoclaving. Crude oil (as carbon source) was supplied
by vapour phase transfer as described by Raymond et al.
(1976). Sample dilution and plate inoculation were
handled as described for TVC analysis while
inoculation was done for one week.
49
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
Isolation of HC-degrading bacteria and development
of defined mixed cultures
The isolation of HC-degrading bacterial species
was performed by enrichment on crude oil in an MSM
described above. To isolate the organisms 2 mL of
water sample was placed in 250 mL conical flask
containing 99 mL of MSM. The medium was
supplemented with 1% (v/v) crude oil as the sole source
of carbon and energy and incubated with shaking at
room temperature for 12 days after three repeated
transfers, aliquots of appropriate dilution of the enriched
culture was plated on MSM agar wherein crude oil was
supplied by vapour phase transfer and incubated for one
week. The resulting colonies were purified on TSA and
screened for HC utilization. The mixed culture was
developed by assemblage of the isolated pure strains.
HC degradation studies
Growth of the defined microbial consortium and
three pure isolates was monitored in 250-mL flasks
containing 99 ml MSM with 1mL of sterile crude oil as
substrate. Both the consortium and pure strains were
pre-grown in peptone water for 18 h before seeded into
the flasks. The experiments were carried out in two
replicates. Flasks containing crude oils but without
inoculation served as control. Utilization of petroleum
cuts, kerosine and diesel was also setup in similar
manner. The pH, TVC and residual oil analysis served
as biodegradation indices and were all monitored at
determined intervals of time.
Extraction of residual oil
Undegraded oil (residual oil) was extracted from
the culture fluids with ethylene chloride. Extraction was
done by adding 10 mL of thoroughly shaken culture to a
separating funnel. To this, was added 10 mL of ethylene
chloride. The funnel was vigorously shaken, after which
contents were allowed to settle in order for the phases to
separate. The organic phase was drawn off, and
thereafter quantified gravimetrically and
chromatographically. The control flasks were also
extracted similarly.
Gas chromatographic (GC) analysis of oil
Fresh and residual oils (1.0 µL) were analyzed by
GC (Hewlett Packard 5890 Series II) fitted with flame
ionization detector, and AJ & W Scientific DB-1 fused
silica 15 m long column (internal diameter, 0.32 mm;
film thickness, 1.0 µm). The injector and detector
temperatures were maintained at 300oC and 325oC
respectively. The column temperature was programmed
to rise from 50 – 500oC for 27 min.
Results
Enumeration of microbial populations
The frequency of occurrence of HC utilizes relative
to the total heterotrophs is presented in Table 1. It was
observed that the proportions of the HC-utilizing
bacteria within the heterotrophic communities were
generally less than 1.0%. However, the highest
population density of heterotrophs and HC-utilizers
were obtained from sample originating from station I,
but sample III gave the highest percent of degraders.
Utilization of crude oil by indigenous microflora
Figure 1 illustrates the growth dynamics and
population increase of microbial communities
indigenous to the various samples polluted with crude
petroleum as well as the undisturbed stream water. The
layout of the growth patterns indicates that the
population of the microbial communities increased
consistently for the next 12 days before declining.
Samples obtained from sites I and II attained relatively
similar population densities before falling to 1.91 × 107
and 1 × 1010 cfu/mL (cfu = colony forming units)
respectively. However, the highest cell increase was
obtained from the sample originating from site III. The
microbial counts of this sample peaked at day 12 at 1.12
× 1011 cfu/mL and thereafter decreased to 9.12 × 109 on
the 15th day. In the case of the undisturbed control
samples, a consistent decrease in population size was
observed for sample III from the onset of experiment to
the end (Figure 1). In the case of the other samples, a
slight growth was observed between day 0 and day 6
after which it dropped sharply. This is likely due to
continued cell division by the robust inoculum or
utilization of endogenous substrates or exogenous
nutrients in the water. In the experimental samples, the
increase in population of microbial communities was
accompanied by visual gradual decrease in crude oil and
total disappearance on day 15.
Identification of bacterial strains
The enrichment of the water samples with crude oil
resulted in the isolation of nine bacterial strains. The
organisms were identified by morphological and
biochemical techniques using the taxonomic scheme of
Bergey’s Manual of Determinative Bacteriology (Holt
et al., 1994), as Pseudomonas fluorescens, P.
aeruginosa, Bacillus subtilis, Bacillus sp., Alcaligenes
sp., Acinetobacter lwoffi, Flavobacterium sp.,
50
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
Micrococcus roseus, and Corynebacterium sp.
However, only three of these isolates namely
Corynebacterium, Acinetobacter lwoffi and
Pseudomonas aeruginosa were selected for further
studies as representative pure cultures. The mixed
culture used for the biodegradation studies consist of an
assemblage of the nine HC-degrading bacterial isolates.
Growth characteristics on petroleum hydrocarbons
The ability of the selected strains and the defined
consortium to grow on spectrum of hydrocarbon
substrates was tested in MSM amended with selected
carbon substrate as the sole source of carbon and
energy. Incubation was carried out at room temperature
on a gyrating shaker incubator for 7 – 14 days. In our
systems, growth was defined as increase in turbidity and
TVC, reduction in residual oil concentration determined
gravimetrically as well as disappearance of individual
HC peaks by GC analysis. The mixed bacterial culture
grew on all the HCs tested though to varying degrees. In
the case of pure strains, growth was sustainable on long
chain n-alkane, dodecane; petroleum fractions including
kerosene, diesel, AP SAE 40 lubricating oil and crude
oil. All the strains failed to utilize, benzene, naphthalene
and toluene with exception of A. lwoffi which showed a
slight growth on the former aromatic. Similarly, growth
of the isolates was not sustainable on hexane and
cyclohexane.
Changes in pH and population counts of bacterial
stains as well as percent degradation in respect of
residual oil concentration during utilization of crude oil
and petroleum cuts are illustrated in Figures 2 – 4. All
the organisms including the mixed cultures grew on
these substrates without lag phase. In the mixed culture
system, relatively high turbidity was observed in less
than three days of incubation while it was longer in the
pure culture systems. The oil layers in all cases were
slowly emulsified and eventually disappeared with
incubation. The growth of the organisms on the PHC
substrates generally resulted in a gradual decrease in pH
of the culture medium contaminant with a systematic
increase in TVC with P. aeruginosa exhibiting it
characteristic pigment on diesel and kerosene.
During degradation of crude oil, more than 60% of
the oil was depleted by the pure strains during the 20-
day incubation period. Mean generation times ranged
between 1.97 and 2.92 days (Table 2). Expectedly, a
more extensive utilization was accomplished by the
mixed bacterial culture. The defined consortium
achieved the highest growth yield (0.49 day-1) while
nearly 90% of the crude petroleum was utilized (Figure
2, Table 2) within the 14-day cultivation period.. The
results obtained from degradation of diesel followed
almost similar sequence with those of crude oil. The
mean generation time ranged insignificantly (P < 0.05)
from 2.03 to 2.27 days for the pure strains (Table 2).
The growth dynamics show a consistent increase in
TVC from the day 0 until day 14 (Figure 4) after which
it declined. For instance, cell of A. lwoffi increased from
1.2 × 105 to 7.59 × 108 cfu/mL at day 14 after which it
decreased gradually apparently due to depletion in
substrate level or accumulation of toxic metabolites. In
the case of kerosene, generation times were slightly
higher than crude and diesel oils. Similarly, cell increase
was relatively less (i.e., 3 – 4 order of magnitude) while
percent degradation obtained was below 80%. Growth
rates obtained for the consortium and pure cultures
ranged from 0.32 – 0.35 day-1 with the highest obtained
for the mixed culture system. Generally, it would appear
that diesel was utilized best on the basis of growth data
obtained even though the highest degradation rate was
obtained when isolates were grown with crude oil. One
phenomenon worthy of note is that in spite of the
apparent decrease in cell densities observed in all flasks,
percent degradation increased consistently (Figures 2
and 3).
Also biomarkers namely, nC17/pristane and
nC18/phytane ratios extrapolated from GC profiles
decreased at a faster rate in the flasks containing mixed
cultures than pure cultures (Table 3). The former ratio
decreased by 89% between 0 and 20 days during growth
of P. aeruginosa with crude oil. The equivalent decrease
in A. lwoffi for the same period was 30%. For the latter
ratio, it was initially 6.14 but decreased to 0.38 and 2.41
respectively in flasks inoculated with P. aeruginosa and
A. lwoffi. The corresponding values for diesel were 1.07
and 0.24 respectively for both isolates (Table 3). In
contrast to these observations, growth of the consortium
on both crude and diesel oils resulted in complete lost of
all alkane peaks including the isoprenoids in 14 days.
Although complete lost of n-alkane and isoprenoid
peaks was also observed during growth of
Corynebacterium sp. with crude oil, however, this was
not achieved until after 20 days of incubation (Table 3).
The qualitative changes in the HC profiles inherent in
kerosene were also revealed by GC analysis. When the
mixed culture was grown with this substrate, similar to
observations on crude and diesel oils, there was total
disappearance of all detectable alkane peaks in less than
14 days (Chromatogram not shown) while
representative peaks were still detectable after 20 days
of growth with pure strains though significant reduction
in concentration was nonetheless obtained.
51
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
52
Table 1. Population counts of heterotrophic and HC-utilizers in water samples
Sample Total heterotrophs
(cfu × 107)
Total HC-utilizers
(cfu × 104)% HC counts
I 4.5 5.7 0.124
II 2.53 1.3 0.051
III 1.53 5 0.327
Table 2. Growth potentials of hydrocarbon-utilizing bacterial strains
Crude oil Diesel Kerosine
Isolate Tg
a
µ
b
% oil
degradation Tg
a
µ
b
% oil
degradation Tg
a
µ
b
% oil
degradation
Corynebacterium
sp. 1.97 0.35 62.8 2.03 0.34 61.43 2.09 0.33 61
Acinetobacter
lwoffi 2.07 0.34 64.7 2.11 0.33 64.03 2.39 0.32 63.3
Pseudomonas
aeruginosa 2.92 0.32 65.8 2.27 0.31 60 2.07 0.34 65.4
Mixed culture 1.4 0.49 88.1 1.21 0.51 85.3 1.99 0.35 77.8
aMean generation time; bspecific growth rate.
Table 3. nC17/Pristane and nC18/Phytane Ratios of Recovered Hydrocarbons During Growth of Bacterial Strains
Crude oil Diesel
Day 14 Day 21 Day 14 Day 21
Isolate nC17/pris
a
ratio
nC18/phy
b
ratio
nC17/pris
ratio
nC18/phy
ratio
nC17/pris
ratio
nC18/phy
ratio
nC17/pris
ratio
nC18/phy
ratio
Corynebacterium
sp. 1.76 (18.56) 4.46 (27.36) 0 (100) 0 (100) 0.67 (50.74) 1.11 (67.73) 0.34 (75) 1 (70.93)
Acinetobacter
lwoffi 1.77 (18.09) 2.91 (52.61) 1.51 (30.13) 2.41 (60.75) 1.07 (21.32) 2.37 (31.11) 0.06 (95.59) 1.07 (68.9)
Pseudomonas
aeruginosa 0.87 (59.74) 0.76 (87.62) 0.24 (88.89) 0.38 (93.81) 0.4 (70.59) 0.67 (80.52) 0.1 (92.65) 0.24 (93.02)
Mixed culture 0 (100) 0 (100) 0.08 (94.12) 0.11 (96.8)
anC17/pristane ratio; bnC18/phytane ratio. Percent reduction of ratios (written in parentheses) have been calculated
with reference to the amount recovered from uninoculated control tubes.
0 3 6 9 12 15 18
2.2
4.0
5.8
7.6
9.4
11.2
Time, day
Log , TVC (c fu/ml)
Figure 1. Crude Oil Degradation by Microbial Communities in the
Polluted Streams. ■, I; ▲, II; ● III. Respective control samples are
represented with open symbols.
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
53
0246810 12 14
0.0
1.8
3.6
5.4
7.2
9.0
10.8
0
20
40
60
80
100
Time, day
Log, TVC (cfu/ml); pH
% degradation
0246810 12 14
0.0
1.8
3.6
5.4
7.2
9.0
10.8
0
20
40
60
80
100
Time, day
Log, TVC (cfu/ml); pH
% degradation
B
0246810 12 14
0.0
1.8
3.6
5.4
7.2
9.0
0
20
40
60
80
100
Time, day
Log, T VC (cfu/ml); pH
% degradation
C
Figure 2. Degradation of Crude Oil (A), Diesel (B) and Kerosene (C) by Defined Microbial Consortium. ●, log TVC
(cfu/ml); ▲, percent degradation; ■, changes in pH.
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
54
036912 15 18 21
0.0
1.8
3.6
5.4
7.2
9.0
0
20
40
60
80
100
Time, day
Log, TVC (cfu/ml); pH
% degradation
A
0 3 6 9 12 15 18 21
0.0
1.8
3.6
5.4
7.2
9.0
0
20
40
60
80
100
Time, day
Log, T VC (cfu/ml); pH
% degradation
B
0 3 6 9 12 15 18 21
0.0
1.8
3.6
5.4
7.2
9.0
0
20
40
60
80
100
Time, day
Log, TVC (cfu/ml); pH
% degradation
C
Figure 3. Degradation of Crude Oil by Pure Cultures of Acinetobacter lwoffi (A), Corynebacterium sp. (B) and
Pseudomonas aeruginosa (C). ●, log TVC (cfu/ml); ▲, percent degradation; ■, changes in pH.
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
0 3 6 9 12 15 18 21
4.5
5.5
6.5
7.5
8.5
9.5
Time, day
Log, TVC (cfu/ml); pH
A
0 3 6 9 12 15 18 21
4.5
5.5
6.5
7.5
8.5
9.5
Time, day
Log, TVC (cfu/ml); pH
B
Figure 4. Growth Dynamics of Pure Cultures on Diesel (A) and Kerosene (B). Log TVC (cfu/ml) of Pseudomonas
aeruginosa, ●; log TVC of Acinetobacter lwoffi, ▲; open symbols represent respective changes in pH values of the
culture media.
Discussion Discussion
Land polluted with petroleum hydrocarbons (PHCs)
is a major problem throughout the world. Apart from
forming breeding centres for mosquitoes, the vector of
the causative agent of many tropical diseases such as
malaria, many economic plants and marine lives are
destroyed by oil pollution (Nwankwo and Irrechukwu,
1987). The need to clean-up these sites has led to the
development of effective bioremediation techniques
(Hopper, 1989). One of such involves the use of
competent microbial flora as inoculum to degrade the
contaminant by a process referred to as
bioaugmentation. Crude oil in the environment is
primarily biodegraded by bacteria and fungi, which all
appear to be ubiquitously distributed in aquatic and
terrestrial ecosystems. Microbial degradation of crude
oil is often shown to occur by attack on alkanes or light
aromatic fractions, while the higher molecular weight
aromatics, resins and asphaltenes are considered
recalcitrant (Lal and Khanna, 1996). The results
obtained during substrate spectrum analysis and growth
studies are comparable to these findings. The three
isolates were all long-chain n-alkane utilizers. Usually,
the use of crude oil or refined petroleum as substrates
for enrichment has often led to the isolation of
microorganisms that metabolize n-alkanes (Atlas,
1992). Although other substrates such as naphthalene,
cyclohexane, toluene that readily supported the growth
of the mixed culture were recalcitrant to the pure strains,
however, studies on individual HCs has been shown not
to be necessarily a good guide to the microbial response
to mixtures as obtained in crude oil and other petroleum
cuts, since there are several reports of co-metabolism
(Mechalas et al., 1973; Atlas, 1995; Ko and Lebeault,
1999).
Land polluted with petroleum hydrocarbons (PHCs)
is a major problem throughout the world. Apart from
forming breeding centres for mosquitoes, the vector of
the causative agent of many tropical diseases such as
malaria, many economic plants and marine lives are
destroyed by oil pollution (Nwankwo and Irrechukwu,
1987). The need to clean-up these sites has led to the
development of effective bioremediation techniques
(Hopper, 1989). One of such involves the use of
competent microbial flora as inoculum to degrade the
contaminant by a process referred to as
bioaugmentation. Crude oil in the environment is
primarily biodegraded by bacteria and fungi, which all
appear to be ubiquitously distributed in aquatic and
terrestrial ecosystems. Microbial degradation of crude
oil is often shown to occur by attack on alkanes or light
aromatic fractions, while the higher molecular weight
aromatics, resins and asphaltenes are considered
recalcitrant (Lal and Khanna, 1996). The results
obtained during substrate spectrum analysis and growth
studies are comparable to these findings. The three
isolates were all long-chain n-alkane utilizers. Usually,
the use of crude oil or refined petroleum as substrates
for enrichment has often led to the isolation of
microorganisms that metabolize n-alkanes (Atlas,
1992). Although other substrates such as naphthalene,
cyclohexane, toluene that readily supported the growth
of the mixed culture were recalcitrant to the pure strains,
however, studies on individual HCs has been shown not
to be necessarily a good guide to the microbial response
to mixtures as obtained in crude oil and other petroleum
cuts, since there are several reports of co-metabolism
(Mechalas et al., 1973; Atlas, 1995; Ko and Lebeault,
1999).
As depicted in Figures 2 – 4, all the isolates
including the consortium grew on all the HC substrates
logarithmically concomitant with reduction in residual
oil concentration and pH of the culture fluids. The
growth dynamics may either be due to the constitutive
nature of HC assimilating capabilities in the organisms
or reflects the adaptation of the strains as a result of
previous exposure to exogenous HCs. This may be
followed by a concomitant development of the ability to
use the oil and/or its catabolic products as carbon and
energy sources. Usually microbial utilization of HCs
often leads to production of organic acids (Amund and
Adebiyi, 1991; Okpokwasili and James, 1995). Thus,
the acids probably produced account for the reduction in
pH levels. The mixed culture exhibited a superior
degradative competence on all the HC substrates tested
than the pure bacteria strains (Figs. 2 – 4, Tables 1 and
2). Generally mixed cultures have been most commonly
found to degrade oil (Leahy and Colwell, 1990). The
degradation of crude oil by these mixed populations was
reported to be in the range of 21 – 68% using 1g/L of
the crude oil. In the present study, nearly 90%
degradation has been reported at 1 g/L concentration
under 14 days. Hydrocarbon degradation particularly of
crude oil, by heterotrophic consortia has been
As depicted in Figures 2 – 4, all the isolates
including the consortium grew on all the HC substrates
logarithmically concomitant with reduction in residual
oil concentration and pH of the culture fluids. The
growth dynamics may either be due to the constitutive
nature of HC assimilating capabilities in the organisms
or reflects the adaptation of the strains as a result of
previous exposure to exogenous HCs. This may be
followed by a concomitant development of the ability to
use the oil and/or its catabolic products as carbon and
energy sources. Usually microbial utilization of HCs
often leads to production of organic acids (Amund and
Adebiyi, 1991; Okpokwasili and James, 1995). Thus,
the acids probably produced account for the reduction in
pH levels. The mixed culture exhibited a superior
degradative competence on all the HC substrates tested
than the pure bacteria strains (Figs. 2 – 4, Tables 1 and
2). Generally mixed cultures have been most commonly
found to degrade oil (Leahy and Colwell, 1990). The
degradation of crude oil by these mixed populations was
reported to be in the range of 21 – 68% using 1g/L of
the crude oil. In the present study, nearly 90%
degradation has been reported at 1 g/L concentration
under 14 days. Hydrocarbon degradation particularly of
crude oil, by heterotrophic consortia has been
55
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
extensively reported (Leahy and Colwell, 1990;
Okpokwasili and James, 1995). It has been observed
that individual organisms could metabolize only a
limited range of HC substrates (Britton, 1984). This has
also been shown in Brevibacterium and
Flavobacterium, which degrade 40 and 75% of the
crude oil in 12 days respectively, analysis by GC
revealed both isolates were capable of degrading the
aliphatic fractions only (Atlas and Bartha, 1972).
Although our isolates degraded between 60 and 66% of
the crude oil and petroleum cuts, GC analysis showed
that significant peaks of alkane were left undegraded
even after 20 days incubation whereas, all the detectable
HC peaks inherent in crude oil, diesel and kerosene
were completely utilized within 14 days by the defined
bacterial consortium.
In conclusion, this work has shown the occurrence of
various strains of bacteria with capability to utilize
crude oil and petroleum cuts in a polluted tropical
stream. The data reported here supported the premise
that faster rate of degradation of HCs is achieved by the
action of assemblages of pure strains of microorganisms
with overall broad enzymatic capabilities rather than by
a single versatile organisms as seen in Pseudomonas
aeruginosa, Acinetobacter Iwoffi and Corynebacterium
sp. and mixed cultures of unknown composition.
Consortia of unknown composition might result in poor
growth and hence less degradation as documented by
Okpokwasili and James (1994), Lal and Khanna (1996)
and recently by Okerentugba and Ezeronye (2003).
More importantly, the release of enrichment cultures of
unknown composition for bioremediation would have
licensing difficulties due to their effect on the receiving
ecosystem. Therefore it necessary to construct effective
mixed cultures of known microorganisms as
demonstrated in this study for effective bioremediation
strategy.
It can be inferred from the growth data that growth
of the isolates and the mixed culture was most
abundantly supported by crude and diesel oils than
kerosene. Preference for higher molecular weight HCs
has been reported previously (Amanchukwu et al.,
1989; Okpokwasili and James, 1995). The reason for
better growth on crude oil than kerosene in part may be
attributed to the more complex chemical composition of
the oil than diesel and kerosine. A refined petroleum
product such as kerosine contain short chain HCs of 5 –
14 carbon atoms in length, and is less likely to support
growth than crude and diesel oils.
Acknowledgements
We are grateful to Chevron Nigeria Limited for the
opportunity to use their facilities particularly, Mr. Brook
Patterson for his technical assistance. Adebusoye S.A.
gratefully acknowledges Federal Institute of Industrial
Research, Oshodi, Nigeria for a laboratory space.
From the GC profiles of residual oils, reductions in
peaks and in values for biomarkers namely
nC17/pristane and nC18/phytane ratios were much more
pronounced in Pseudomonas aeruginosa than
Acinetobacter lwoffi and Corynebacterium sp. with the
exception of the latter on crude oil where total
disappearance of alkane peaks was obtained after 20-
days cultivation (Table 2). Most importantly, these
chromatographic characteristics correspond to criteria
used to quantify petroleum degradation due to microbial
activities (Wang et al., 1994; Yveline et al., 1997). The
growth of mixed bacteria culture with crude and diesel
oils resulted in the total disappearance of all the
representative HC peaks including pristane and phytane
peaks in 14 days. This revelation however, shows that
these compounds are not as resistant to biodegradation
as once believed. Previously, Ward et al. (1980) also
observed rapid degradation of pristane and phytane by
microorganisms during growth on Amoco Cadiz oil and
therefore cautioned against using them as internal
standards for biodegradation. Similarly in a recent
publication, Ko and Lebeault (1999) observed rapid
degradation of these isoprenoids in an HC mixture by
co-culture of Pseudomonas aeruginosa K1 and
Rhodococcus equi P1, than by pure cultures of these
organisms.
Corresponding Author:
Sunday A. Adebusoye
Faculty of Science
Department of Botany and Microbiology
University of Lagos
Akoka, Yaba, Lagos, Nigeria
Phone: (234) 80-3438-8871
Email: sadebusoye@yahoo.com
Information of Authors
Prof. N. A. Olasupo
Dept of Microbiology,
Lagos State University,
Ojo, Lagos. Nigeria
Tel: +234 802 319 8533
E mail: naolasupo@yahoo.com
Dr. A. O. Olaniran
Dept of Microbiology,
University of Durban – Westville,
P. Bag X 54001, Durban 4000.
South Africa.
Tel: +27 31 204 4401
E mail: adeolaniran@yahoo.com
Dr G. Soberon – Chavex
56
The Journal of American Science, 2(3), 2006, Adebusoye, et al, Microbial Degradation of Hydrocarbons
Dept de Microbiologia Molecular,
Instituto de Biotecnologia,
Universidad Nacional Autonoma de Mexico
Apdo. Postal 510-3, Cuernavaca, Mor 62250, Mexico
Tel: +52 7 3291634
E mail: gloria@ibt.unam.mx
Dr R. Swannell,
National Environmental Technology centre
AEA Technology, 353 Harwell,
Didcot, Oxon OX11 ORA, UK.
Tel: +44 1235 463974
E mail: richard.swannell@aeat.co.uk
Sunday A. Adebusoye
Faculty of Science
Department of Botany and Microbiology
University of Lagos
Akoka, Yaba, Lagos, Nigeria
Phone: (234) 80-3438-8871
Received: June 30, 2006
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