Petroleum Degrading Potentials of Bacterial Isolates from River Ethiope, Delta State, Nigeria

Full text

_____________________________________________________________________________________________________
*Corresponding author: Email: emmaidise@yahoo.com;
British Microbiology Research Journal
6(3): 175-184, 2015, Article no.BMRJ.2015.070
ISSN: 2231-0886
SCIENCEDOMAIN international
www.sciencedomain.org
Petroleum Degrading Potentials of Bacterial Isolates
from River Ethiope, Delta State, Nigeria
Idise Okiemute Emmanuel
1*
, Owhe-Ureghe
Ubreye Benjamin
1
1
Department of Microbiology, Delta State University, Abraka, Nigeria.
Authors’ contributions
This work was carried out in collaboration between all authors. Author IOE designed the study,
performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Author
OUUB managed the analyses of the study as well as the literature searches. All authors read and
approved the final manuscript.
Article Information
DOI: 10.9734/BMRJ/2015/8111
Editor(s):
(1) Giuseppe Blaiotta, Department of Food Science, Via Università, Italy.
Reviewers:
(1) Anonymous, India.
(2)
Boniface Chidi Okoro, Civil Engineering Dept., Federal Univ. of Technology Owerri, Imo state, Nigeria And Head of Dept.,
Building Technology Dept., Sch. Of Environmental Tech., Federal Univ. of Technology Owerri, Imo State, Nigeria
Complete Peer review History:
http://www.sciencedomain.org/review-history.php?iid=829&id=8&aid=7743
Received 28
th
November 2013
Accepted 3
rd
July 2014
Published 10
th
January 2015
ABSTRACT
Water samples were collected from twelve locations along the Ethiope River, Delta State, Nigeria
to determine the petroleum degrading potentials of bacterial isolates and their x-ray mutants using
standard methods. Bacteria isolated were Bacillus sp, Klesiella sp, Escherichia sp, Micrococcus
sp, Pseudomonas sp, Staphylococcus sp and Streptococcus sp. All the isolates and their x-ray
mutants possessed ability to degrade 1% (v/v) Forcados crude-oil with Bacillus sp, Escherichia sp
and Pseudomonas sp isolated from a location, with reported crude-oil spillage, degrading 2.5%
(v/v). X-ray mutation led to % kill that ranged from 3.58 (Micrococcus sp) to 11.59 (Streptococcus
sp). There were reduction in crude-oil degradation in mutants from 0.33% (Escherichia sp) to
6.31% (Pseudomonas sp) with parent Pseudomonas sp (1.680 ppm) and mutant Streptococcus sp
(1.474 ppm) being highest and least degraders respectively. Statistically significant differences
(t= 0.95) in crude-oil degradation were observed for parents and mutants of Streptococcus sp,
Escherichia sp and klebsiella sp but not for Bacillus sp, Staphylococcus sp, Pseudomonas sp and
Micrococcus sp. Thus, irrespective of source of isolation on the Ethiope River, crude-oil degrading
bacteria abound with increased ability on exposure to crude-oil pollution and reduced ability on x-
ray mutation. The bacteria encountered in this study could be employed in bioremediation of crude-
oil polluted environments.
Original Research Article

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
176
Keywords: Bacteria; degradation; mutants; bioremediation.
1. INTRODUCTION
River pollution occurs as a result of
contamination of flowing surface water (s) that
originate from a source. It occurs when waste
and other different pollutants are discharged into
a river without treatment and/or improper
treatment. It has negative effects on aquatic
ecosystem. The negative impacts on plants and
animals often lead to decline in species and
sometimes even to extinctions of entire species.
Water is the best known and most abundant of
all chemical components, which occur naturally
on the surface of the earth or in aquifers under
the ground. Most surface waters are almost
always polluted because they are exposed to
direct contamination from external sources or soil
and basement rocks as it flow from the source
downstream. Biologically, most natural waters
contain a range of microorganisms, plants and
animals that exist in a balanced ecological state.
When this state is altered, it results in fatal
consequences [1].
River Ethiope, a fresh water river in Delta State,
flows through several local Government areas
beginning from Umuaja in Ukwuani, through
Ethiope East, Okpe, Ethiope West, and Sapele
Local Government Areas where it empties into
River Jemison which ultimately discharges into
Benin River. At the Sapele end of the river, it is
deep enough to provide a harbor for ocean-going
vessels. The river serves as a major source of
water for drinking, agriculture and transportation.
Other purposes include fishing, recreation
(beaches) and other domestic purposes at
various locations as there are different human
settlements along the bank of the river. The
communities, through which the Ethiope River
traverses, are mainly peasant farmers whose
food products include yams, corn, vegetables,
cassava, plantain and fruits. Agricultural activities
in the area are also carried out along the bank of
the Ethiope River and agricultural wastes are
discharged into the river as runoffs after rainfalls
[2]. The river also re-distributes these wastes
which may serve as plants/aquatic animal
nutrients. However, a host of these pollutants are
inimical to the ecosystem which may also have
direct or indirect negative impacts on man as a
result of bioaccumulation and/or
biomagnifications [3].
Recently, the river has been reported to be
polluted with crude oil believed to be leaking from
an oil pipeline belonging to NNPC which runs
across the river at Okirighre, near Sapele [4,5].
This and other pollutants may have altered the
bacterial qualities of the river [6]; [7]. It has been
reported that in the Niger-Delta region of Nigeria,
bacterial isolates possessed the ability to
degrade crude oil irrespective of location of
isolation apparently due to flooding of unpolluted
plains with contaminated surface waters with
concomitant exposure of the bacteria therein to
petroleum pollutants during the wet season [8].
The aim of this study therefore was to determine
the potentials of some bacterial isolates of River
Ethiope and their x-ray modified mutants to
degrade Forcados crude oil.
2. MATERIALS AND METHODS
2.1 Collection of Samples
Water samples were collected into sterilized 2L
plastic containers from twelve (12) locations
along the River Ethiope, Delta State, Nigeria for
three months (April – June, 2012) in accordance
with the procedures reported in APHA [9]. The
stations were Umutu Bridge, Umutu-Agbor Road,
Umutu; Obiaruku Bridge, Obiaruku; Uruoka
Bridge, Abraka; Okotie River Resorts, Abraka;
Abraka River Motel, Abraka; Swimming Pool at
the Delta State University, Abraka; Mudi Beach,
Abraka; Canoe anchor points at Sanubi, Eku,
Igun and Okpara Waterside and Okirighre Bridge
near Sapele (See Fig. 1 below, map of study
area). All collected samples were transported in
ice-chest to the Microbiology laboratory at the
Delta State University, Abraka for analyses within
an hour of collection.
2.1.1 Isolation of bacteria
The procedures of pour plate technique reported
in 2004 [10] on Nutrient and Mac Conkey agar
were used. Plates were incubated aerobically in
an incubator (Model TT-9082, Techmel and
Techmel, U.S.A.) at 37°C for 24hours. Pure
cultures obtained were stored at +4°C on agar
slants until required.
2.1.2 Identification of isolates
These were carried out in accordance with the
procedures reported by Cowan and Steel [11] in
which isolates were biotyped for their: Gram
reaction, growth under aerobic and anaerobic
conditions, ability to form endospores, motility,
possession of Oxidase, Catalase and Coagulase

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
177
enzymes, ability to produce H
2
S, ability to utilize
citrate, ferment sucrose, lactose and glucose
with production of gas. Results obtained were
matched with the isolates described by Cowan
and Steel [11].
2.1.3 Determination of crude-oil degrading
potentials of isolates
The procedures reported by Idise et al. [12] were
adopted using 0.1, 0.5, 1.0, 1.5 and 2.5% (v/v)
Forcados crude in minimum basal medium
(MBM). The MBM, made up of Glucose 25 g,
NaNO3 15 g, MgSO4.7H2O 1.3 g, KCl 1.3 g,
KH2PO4 3.8 g, FeSO4. 7H2O 10 mg, ZnSO4.
7H2O 1.0 mg, Agar 37.5 g and 2.5 litres of
distilled water, was prepared and the pH
adjusted from 3.08 to 6.93 and sterilized. 0.1,
0.5, 1.0, 1.5 and 2.5mL of Forcados crude oil
were respectively (aseptically) dispensed into
99.9, 99.5, 99.0, 98.5 and 97.5 mL of molten
MBM in five separate conical flasks. The flasks
were swirled to homogenize the mixtures. Ten
milliliters (10 mL) of the mixtures of each flask
was dispensed into sterile Petri dishes in
duplicates and labeled for each isolate. Lawn
cultures of isolates were prepared in accordance
with procedure adopted by Sharma [13].
Inoculated plates were incubated aerobically at
37°C for 24h and observed for growth.
2.1.4 Mutagenesis of isolates with x-rays
A modification of the procedures reported by
Idise and Ameh [14] was used in which 10 mL of
1.725 Log
10
cfu/mL 24 h-cultures of isolates were
exposed to x-ray (UX Universal mobile X-ray
Machine, Made in England) flashes for five times.
X-ray treated plates were, after each flash,
wrapped in aluminium foil to avoid photo-
oxidation and immediately incubated aerobically
at 37°C for 24 hours to determine viable counts.
2.1.5 Determination of crude-oil degrading
potentials of parents and x-ray mutants
These were carried out in accordance with the
procedures reported by Idise and Ameh [14].
Using one hundred and sixty milliliters of 1% (v/v)
Forcados crude in MBM dispensed into each 250
mL conical flask, 40 mL of 1.685 Log
10
cfu/mL
24h-cultures of each isolate was added to the
respective flasks and incubated aerobically at
room temperature (28±2°C). Separate flasks
were used for parents and their x-ray mutants.
Growth was determined at day zero (one hour
after incubation) and at three-day intervals for 30
days using a spectrophotometer (Model 721-
2000 vmcs, Made in China) at 560 nm.
2.1.6 Determination of total petroleum
hydrocarbons (TPH)
These were determined using ASTM D3921-96
in ASTM Vol. 11 [15] thus: Thirty milliliters of
carbon tetrachloride (CCl
4
) was added to one
liter of the water sample to dissolve the
hydrocarbons and shaken vigorously for two
minutes. The mixture was poured into a
separating funnel and shaken intermittently for
three minutes and expelled the air by releasing
the valves of the funnel. The funnel was then
rinsed with twenty milliliters of carbon
tetrachloride (CCl
4
). The resulting oil was drained
into a fifty milliliter volumetric flask through a
funnel containing cotton wool to which had been
added anhydrous sodium sulphate
(Na
2
SO
4.
7H
2
O) to remove traces of water from
the oil. The separating funnel was then rinsed
with ten milliliters of CCl
4
to remove all the oils
.
The Infra-red spectrophotometer was calibrated
at 420nm using dry Forcados Dry crude oil with
concentrations between 100 and 500 mg/L to
obtain a standard curve. To ten milliliters of the
extracted oil was added 0.5 grams of deactivated
floriscil. The resultant supernatant contained the
total petroleum hydrocarbons (TPH) while the
sediment contained the biogenic hydrocarbons.
The TPH was then determined using the
supernatant on the Infra-red spectrophotometer
(Model Cecil CE 7400, Made in England) at
Optical Density of 420 nm. The concentration of
the sample was extrapolated from the standard
curve. The values were obtained using the
following relation:
TPH (ppm) = Absorbance x DF x 50
Volume of sample
Where:
DF = Dilution factor
50 = initial extraction volume
2.2 Statistical Analyses
Descriptive statistics and t-tests were carried out
on the data obtained using Microsoft excel 1998-
2003 Package.
3. RESULTS
The results of this study (Table 1) show that
seven bacterial genera were isolated from the
different locations along the Ethiope River, Delta

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
178
State, Nigeria. These were Bacillus sp, Klesiella
sp, Escherichia sp, Micrococcus sp,
Pseudomonas sp, Staphylococcus sp and
Streptococcus sp. Two to five genera were
isolated from each sample location.
The crude-oil degrading ability of the bacterial
isolates is presented in Table 2. It was observed
that all the isolates from the sample locations
possessed the ability to degrade 1% (v/v) crude
oil except Bacillus sp, Escherichia sp and
Pseudomonas sp that were isolated from
Okirighre Bridge, Sapele that degraded 2.5%
(v/v) crude oil.
The effects of x-ray mutagenic treatment on
bacterial isolates are presented in Table 3. It was
observed that the % kill ranged from 1.14
(Bacillus sp) to 9.78 (Pseudomonas sp) for one
x-ray flash; 0.65 (Pseudomonas sp) to 6.32
(Klebsiella sp) for two x-ray flashes; 1.21
(Pseudomonas sp) to 8.67 (Klebsiella sp) for
three x-ray flashes; 2.50 (Staphylococcus sp) to
6.18 (Streptococcus sp) for four x-ray flashes
and 0.00 (Micrococcus sp and Pseudomonas sp)
to 13.07 (Staphylococcus sp) for five x-ray
flashes.
The crude-oil degrading potentials of the x-ray
modified bacterial isolates are presented in
Table 4. It was observed that while Escherichia
sp, Bacillus sp and Klebsiella sp degraded 2.5%
(v/v) crude oil, other isolates degraded 1% (v/v)
crude oil.
The mean values of crude-oil degraded by
isolates are presented in Table 5 while the
spectroscopic values are presented in Table 6. It
was observed in Table 5 that parents degraded
more than their mutants with reductions that
ranged from 0.14% in Streptococcus sp to 6.31%
in Pseudomonas sp. Parent Pseudomonas sp
(1.680 ppm) was highest degrader while mutant
Streptococcus sp (1.474 ppm) was least
degrader. While the SD ranged from 0.001 to
0.075, the SE ranged from 0.001 to 0.15.
The values for TPH in Table 6 ranged from 8.378
in parent Staphylococcus sp to 8.526 in parent
Streptococcus sp while it ranged from 8.320 in
mutant Pseudomonas sp to 8.526 in mutant
Streptococcus sp.
The crude oil degradation by parents and
mutants are presented in Table 7a below while
the values of t-test are presented in Table 7b
below. It was observed that mutant
Streptococcus sp (1.47±0.005) was the least
degrader while parent Pseudomonas sp
(1.68±0.05) was the highest degrader. The
statistical analyses in Table 7b below showed
that while parents and mutants of Bacillus sp,
Staphylococcus sp, Pseudomonas sp and
Micrococcus sp showed no statistically significant
differences in crude oil degradation, there were
in parents and mutants of Streptococcus sp,
Escerichia. coli and Klebsiella sp.
Fig. 1. Map of the River Ethiope

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
179
Table 1. Bacteria isolated from locations
S/N
Location
Bacteria
No. of
genera
1. Umutu bridge, Umutu Bacillus sp, Escherichia sp, 2
2. Obiaruku Bridge, Obiaruku Bacillus sp, Escherichia sp 2
3. Uruoka Bridge, Abraka Bacillus sp, Escherichia sp 2
4. Okotie River Resorts, Abraka Klebsiella sp, Micrococcus sp,
Staphylococcus sp, Streptococcus sp
4
5. River Motel, Abraka Bacillus sp, Escherichia sp, Pseudomonas sp,
Staphylococcus
sp,
Streptococcus
sp
5
6. Delta State University Swimming Pool,
Abraka
Escherichia sp Klebsiella sp, Pseudomonas sp,
Staphylococcus sp, Streptococcus sp
5
7. Mudi Beach, Abraka Escherichia sp Klebsiella sp, Pseudomonas sp,
Staphylococcus sp, Streptococcus sp
5
8. Canoe Anchor point Sanubi-Eku Bacillus sp, Escherichia sp 2
9. Canoe Anchor point, Eku Bacillus sp, Escherichia sp 2
10. Canoe Anchor point, Igun Escherichia sp Klebsiella sp 2
11. Canoe Anchor point, Okpara Waterside Bacillus sp, Escherichia sp 2
12. Okirighre Bridge, Sapele. Bacillus sp, Escherichia sp, Pseudomonas sp 3
Table 2. Crude-oil degrading ability of the bacterial isolates
Location
Isolates
0.1%
(v/v)
crude
0.5%
(v/v)
crude
1%
(v/v)
crude
1.5%
(v/v)
crude
2.5%
(v/v)
crude
Umutu Bridge, Umutu Staphylococcus sp
Escherichia coli
Bacillus sp
Streptococcus sp
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
Obiaruku Bridge, Obiaruku Staphylococcus sp,
Bacillus sp
Streptococcus
sp
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
Uruoka Bridge, Abraka Staphylococcus sp,
Bacillus sp
Streptococcus sp
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
Okotie River Resorts, Abraka Staphylococcus sp,
Klebsiella sp
Micrococcus sp
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
River Motel, Abraka Staphylococcus sp,
Escherichia coli,
Bacillus sp
Streptococcus sp
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
Delta State University
Swimming Pool, Abraka
Klebsiella sp
Pseudomonas sp
+
+
+
+
+
+
-
-
-
-
Mudi Beach, Abraka Klebsiella sp,
Escherichia sp
Streptococcus sp
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
Canoe Anchor point Sanubi-
Eku
Staphylococcus sp,
Escherichia coli,
+
+
+
+
+
+
-
-
-
-
Canoe Anchor point, Eku Bacillus sp
Streptococcus sp
+
+
+
+
+
+
-
-
-
-
Canoe Anchor point, Igun Staphylococcus sp
Klebsiella sp
+
+
+
+
+
+
-
-
-
-
Canoe Anchor point, Okpara
Waterside
Bacillus sp
Streptococcus sp
+
+
+
+
+
+
-
-
-
-
Okirighre Bridge, Sapele. Pseudomonas sp
Bacillus sp
Escherichia sp
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Key: + = growth, - = no growth

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
180
Table 3. Effects of x-ray treatment on isolates
Isolate Number of x-ray flashes SD SE
1 2 3 4 5
Bacillus sp
Initial 1.76 1.77 1.77 1.78 1.77
0.007
0.003 Final 1.74 1.66 1.74 1.72 1.73
% kill 1.14 6.22 1.70 3.37 2.26
Klebsiella sp
Initial 1.75 1.74 1.73 1.74 1.73
0.008
0.004 Final 1.60 1.63 1.58 1.65 1.64
% kill 8.57 6.32 8.67 5.17 5.20
Escherichia sp
Initial 1.79 1.80 1.80 1.78 1.81
0.19
0.009 Final 1.71 1.73 1.68 1.71 1.64
% kill 4.47 3.89 6.67 3.93 9.39
Micrococcus sp
Initial 1.76 1.78 1.77 1.79 1.76 0.013 0.006
Final 1.73 1.70 1.73 1.74 1.76
% kill 1.71 4.49 2.26 2.79 0.00
Pseudomonas sp
Initial 1.84 1.55 1.65 1.54 1.70 0.123 0.055
Final 1.66 1.54 1.63 1.50 1.70
% kill 9.78 0.65 1.21 2.60 0.00
Staphylococcus sp
Initial 1.80 1.80 1.60 1.60 1.50 0.134 0.06
Final 1.70 1.70 1.75 1.56 1.76
% kill 5.56 5.56 3.13 2.50 2.67
Streptococcus sp
Initial 1.73 1.82 1.80 1.78 1.76 0.035 0.016
Final 1.72 1.75 1.70 1.67 1.53
% kill 0.58 3.85 5.56 6.18 13.07
Key: SD = Standard deviation, SE = Standard error
Table 4. Crude-oil degrading potentials of the x-ray modified bacterial isolates
Isolate % crude oil degraded Isolate % crude oil degraded
Escherichia sp 2.5 Micrococcus sp 1.0
Bacillus sp 2.5 Klebsiella sp 1.0
Pseudomonas sp 2.5 Staphylococcus sp 1.0
Streptococcus sp 1.0
SE = 0.00 and SD = 0.00
Table 5. Mean values of crude-oil (ppm) degraded by parents and mutants of isolates
Isolate Parent
(ppm)
Mutant
(ppm)
% decrease
SD SE
Bacillus sp 1.579 1.496 5.26 0.006 0.04
Staphylococcus sp 1.652 1.622 1.82 0.02 0.15
Pseudomonas sp 1.680 1.574 6.31 0.075 0.053
Streptococcus sp 1.476 1.474 0.14 0.001 0.001
Escherichia sp 1.536 1.531 0.33 0.004 0.003
Micrococcus sp 1.590 1.538 0.63 0.037 0.026
Klebsiella sp 1.578 1.528 3.17 0.035 0.025
4. DISCUSSION
River Ethiope, a clear fresh water body, which
flows for over 100km to empty into the Benin
River [16], is used extensively for recreational
and occupational activities such as fishing,
swimming, washing, bathing and for domestic
purposes by the inhabitants of villages and towns
along its bank. Aside these, River Ethiope
overflow its bank annually carrying all the
domestic wastes into the river in addition to those
attributable to runoffs during rains. These
wastes, in addition to accidental release of spent
oil and crude oil from spills into the Ethiope
River, probably accounts for the variation in the
number of bacterial genera from the sampled
locations (Table 1). The numbers of isolates

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
181
(genera) were found to be higher in those
locations where recreational activities were high
i.e. in beaches and swimming pools established
along the river bank. The bacterial isolates
obtained in this study - Bacillus sp, Klesiella sp,
Escherichia sp, Micrococcus sp, Pseudomonas
sp, Staphylococcus sp and Streptococcus sp
(Table 1), have previously been reported in fresh
water bodies from Nigeria by Ekhaise and Anyasi
[3], Aluyi et al. [1,6] Duru and Nwanekwu [17]
and Agbabiaka and Oyeyiola [7].
All the isolates obtained were able to degrade
1% (v/v) crude-oil (Table 2) with isolates from
Okirighre bridge, Sapele, where there was a
report of oil spillage, being able to degrade 2.5%
(v/v) crude-oil. These results agree with the
reports by Okoh [8] Addeh [4] of widespread
bacterial species with the ability to degrade
crude-oil, even in unpolluted environments in the
Niger-Delta region of Nigeria. The results also
agree with the observations of Benter-Coker and
Ekundayo [18], Idise and Ameh [14,19], Idise et
al. [12,14,19,20] that microbial isolates from
petroleum oil-polluted soils and water possessed
the metabolic potentials to degrade oil.
The observed reduction in counts, with exposure
to increasing number of x-ray flashes in Table 3,
as well as the percentage of kill of isolates
agrees with reports of Idise and Ameh and Idise
et al. [14,19,20] of same trend with mutational
treatments using x-rays. This could apparently be
due to damages in the DNA of the isolates after
mutation leading to an inversely proportional
relationship between the number of x-ray flashes
and the bacterial counts. Statistical analyses in
Table 3 showed that SD ranged from 0.007
(Bacillus sp) to 0.19 (Escherichia coli) while SE
ranged from 0.003 (Bacillus sp) to 0.055
(Pseudomonas sp).
The observation that the x-ray modified isolates
(mutants) in Table 4 possessed the potentials to
degrade crude-oil, with isolates from the oil-
polluted location having increased potentials,
corroborates the reports of other workers on
increased abilities of isolates after mutational
treatment, as well as increased ability of mutants,
due to enzyme induction Idise and Ameh, [19];
Idise et al. [14,20]. The observed reduction in
ability of mutants to degrade 1% (v/v) crude-oil in
Table 5 could be due to reversal of x- rays
mutational effects by the isolates apparently due
to DNA repair mechanisms. The statistical
analyses in Table 5 supports above results as
while the SD ranged from 0.001 to 0.075, the SE
ranged from 0.001 to 0.053 in Streptococcus sp
and Pseudomonas sp respectively. The
spectroscopic values in Table 6 also corroborate
this reduction in crude-oil degradation by
mutants. The crude-oil degradation by parents
and mutants in Table 7a below as well as their
statistical analyses in Table 7b below both also
support the above findings. These results agree
with the findings of Idise and Ameh [14] who
reported reduced crude-oil degradation by x-ray
mutants of Bacillus sp obtained from motor
spent-oil contaminated soil in Abraka. The results
also support the findings of reduced crude-oil
degradation from 0.14% in Streptococcus sp to
6.31% in Pseudomonas sp (Table 5) with
Pseudomonas sp (1.680 ppm) and mutant
Streptococcus sp (1.474 ppm) being highest and
least crude-oil degraders respectively. However,
while there were no statistically significant
differences in crude-oil degradation by parents
and mutants of Bacillus sp, Staphylococcus sp,
Pseudomonas sp and Micrococcus sp, there
were in parents and mutants of Streptococcus
sp, Escherichia coli and Klebsiella sp.
Table 6. Spectroscopic values
Organism Control Sample TPH
Bacillus sp parent
Bacillus sp mutant
0.00
0.00
16.842
17.042
8.504
8.421
Staphylococcus sp parent
Staphylococcus sp mutant
0.00
0.00
16.696
16.756
8.378
8.348
Pseudomonas sp parent
Pseudomonas sp mutant
0.00
0.00
16.640
16.852
8.426
8.320
Streptococcus sp parent
Streptococcus sp mutant
0.00
0.00
17.048
17.052
8.526
8.524
Escherichia sp parent
Escherichia sp mutant
0.00
0.00
16.928
16.938
8.469
8.464
Micrococcus sp parent
Micrococcus sp mutant
0.00
0.00
16.820
16.924
8.462
8.410
Klebsiella sp parent
Klebsiella sp mutant
0.00
0.00
16.844
16.944
8.472
8.422
Key: TPH = Total petroleum hydrocarbons

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
182
Table 7a. Crude oil degradation by parents and mutants of isolates
Parent
bacillus
Mutant
bacillus
Parent
staph
Mutant
staph
Parent
pseud
Mutant
pseud
Parent
strep
Mutant
strep
Parent
esch
mutant
esch
Parent
micro
Mutant
micro
Parent
kleb
Mutant
kleb
Day 1 1.486 1.523 1.343 1.309 1.339 1.379 1.329 1.419 1.279 1.36 1.516 1.516 1.481 1.515
4 1.28 1.342 1.976 1.997 1.956 1.934 1.087 1.287 1.884 1.158 1.417 1.462 1.275 1.267
7 1.618 1.437 1.432 1.439 1.465 1.382 1.338 1.333 1.282 1.544 1.61 1.682 1.719 1.679
11 1.972 1.826 1.743 1.749 1.803 1.706 1.726 1.704 1.734 1.739 1.745 1.826 1.749 1.702
14 1.633 1.596 1.753 1.77 1.752 1.707 1.716 1.678 1.753 1.712 1.651 1.586 1.664 1.516
17 1.677 1.651 1.69 1.55 1.643 1.51 1.539 1.281 1.426 1.503 1.723 1.583 1.701 1.518
21 1.722 1.647 1.558 1.488 1.74 1.446 1.529 1.439 1.367 1.488 1.735 1.672 1.794 1.772
24 1.557 1.252 1.692 1.649 1.644 1.507 1.25 1.446 1.446 1.533 1.771 1.348 1.539 1.444
27 1.253 1.25 1.53 1.507 1.621 1.448 1.436 1.433 1.46 1.516 1.121 1.255 1.285 1.401
30 1.533 1.401 1.747 1.7 1.743 1.692 1.678 1.608 1.688 1.669 1.504 1.433 1.462 1.403
33 1.638 1.527 1.705 1.683 1.772 1.604 1.626 1.59 1.574 1.615 1.701 1.55 1.689 1.562
N 11 11 11 11 11 11 11 11 11 11 11 11 11 11
Mean (X) 1.58 1.50 1.65 1.62 1.68 1.57 1.48 1.47 1.54 1.53 1.59 1.54 1.58 1.53
SD 0.20 0.18 0.18 0.19 0.17 0.17 0.21 0.15 0.20 0.17 0.19 0.16 0.18 0.15
X±SE 1.58±0.06 1.5±0.05 1.65±0.05 1.62±0.06 1.68±0.05 1.57±0.05 1.48±0.06 1.47±0.05 1.54±0.06 1.53±0.05 1.59±0.06 1.54±0.05 1.58±0.06 1.53±0.05
Key: Staph = Staphylococcus sp, Pseud = Pseudomonas sp, Strep = Streptococcus sp, Esch = Escherichia sp, Micro = Micrococcus sp, Kleb = Klebsiella sp

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
183
Table 7b. Values of t-test for Crude oil degradation by parents and mutants of isolates
Isolate
t-cal
t-crit
Decision
Pearson
correlation
Bacillus sp 2.5976 1.1825 H
o
accepted 0.85
Staphylococcus sp 2.1199 1.1825 H
o
accepted 0.97
Pseudomonas sp 3.8939 1.1825 H
o
accepted 0.86
Streptococcus sp 0.0834 1.1825 H
A
accepted 0.83
E. coli
0.0669
1.1825
H
A
accepted
0.08
Micrococcus sp 1.1413 1.1825 H
o
accepted 0.64
Klebsiella sp 2.0499 1.1825 H
A
accepted 0.89
5. CONCLUSION
Two to five of seven bacteria (Bacillus sp,
Klesiella sp, Escherichia sp, Micrococcus sp,
Pseudomonas sp, Staphylococcus sp and
Streptococcus sp) were isolated from each of the
twelve locations along the River Ethiope. All the
isolates possessed the ability to degrade 1% (v/v)
crude-oil even after mutation using x-rays, with
increased ability on exposure to crude-oil
pollution. However, there were reduced potentials
to degrade crude-oil after x-ray mutation. There
were observed statistically significant differences
in crude-oil degradation by parents and mutants
of Bacillus sp, Staphylococcus sp, Pseudomonas
sp and Micrococcus sp, while no differences were
observed in parents and mutants of
Streptococcus sp, Escherichia coli and Klebsiella
sp. Thus, irrespective of source of isolates on the
Ethiope River, Nigeria, crude-oil degrading
bacteria abound and the bacteria obtained in this
study could be employed in bioremediation of
crude oil pollution on the Ethiope River and/or
other environments.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
REFERENCES
1. Aluyi HSA, Atuanya EI, Amoforitse SC.
Bacteriological and physico-chemical
investigations of Ethiope River, Delta
State, Nigeria. African Journal of Applied
Zoology and Environmental Biology,
2003;5:29–36.
2. Agbaire PO, Obi CG. Seasonal variations
of some physico-chemical propertiesof
River Ethiope Water in Abraka, Nigeria.
Journal of Applied Science
and Environmental Management.
2009;13(1):55-57.
3. Ekhaise FO, Anyasi CC. Influence of
breweries effluent discharge on
the microbiological and physico-chemical
quality of Ikpoba River, Nigeria. African
Journal of Biotechnology. 2005;4(10):1062
–1065.
4. Addeh E. River Ethiope polluted by
massive oil spill. The Punch Newspaper;
2012.
5. Oghre G. River Ethiope polluted by
massive oil spill. In The Vanguard
Newspaper. 2012;10-11.
6. Aluyi HSA, Ekhaise FO, Adelusi DM. Effect
of human activities and oil pollution on the
microbiological and physico-chemical
quality of Udu River, Warri, Nigeria.
Journal of Applied Sciences.
2006;6(5):1214–1219.
7. Agbabiaka TO, Oyeyiola GP. Microbial and
physico-chemical assessment of Foma
River, Ittanmo, Ilorin, Nigeria: An important
source of domestic water in
Ilorinmetropolis. International Journal of
Plant, Animal and Environmental Sciences.
2012;2(1):209–216.
8. Okoh AI. Biodegradation alternative in the
cleanup of petroleum hydrocarbon
pollutants. Biotechnology and Molecular
Biology Review. 2006;1(2):38-50.
9. American Public Health Assessment
(APHA). Standard methods for the
examination of water and waste water, 19
th
edition. Washington DC. 1995;125.
10. Cheesbrough M. District laboratory
practice in Tropical Countries Part II.
Cambridge Low Price Edition. Cambridge
University Press, London. 2004;434.
11. Cowan ST, Steel KT. Manual for
identification of medical bacteria. 2
nd
Edition. Cambridge University Press,
London. 2004;185.
12. Idise OE, Ameh JB, Ado SA.
Petroleum degrading potentials of wild
Pseudomonas aeruginosa and its mutant
strains. Biological and Environtental

Emmanuel et al.; BMRJ, 6(3): 175-184, 2015; Article no.BMRJ.2015.070
184
Sciences. Journal for the Tropics. 2009;
6(1):26-31.
13. Sharma K. Manual of Microbiology: Tools
& Techniques. 2
nd
Ed. Ane books Pvt. Ltd.
New Delhi, India. 2009;405.
14. Idise OE, Ameh JB. Studies on petroleum-
degrading potentials of wild and mutant
strains of Bacillus sp isolated from an oil-
contaminated soil in Abraka, Delta
State, Nigeria. Bayero Journal of Pure and
Applied Sciences. 2010;3(1):176 –179.
15. American Standard Methods (ASTM);
2002;11. Accessed 20/05/13.
Available: http//www.ASTM.com.
16. Omo-Irabor OO, Olobaniyi SB.
Investigation of the hydrological quality of
Ethiope River watershed, Southern
Nigeria. Journal of Applied Science,
Environment and management.
2007;11(2):13–19.
17. Duru M, Nwanekwu K. Physico-chemical
and microbial status of Nworie River,
Owerri, Imo State, Nigeria. Asian Journal
of Plant Science and Research.
2012;2(4):433– 436.
18. Benter-Coker MO, Ekundayo JA.
Applicability of evaluating the ability of
microbes isolated from an oil spill site to
degrade oil. Environmental Monitoring and
Assessment. 1997;45:259-272.
19. Idise OE, Ameh JB, Yakubu SE, Okuofu
CA. Modification of Bacillus cereus and
Pseudomonas aeruginosa from a
petroleum refinery effluent for
increased petroleum product degradation.
African Journal of Biotechnology.
2010;9(20):3303–3307.
20. Idise OE, Ameh JB, Yakubu SE, Okuofu
CA. Assessment of physico- chemical and
microbial characteriustics of refinery
effluent after treatment. Journal of Applied
Science, Environment and Management.
2012;16(1):5–10.
_________________________________________________________________________________
© 2015 Emmanuel et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Peer-review history:
The peer review history for this paper can be accessed here:
http://www.sciencedomain.org/review-history.php?iid=829&id=8&aid=7743