Sky Journal of Microbiology Research Vol. 4(4), pp. 021 - 030, June, 2016
Available online http://www.skyjournals.org/SJMR
ISSN 2315-876X ©2016 Sky Journals
Full Length Research Paper
Comparative
bioremediation
of
crude
oil
–
contaminated soil samples using activated soil and
activated cow dung
Ikuesan, Felix Adeleke*, Boboye, Bolatito Esther and Adetuyi, Fatusa Clement
Department of Microbiology, The Federal University of Technology, P. M. B., 704, Akure, Ondo State, Nigeria.
Accepted 27 May, 2016
This research studied the relative potentials of activated soil and activated cow dung in bioremediation of crude
oil contaminated soils. Crude oil degrading microorganisms were isolated and identified from soil and cow
dung samples before activation. Activated soil and activated cow dung prepared with 1% (w/w) crude oil were
evaluated for their potentials in bioremediation of three agricultural soil samples of Igodan - Lisa, Oba-Ile and
Idoani in Ondo State, Nigeria. The activated materials were applied at 5 and 10% (w/w) rates to 10% (w/w)
experimentally crude oil contaminated soil samples. Crude oil loss in samples was estimated fortnightly for 16
weeks by the weight loss method using n-hexane as extractant. Klebsiella pneumoniae, Pseudomonas
aeruginosa, Bacillus subtilis, Klebsiella edwardsii, Escherichia coli, Staphylococcus hominis, Actinomyces
bovis, Trichoderma species, Kodamaea ohmeri, Mucor mucedo, Aspergillus species, Scopulariopsis
brevicaulis, Fusarium species were among the crude oil degrading microorganisms obtained from the samples.
The activated cow dung exhibited higher percentage (52.12 – 60.23%) of crude oil loss than activated soil (39.84
- 44.16%) at both 5 and 10% levels of activated materials addition. The results of this study suggest the relative
potentials of activated cow dung in the enhancement of bioremediation of crude oil polluted agricultural soils.
Key words: Crude oil, contaminated soil, bioremediation, activated cow dung enhancement, agricultural soils, Ondo
State.
INTRODUCTION
Crude oil is the economic mainstay of Nigeria. This is
followed by agriculture which is the main occupation of
the people of Ondo State in the Niger- Delta region of
Nigeria. Crude oil contamination of soils and ground
water is frequent in the Niger-Delta region due to human
activities of crude oil exploration, exploitation and
distribution. The problem has been compounded in the oil
producing region of Nigeria due to sabotage and
vandalisation of well heads and flow lines, oil bunkering
*Corresponding author. E-mail: fadelremiks@yahoo.com. Tel.:
+2348033842588.
and negligence etc (Salam et al., 2011; Etuk et al., 2013;
Odeyemi, 2014; Onuoha et al., 2014) and the lack of
effective regulatory environmental laws. The endless
demand for and indiscriminate use and discharge of
petroleum and its derivatives frequently result in pollution
with its concomitant environmental impacts on both biotic
and abiotic components of the environment (Salami and
Elum, 2010; Salam et al., 2011; Odeyemi, 2014).
Soil is a key receiver of crude oil spill as well as many
different types of products and chemicals. Once these
substances enter the soil, they become part of the
biological cycle, thereby affecting all forms of life
supported by the environment (Mbah et al., 2009). The
22
Sky. J. Microbiol. Res.
discharge of petroleum hydrocarbon or its derivatives into
the environment increase the level of its concentration as
environmental pollutants and consequently affecting the
ecosystem until natural processes or human intervention
restores stability.
The impact of crude oil pollution of soil leads to loss of
soil fertility in terms of productive capacity of the polluted
soil, creating nutritional imbalances at the spilled sites
(Chorom et al., 2010) and thus negatively influencing
plant reproduction, eliminating the vegetational cover and
consequently, triggering soil erosion (Merkl et al.,
2005a,b) with overall implications on living organisms and
on the economy of the communities residing in the
polluted area due to the subsequent high unemployment
and increased poverty rate (Etuk et al., 2013).
Pollution of the soil environment with petroleum
hydrocarbon and its derivatives and the consequent
ecological and environmental impacts is a global
concern. Therefore, soil as an essential component of our
natural environment must be protected and sustained in
order to achieve sustainable ecosystem and improve the
livelihood of the people in the risk areas of crude oil
pollution (Adedokun and Ataga, 2007; Adenipekun, 2008;
Etuk et al., 2013; Obasi et al., 2013).
Several physicochemical methods such as incineration,
thermal desorption, coker, solvent extraction, excavation
etc. have been applied to return polluted sites to their
pre- contamination status (Jain et al., 2011). Each of
these physicochemical approaches has limitations
ranging from ineffectiveness to cost of application etc.
For example, incineration is a very effective soil treatment
method, but it is very expensive, leading to loss of
nutritional value and destruction of soil structure (Jain et
al., 2011). Incineration may also lead to inferno with its
attendant hazards and the smoke generated remains
another form of environmental pollution. Apart from
physicochemical methods of contaminant removal from
the environment, biological approaches are also
employed in the remediation of polluted soils (Jain et al.,
2011; Erdogan and Karaca, 2011; Odeyemi, 2014).
Biological method which is also known as bioremediation
appears to have advantage over the physicochemical
methods because of its environmental friendliness,
simplicity, low cost, ability to prevent the accumulation of
contaminants and applicability over large areas (Bento et
al., 2005). Bioremediation is the use of microorganisms to
detoxify or remove organic and inorganic xenobiotic
compounds from the environment (Onuoha et al., 2014).
In this process, microorganisms with the desired
catabolic activities to transform or remove the pollutant
(s) are purposefully used to degrade or remove the
contaminant from the environment. Bioremediation as a
clean-up strategy is not only useful in the recovery of
crude oil contaminated sites, but also in the restoration of
hazardous waste contaminated sites or media (Akinyele
et al., 2011; Amini et al., 2011)
Petroleum hydrocarbons in the environment can be
degraded by bacteria and fungi (Das and Chandran,
2011; Odeyemi, 2014) and are ubiquitously distributed in
the environment. A number of microorganisms with ability
to degrade a wide array of compounds have been
isolated and characterized. Also, a consortium of
microorganisms or supporting materials can be used to
enhance the biodegradation process (Trejo-Hernandez et
al., 2007; Hii et al., 2009; Wang et al., 2010; Zhang et al.,
2010). Okoh and Trejo- Hernandez (2006) reported that
the microorganisms in soil have a broad array of
catabolic activities, and the simple ways of degrading
pollutants are to add the compounds or materials
containing them to the soils and rely on the indigenous
micro flora.
Consequent upon the high degree of success on the
ability of microorganisms to completely degrade or
mineralize petroleum and the safety of bioremediation
approach, it becomes pertinent to source for materials
containing these microorganisms with high potentials to
bioaugment the degradation of crude oil. The ideal carrier
material transfers the microorganisms without affecting
their population or capacity to degrade chemicals.
Therefore, inoculum carrier is one of the primary factors
that affect bioaugmentation. Carriers include agricultural
by products, peat, commercial mushroom, wheat straw,
corncobs, compost, animal dropping etc (Barbeau et al.,
1997). Cow dung is widely available at almost cost - free
in the environment and has the potentials to supply
additional carbon as source of energy for microbial cell
multiplication. Omotayo et al. (2012) reported that the use
of cow dung on crude oil contaminated soils will also
protect the soil structure, provide utilizable nutrients to
indigenous microorganisms thereby enhancing their
activities and further take advantage of crude oil
degrading microbes in the added materials which are not
culturable but important to enhance hydrocarbon
degradation (Ikuesan et al., 2015). Therefore, the use of
acclimated microbial consortium in activated materials
(soil and cow dung) is proposed in this study to evaluate
their potentials in bioremediation of selected crude oil
contaminated agricultural soil samples in Ondo State,
Nigeria. This is because communities exposed to
hydrocarbon become adapted, exhibiting selective
enrichment and genetic changes thereby increasing the
population of crude oil degraders (Odeyemi, 2014).
Therefore, the objective of this research is to (i) isolate
Ikuesan et al.
and identify the crude oil degrading microorganisms in
soil and cow dung and finally, (ii) compare the use of
activated soil and activated cow dung as inoculum
carriers for bioremediation of crude oil contaminated
soils.
MATERIALS AND METHODS
Sample Collection
i.) Soil and cow dung for activation: The soil sample
activated was collected from
o
o
o
Orioke- Iwamimo (6 11’ 0’’N, 4 41 ’0’’ E) in the Mahin (6
o
10’ 0’’N, 4 48’ 0’’ E) area of Ilaje Local Government Area
of Ondo State. This coastline community had witnessed
over thirty four (34) regular crude oil spills of varying
quantities in the last few years. The cow dung sample
used in this study was collected from a cow ranch at
Shasha located along Akure – Owo Road, Akure, Ondo
State, Nigeria. The samples were collected using the
hand auger at a depth of 15 – 20 cm into sterile black
cellophane bags. The samples were then partially airdried at 28 ± 2°C and sieved to uniform consistency using
2 mm sieve.
ii.) The experimentally crude oil contaminated agricultural
soils used for this bioremediation study were collected
o
o
o
from Igodan-Lisa (6 27’ 0’’N, 4 47’ 0’’E), Oba-Ile (7 16’
o
o
o
0’’N, 5 15’ 0’’E) and Idoani (7 17’ 0’’N, 5 52’ 0’’E), all in
Ondo State, Nigeria. Samples were also air-dried and
sieved as earlier described. The physicochemical
properties of the soils and cow dung are reported in
Ikuesan (2015).
Microbiological analysis of samples
Isolation and identification of crude oil degrading
microorganisms
Bacteria and fungi with ability to degrade crude oil were
isolated from the soil and cow dung samples before
activation. One gram of each sample was serially diluted
10
to 10 using nutrient broth as diluents within 48 h of
collection from the field (Onifade et al., 2007). Each
dilution (1 ml) was cultured using pour plate method on
Nutrient Agar (NA), Malt Extract Agar (MEA) and Mineral
Salt Medium (MSM) for the isolation and identification of
Total Heterotrophic Bacteria (THB), Total Heterotrophic
Fungi (THF) and Crude Oil Degrading Bacteria (CDB)
and Fungi (CDF) respectively. All media and diluents
23
were sterilized by autoclaving at 121°C for 15 min
(Nwaogu, 2008; Onuoha et al., 2011; Nduka et al., 2012)
2
at 1.1 kg/cm pressure. Glasswares were sterilized in a
hot air-oven at 160°C for 2 h. The MEA plates for the
cultivation of total heterotrophic fungi were incubated at
O
28±2 C for 7 days (Onifade et al., 2007) while NA plates
(for bacteria) after gelling were incubated at 35°C for 48
h, thus, isolating only aerobic and facultative
heterotrophic microbes. The MSM used was BushnellHass broth incorporated with 1.5% agar (for bacteria),
1.2% agar (for fungi). The media were also fortified with
fungisol (10 mg/l) for bacteria and 50 mg/l of streptomycin
for fungi after sterilization. Crude oil (2%) sterilized using
0.45 µm Millipore filter served as carbon source. The pH
of the culture medium for bacteria and fungi was adjusted
to 7. 2 and 5.6 respectively. The MS-oil medium for crude
oil degrading bacteria and crude oil degrading fungi was
then incubated at 28±2°C respectively for 14 and 21days.
Purified isolates were then characterized and identified
based on their colonial characteristics, cell morphology
and biochemical tests including sugar fermentation. The
data obtained were compared with standards obtainable
in Bergey’s Manual of Determinative Bacteriology (Holt et
al., 1994). Cultural features and microscopic
characteristics described by Onions et al. (1981) and
Barnett and Hunter (1983) were used for the identification
of fungi.
Bioremediation experiment
Soil and cow dung activation
The air – dried and sieved soil and cow dung were
activated with 1% (w/w) crude oil according to the method
of Omosun et al., (2008); Ekpo and Ebeagwu (2009).
These were then incubated at 28 ± 2°C for 21 days to
develop acclimated microbial consortium and thereafter
called activated soil and activated cow dung (Ikuesan,
2015) for the degradation of crude oil in experimentally
crude oil polluted soil samples
Soil microcosms and treatment
The samples used in this study were the agricultural soils
collected from Igodan-Lisa, Oba-Ile and Idoani, all in
Ondo State, Nigeria. 240 g of air-dried and sieved soil
samples were measured into plastic buckets and then
treated with crude oil according to the method of Omosun
et al. (2008); Ekpo and Ebeagwu (2009) to obtain 10%
(w/w) crude oil contaminated soils.
24
Sky. J. Microbiol. Res.
Table 1. Experimental outlay.
Plan
A
B
Experimental outlay
240g of soil sample + 10% crude oil as control (no amendment)
240g of soil sample + 10% crude oil +5% (w/w) activated soil
240g of soil sample +10% crude oil +5% (w/w) activated cow dung
240g of soil sample + 10% crude oil as control (no amendment)
240g of soil sample +10% crude oil + 10% (w/w) activated soil
240g of soil sample +10% crude oil + 10% (w/w) cow dung
Bioremediation of crude oil contaminated agricultural
soil samples using activated soil and activated cow
dung
This study was undertaken between the month of July
and
October
(raining
season)
to
investigate
bioremediation of 10% (w/w) crude oil contaminated soil
microcosms using activated soil and activated cow dung.
Bioremediation of the experimentally crude oil
contaminated agricultural soils were carried out through
the introduction of inoculum carriers (activated soil and
activated cow dung). The inoculum carriers were added
at 5 and 10% (w/w) rates. The experimental outlay is as
shown in Table 1.
The plastic vessels containing the experimentally crude
oil contaminated soils were left undisturbed for 2days to
allow the volatilization of toxic components of the oil
(Abioye et al., 2012). The inoculum carriers (activated soil
sample, activated cow dung) were dissolved in the
sterilized water used for adjusting the moisture content
(25% w/w) of the soil samples and then separately added
as shown in the experimental outlay A and B in Table 1.
The experimental plastic buckets were not covered and
the contaminated soil moistened weekly by the addition
of 10 ml sterile distilled water until the end of the study
(Bento et al., 2005). Triplicate samples of the various
treatment containers were then incubated at 28 ± 2°C
and tilled twice a week with spatula to provide the
necessary aeration (Ayotamuno et al., 2006; Onuoha et
al., 2014) and proper mixing of the inoculum carriers with
the contaminated soils. Crude oil loss in the samples was
estimated by the weight loss method using n-hexane as
extractant. Periodic sampling for crude oil loss from each
container was carried out at 7days post contamination as
day zero (Onuoha et al., 2014), then at 2 weeks interval
for 16 weeks.
Determination of amount of crude oil in samples
The weight loss method described by Nwaogu et al.
(2008); Njoku et al. (2009) was used to determine the
amount of crude oil in samples. The amount of crude oil
in soil samples was determined using air-dried sample to
which crude oil had been added. 20 g portion of each soil
sample was mixed with 40ml of n- hexane as extractant
in a 250 ml Erlenmeyer flask. The flask was then shaken
vigorously with mechanical shaker for 30 min to extract
the oil (Njoku et al., 2009). The soil-crude oil- n- hexane
mixture was allowed to stand for 10 min and then slowly
filtered into a pre-weighed beaker through a Whatmann
No.1 filter paper. Anhydrous sodium sulphate was spread
over the filter paper to remove any moisture present in
the mixture. The solvent (n - hexane) was allowed to
evaporate by gentle heating at 40°C to a constant weight
and the residual crude oil determined. The amount of
crude oil loss from the soil was then determined as the
amount of crude oil added to the soil minus that
recovered in the soil at the time of analysis (Njoku et al.,
2009). This was then expressed as percentage of crude
oil initially present in sample.
Statistical analysis
Data obtained from this study were analyzed by one way
Analysis of Variance (ANOVA) using SPSS version 18.0
(2010).
RESULTS
Microbiological analysis of soil and cow dung
samples (before activation)
The identities of the intrinsic crude oil degrading bacteria
and fungi in the soil and cow dung are shown in Table 2.
Results revealed that the crude oil degrading bacterial
isolates obtained in this study are predominantly gram
negative. Staphylococcus hominis isolated from the cow
faecal deposit was the only cocci crude oil degrader from
the study. Similarly, except for Kodamaea ohmeri and
Ikuesan et al.
25
Table 2. Identities of crude oil degrading microorganisms associated with the
soil and cow dung.
Sample
Soil
Cow dung
Microorganisms
Bacteria
Fungi
Klebsiella pneumoniae
Trichoderma species
Pseudomonas aeruginosa
Mucor mucedo
Flavobacterium species
Pullularia pullulans
Proteus mirabilis
Aspergillus flavus
Bacillus subtilis
Kodamaea ohmeri
Klebsiella edwardsii
Escherichia coli
Mucor mucedo
Enterobacter spp.
Aspergillus flavus
Pseudomonas aeruginosa
Aspergillus glaucus
Pseudomonas pseudomallei Penicillium citrium
Staphylococcus hominis
Aspergillus niger
Bacillus subtilis
Scopulariopsis brevicaulis
Klebsiella pneumoniae
Fusarium species.
Klebsiella edwardsii
Aspergillus terreus
Actinomyces bovis
Meyerozyma carribbica
Note: Detailed characteristics not shown.
Meyerozyma carribbica which are yeasts, the other
fungal isolates are molds.
Bioremediation of crude oil contaminated agricultural
soil samples using activated soil and activated cow
dung
Figures 1, 2 and 3 respectively show the percentage of
crude oil degraded when Igodan - Lisa, Oba - Ile and
Idoani soil samples polluted with 10% (w/w) crude oil
were
variously
bioaugmented
with
inoculum
carriers(activated soil and activated cow dung) containing
directly enriched crude oil degrading microorganisms at 5
and 10% (w/w) application rates. At the end of the
bioremediation experiment of 16 weeks, results revealed
higher amount of crude oil loss of 54.97%, 52.12% and
55.20% (for 5% activated cow dung) and 59.78%,
58.97% and 60.23% (for 10%cow dung) respectively for
Igodan – Lisa, Oba – Ile and Idoani than the 39.84 44.16% obtained with the application of activated soil at
both 5 and 10% application rates and 23.68 – 27.90% of
control samples.
DISCUSSION
The soil and cow dung activated for this bioremediation
study contained diverse genera of crude oil degrading
bacteria and fungi. This suggests that fungi as well as
bacteria are associated with crude oil degradation. The
bacterial isolates obtained in this study belong to both the
gram positive and gram negative groups, although, the
gram negative bacteria predominate in all the samples.
These findings corroborate the report of Salam et al.
(2011) that both gram negative and positive bacteria
have been implicated in the mineralization of
hydrocarbon pollutants. The dominance of gram negative
bacteria in all the samples also agrees with the report of
Kaplan and Kitts (2004) that gram positive bacteria if
detected in bioremediation are never diverse and
dominant. This is suggestive of the fact that the soil and
cow dung are carriers of crude oil degrading
microorganisms and can thus be applied as inoculum
carriers for bioremediation.
The use of activated soil and activated cow dung in
bioremediation showed that more crude oil degradation
occurred in soils both at 5 and 10% (w/w) amendment
rates than unamended samples. This may be due to soils
and cow dung serving as carrier materials that retain
microorganisms in required places to enhance
degradation.
The effects of the amount of supporting materials
(activated soil and activated cow dung) and time were
significant on crude oil degradation. Degradation at 10%
(w/w) amendment rate was higher than in 5% (w/w) for
the activated cow dung. Conversely, the amount of crude
oil degraded was higher in 5% (PSAS) than 10%
(PSAS2) addition for activated soil. This may be due to
26
Sky. J. Microbiol. Res.
Figure 1. Percentage crude oil degraded in Igodan – Lisa soil sample. UPS; Unamended polluted soil, PSAS; Polluted soil +
5% activated soil, PSACD; Polluted soil + 5% activated cow dung, PSAS(2); Polluted soil + 10% activated soil, PSACD(2);
Polluted soil + 10% activated cow dung.Values plotted are means ± standard deviation of triplicate determinations.
Ikuesan et al.
27
Figure 2. Percentage crude oil degraded in Oba – Ile soil sample. UPS; Unamended polluted soil, PSAS;
Polluted soil + 5% activated soil, PSACD; Polluted soil + 5% activated cow dung, PSAS(2); Polluted soil + 10%
activated soil, PSACD(2); Polluted soil + 10% activated cow dung. Values plotted are means ± standard
deviation of triplicate determinations.
increased quantity of clay in the 10% activated soil which
prevents microbial access to the contaminant. Odeyemi
(2014) reported that clay soil protects contaminant and
makes them unavailable for microbial activities. This
progressive increase in the amount of crude oil degraded
could be due to increased microbial population in the
added materials as inoculum carriers. This assertion is in
line with the report of Odeyemi, (2014) that the lack,
scarcity or low population of hydrocarbon degrading
microorganisms in an oil polluted site is a limiting factor to
biodegradation. This implies that the success of
bioremediation of hydrocarbon polluted environments has
a direct relationship with the biodegrading capabilities of
native
microbial
populations
or
exogenous
microorganisms used as inoculants. This result suggests
that addition of supporting materials with directly enriched
acclimated consortium of microorganisms to crude oil
polluted soils enhanced the degradation of crude oil in
the soils. It is therefore suggestive of the fact that the
inherent crude oil degrading population of microbes in the
inoculum carriers including those that are non - culturable
may also have played a role in the enhancement of crude
oil degradation by synergizing with the intrinsic
microorganisms in the polluted soil.
Similarly, there were significant differences among the
various treatments. Degradation of crude oil was higher
in samples with activated cow dung than soils with
activated soil. The enhancement of degradation by
28
Sky. J. Microbiol. Res.
Figure 3. Percentage crude oil degraded in Idoani soil sample. UPS; Unamended polluted soil, PSAS; Polluted
soil + 5% activated soil, PSACD; Polluted soil + 5% activated cow dung, PSAS(2); Polluted soil + 10% activated
soil, PSACD(2); Polluted soil + 10% activated cow dung.Values plotted are means ± standard deviation of
triplicate determinations.
activated cow dung over activated soil may be suggestive
of the fact that cow dung is a better enrichment medium
than soil due to the presence of wide variety and
abundance of crude oil degrading microorganisms, which
developed into acclimated consortium for the degradation
of crude oil and the supply of supplemental nutrients
used for the growth of crude oil degrading microbes,
especially nitrogen, phosphorous and organic carbon
which Ikuesan (2015) reported to be of higher values in
cow dung (N; 3.24%, P; 36.95%, Organic carbon;
30.46%) than the Orioke –Iwamimo soil (N; 1.27%, P;
10.80%, Organic carbon; 0.54%). This is because these
nutrients among other requirements enhance the growth
of microorganisms which leads to more rapid
decomposition of contaminants (Machida and Gomi,
2010). Also, cow dung (the undigested plant matter which
has passed through the animal guts) is known to be rich
in mineral and often used as agricultural manure,
providing food for a wide range of animals and fungus
species and has the abilities to supply additional carbon
as source of energy for cell multiplication, thereby
enhancing microbial degradation.
Conclusion and recommendation
The ability to isolate high number of certain crude oil
degrading microorganisms from the soil and cow dung is
indicative that those microorganisms are the most active
degraders in that environment and can be used in the
bioremediation of petroleum oil contaminated sites.
Therefore, these microbial isolates can be applied singly
or as a consortium for the enhancement of degradation of
crude oil when produced and harvested in large number
as biomass. Also, these carrier materials can be applied
directly (soil and cow dung) as inoculum carriers for the
Ikuesan et al.
enhancement of crude oil bioremediation thereby saving
time, effort and economy.
In the present study, the crude oil removal efficiency of
activated soil and activated cow dung as supporting
materials or inoculum carriers to bioaugment the
remediation of crude oil polluted agricultural soils were
evaluated. The result of bioremediation experiment which
is the main focus of this research shows the effect of the
application of activated soil and activated cow dung as
inoculum carriers on the remediation of three selected
agricultural soil of Igodan- Lisa, Oba-Ile and Idoani
contaminated with 10% (w/w) crude oil. The results
shows that the use of activated cow dung as inoculum
carrier is more effective than activated soil for
bioremediation of crude oil polluted agricultural soil. The
application of this finding will reduce the impact of crude
oil on soil and can help in environmental restoration
measures using cow dung which is widely available to a
large population of farmers almost at a lower cost relative
to other techniques which might be expensive,
unaffordable and difficult to adapt. The use of activated
cow dung takes advantage of indigenous population in
the added materials which are otherwise non- culturable
(unidentified consortia of microbes), but possess the
desired metabolic ability to participate in petroleum
hydrocarbon degradation, has the advantage of time and
economics required for the isolation of degraders as well
as protecting the soil structure, providing utilizable
nutrients for native soil microorganisms for microbial cell
multiplication.
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