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*Corresponding author: E-mail: herbert.stanley@uniport.edu.ng;
Asian Journal of Advanced Research and Reports
2(1): 1-7, 2018; Article no.AJARR.44326
Crude Oil Degradation Using Spent Mushroom
Compost (SMC) of Pleurotus florida
H. O. Stanley
1*
, S. N. Maeba
1
, D. K. Gbenekanu
2
and C. J. Ugboma
3
1
Department of Microbiology, University of Port Harcourt, P.M.B 5232, Port Harcourt, Nigeria.
2
Wigweh Transnational Limited, Port Harcourt, Nigeria.
3
Department of Microbiology, Rivers State University, Nkpolu, Port Harcourt, Rivers State, Nigeria.
Authors’ contributions
This work was carried out in collaboration between all authors. All authors read and approved the final
manuscript.
Article Information
DOI: 10.9734/AJARR/2018/44326
Editor(s):
(1) Dr. Sobia Chohan, Assistant Professor, Department of Plant Pathology, Bahauddin Zakariya University, Multan, Pakistan.
(2) Dr. Shu-Lung Kuo, Associate professor, Engineering Consultant, Kelee environmental consultant corporation, Kaohsiung
City, Taiwan And Department of Technology Management, The Open University of Kaohsiung, Kaohsiung City, Taiwan.
Reviewers:
(1)
Debra Jene Kirkconnell Reyes, Mexico.
(2) Graciela Pucci, Universidad Nacional de la Patagonia San Juan Bosco, Argentina.
(3) P. Saravana Kumari, Rathnavel Subramaniam College of Arts and Science, Bharathiar University, India.
Complete Peer review History:
http://www.sciencedomain.org/review-history/26510
Received 11 June 2018
Accepted 28 September 2018
Published 04 October 2018
ABSTRACT
This paper investigated the effectiveness of spent mushroom compost (SMC) of Pleurotus florida in
the biodegradation of crude oil contaminated soil for a period of 42 days. The crude oil contaminated
soil was supplemented with different concentrations of the SMC of P. florida throughout the period of
study. Microbiological and physicochemical parameters including Total Petroleum Hydrocarbon
(TPH) content were monitored from the baseline to the 42
nd
day. Results showed significant
decreases in the physicochemical parameters during the study period. The percentage loss of TPH
at the end of the investigation was 90.09%. The hydrocarbon utilising bacterial isolates were
Bacillus sp, Pseudomonas sp, Flovobacterium sp, Micrococcus sp and Arthrobacter sp. The
hydrocarbon utilizing fungal isolates were Penicillium sp, Fusarium sp, Sacchoromyces sp,
Microsporum sp, Cryptococcus sp and Mucor sp. This study showed that SMC of Pleurotus florida is
an effective nutrient source for biodegradation.
Original Research Article
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
2
Keywords: Biodegradation; total petroleum hydrocarbon; spent mushroom compost; Pleurotus florida.
1. INTRODUCTION
Crude oil exploration has been on-going for
about six decades in the oil-rich Niger Delta
region of Nigeria. Several land sites close to oil
industry facilities in this region have their soil
contaminated beyond the Nigerian national
standards, as set out in the Environmental
Guidelines and Standards for the Petroleum
Industries in Nigeria (EGASPIN). The UNEP [1]
Environmental Assessment of Ogoniland report
revealed that the pollution of soil by
hydrocarbons in Ogoniland is extensive in land
areas, sediment and swampland. Oil spills have
had adverse effects on the region’s ecosystem
leading to adverse environmental pollution and
economic hardship. Oil spill in an area usually
results to an imbalance in the inorganic
components of the affected soil [2].
Bioremediation, a process involving the use of
microorganisms can be adopted to reduce,
eliminate or transform contaminated soils,
sediment, air and/or water [3]. The technology
involves the manipulation of microbial metabolic
processes and enzymatic actions to degrade
compounds of concern in polluted sites. Due to
the ubiquity of hydrocarbon-degradation
microorganisms in the soil, bioremediation of
crude oil contaminated soil is considered of
greater economic value in terms to the cost and
effectiveness.
The use of spent mushroom substrate has been
proposed in bioremediation, as it helps to
stimulate the population of indigenous
microorganisms which breakdown hydrocarbon
compounds [4-6]. Mushrooms make use of
several agro-waste products as growth substrate
leaving behind a nutrient-rich substance that can
be used to promote the growth of crude oil
degrading microorganisms in the soil. This paper
investigated the effectiveness of bioremediation
of crude oil contaminated soil using different
concentrations of SMC of P. florida.
2. MATERIALS AND METHODS
2.1 Study Area
The soil used for this study was collected from K-
Dere community in Gokana Local Government
Area of Rivers State, Ogoniland in the Niger
Delta region of Nigeria. Farming and fishing are
the predominant occupation of the people. There
have been various cases of crude oil pollution in
this area as reported in the Ogoni environmental
assessment report by United Nations
Environmental Programme [1].
2.2 Collection of Samples
Soil samples from crude oil contaminated sites
were collected at about 0-15cm depth, using soil
augar at four different locations within the
community. The four samples were lumped
together in a plastic bag and transported to the
Environmental Microbiology Laboratory of the
University of Port Harcourt for bioremediation
studies. The Spent mushroom compost of
P. florida was collected from the University of
Port Harcourt Demonstration Farm, Choba,
Rivers State, Nigeria.
2.3 Experimental Design
Three treatment options with varying
concentrations of P. florida SMC in polluted soil
and a control were set up. Biodegradation under
a controlled environmental condition was
monitored for 42-days of study. The experimental
design is as shown in Table 1.
2.4 Physicochemical Analysis of Soil
The pH of the soil sample was measured in 1:1
(soil: water) ratio using Winlab digital pH meter.
Moisture content and total organic carbon
content were determined following the methods
of Walkey and Black [7]. Soil Nitrogen and
phosphate were determined quantitatively
following the methods of America Public Health
Association [8]. Gas chromatographic analysis
was conducted to determine the residual total
petroleum hydrocarbon over a 42-day study
period.
2.5 Microbiological Analysis
2.5.1 Enumeration of total heterotrophic
bacteria and fungi
Ten-fold serial dilution of the samples was done;
with one gram of the soil samples weighed out
and dispensed into test tubes containing normal
saline. Each mixture in the test-tube was shaken
thoroughly for proper mixing. 1.0 ml of the
aliquots was pipette into another test tube
containing 9.0 ml of normal saline to give a
dilution of 10
-1
. The sample was diluted up to 10
-
5.
All test tubes were covered with cotton plug to
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
3
Table 1. Experimental design
Experimental set
Test Experiment
Set-up A 1000 g polluted soil + 250 g SMC of P. florida
Set-up B 1000 g polluted soil + 500 g SMC of P. florida
Set-up C 1000 g polluted soil + 1000 g SMC of P. florida
Set-up D (Control) 1000 g polluted soil
prevent further contamination. Aliquots of 0.1 ml
serially diluted soil samples were plated in
duplicates on nutrient agar and potatoes
dextrose agar using spread plate technique. The
plates were incubated at 35±2°C for 24 hours for
bacterial count and between 5-7days fungal
count. Incubation was within 5-7days at 28±2°C
2.5.2 Enumeration of total hydrocarbon
utilizing Bacteria and fungi
Vapour-phase method was adopted to estimate
the population of total hydrocarbon utilizing
bacteria (THUB), on a modified mineral salt agar
(MSA) with the following composition: NaCl=
10.0 g, MgSO
4
= 0.42 g, KH
2
PO
4
= 0.83 g, KCl=
0.29 g, NaNO
3
= 0.42 g, K
2
HPO
4
= 1.25 g, agar =
15.0 g in one litre of distilled water as described
by Chikere and Okpokwasili [9]. A sterile filter
paper was saturated with sterile crude oil and
placed inside the cover of each petri dish, kept in
an inverted position, the plates (containing 0.1 ml
of aliquots of serially dilute soil samples) were
incubated at 35- 37°C for 5-7 days. The crude oil
served as the only source of carbon and energy
for the growing culture. After incubation, the
colonies were counted and the mean counts
were recorded. The same procedure used for the
enumeration of hydrocarbon utilizing bacteria
was adopted for the enumeration of hydrocarbon
utilizing fungi with addition of 1.0 ml lactic acid for
the inhibition of the growth of hydrocarbon
utilizing bacteria.
2.6 Purification of Isolates
Pure cultures were isolated from discrete
colonies by sub-culturing on nutrient agar and
incubated at 35±2°C for 24 hours for bacteria
and 5-7 days for fungi. The isolates were
preserved in slants at 4°C for identification.
3. RESULTS
Baseline microbiological and physicochemical
properties of the crude oil contaminated soil and
P. florida SMC are shown in Table 2. The
baseline microbiological parameters were higher
in the crude oil contaminated soil than in
P. florida SMC except for hydrocarbon utilizing
fungi. The baseline physicochemical parameters
were higher in P. florida SMC than in the
contaminated soil except for TPH which was only
present in the contaminated soil and total organic
carbon.
4. DISCUSSION
The amount of hydrocarbon utilizers in the SMC
and the contaminated soil were considerably
high and adequate for bioremediation. The pH of
SMC has been documented by other researchers
to fall within the range of 5.0 – 8.0 (Fig. 1). The
weak acidic nature of the SMC used in this study
was due to decomposition of organic matter
present in the compost material.
Table 2. Baseline Properties of Contaminated Soil and Spent
P. florida
Compost
Parameter Contaminated Soil P. florida SMC
Total Heterotrophic Bacteria (cfu/g) 1.94 X 10
6
1.43 X 10
5
Total Heterotrophic Fungal (cfu/g) 1.23 X 10
6
7.3 X 10
5
Hydrocarbon Utilizing Bacteria (cfu/g)
8.2 X 10
5
6.6 X 10
5
Hydrocarbon Utilizing Fungal (cfu/g) 5.4 X 10
5
9.3 X 10
5
Total Petroleum Hydrocarbon (mg/kg) 13286.3 -
pH 5.6 6.7
Moisture (%) 11.4 56.7
Total Nitrogen (%) 4.0 21.0
Total Phosphorus (%) 0.71 4.45
Total Organic Carbon (%) 11.8 7.56
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
4
Fig. 1. Changes in pH values of the treatment options during remediation study
The pH value of the various treatment set up
were slightly acidic and alkaline. This may be
attributed to the enhancement or enrichment of
the nutrient levels (by supplementing with various
amounts of spent P. florida compost) of the soil.
The total organic carbon (TOC) content of the
contaminated soil prior to amendment was
11.80%. At the end of day 42, the TOC present
in the sample was 7.40% for Set-up A, 7.40% for
Set-up B, 8.61% for Set-up C experiments
respectively as shown in Fig. 2.
There were slight decreases in total organic
carbon (TOC) concentration in the various set
up. The loss in TOC has been correlated with
biomass increase in microbial systems. Ibiene et
al. [10] and Adenipekun and Ogunjobi [11]
however reported slight increases in TOC in their
bioremediation studies. The total nitrogen
content of the contaminated soil prior to
amendment was 4.0% as shown in Table 2. After
amendment of the experimental soil samples
with various concentration of spent P. florida
compost, the total available soil nitrogen at day
42 of the study period was 7.58% for Set-up A,
9.76% for Set-up B, 11.72% for Set-up C
experiments respectively as shown in Fig. 3.
Also, the total phosphate content of the
contaminated soil after amendment with various
concentration of SMC for set-up A, B and C after
42 days of study increased to 3.96 mg/kg, 5.95
mg/kg and 9.156 mg/kg respectively (Fig. 4)
when compared with the baseline of 0.71 mg/kg.
Fig. 2. Changes in Total Organic Carbon (TOC) concentration of the treatment options during
remediation study
0
1
2
3
4
5
6
7
8
0
14
28
42
pH
Time (Days)
Set-Up A
Set-Up B
Set-Up C
0
5
10
15
20
25
0 14 28 42
Totat Organic Carbon
Concentration (mg/kg)
Time (Days)
Set-Up A
Set-Up B
Set-Up C
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
5
Fig. 3. Changes in nitrogen concentration of the treatment options during remediation study
Fig. 4. Changes in Phosphorus concentration of the treatment options during remediation
study
The result for total petroleum hydrocarbon (TPH)
shown in Fig. 5 revealed a total percentage loss
of 90.09% in Set-up C after the 42 days of study,
a significant decrease with respect to time when
compared to the baseline concentration.
Decrease in TPH indicates the effectiveness of
SMC of P. florida as a very useful bioremediating
organic substance. Stanley et al. [12] and
Adenipekun and Ogunjobi [11] in their study also
reported a very high percentage loss of about
90% of TPH.
The hydrocarbon utilizing bacteria isolated were
both Gram positive and negative bacteria.
Okerentugba et al. [13] reported that Gram
negative bacteria have a dominant population in
crude oil contaminated soil. Bioremediation of
crude oil by spent P. florida yielded favourable
results when compared with mycoremediation.
Spent P. florida compost provide great capacity
to remediate polluted soil when compared to
other members of Pleurotus family previously
and commonly used in bioremediation.
Table 3. Microorganisms isolated from the
contaminated soil and
P. florida
SMC
Bacterial isolate
Fungal isolate
Bacillus sp Penicillium sp
Pseudomonas sp Fusarium sp
Micrococcus sp Microsporum sp
Flavobacterium sp Sacchoromyces sp
Arthobacter sp
0
5
10
15
20
25
0
14
28
42
Nitrogern Concentration (mg/kg)
Time (Days)
Set-Up A
Set-Up B
Set-Up C
0
2
4
6
8
10
12
14
16
0 14 28 42
Phosphate Concentration (mg/kg)
Time (Days)
Set-Up A
Set-Up B
Set-Up C
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
6
Fig. 5. Changes in Total Petroleum Hydrocarbon Carbon (TPH) concentration of the treatment
options during remediation study
5. CONCLUSION
This research has shown the effectiveness of
spent mushroom compost of Pleurotus florida in
bioremediation. The result of the total petroleum
hydrocarbon of 1212 mg/kg and a percentage
loss of 92.09% after 42 days of study shows a
significant decrease with time when compared
with the initial TPH concentration of the
contaminated soil of 13286.3 mg/kg. Therefore,
future work should focus on the bioremediation of
crude oil contaminated sites with optimum
combination of spent Pleurotus florida and other
organic wastes.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
REFERENCES
1. United Nations Environment Programme
(UNEP), Environmental Assessment of
Ogoniland Report; 2010.
2. Adoki A, Orugban T. Removal of crude
petroleum hydrocarbon by heterotrophic
bacteria in soils amended with nitrogenous
fertilizer plant effluent. African Journal of
Biotechnology. 2007;6(13):1529-1535.
3. Sihag S, Pathak H. Factors affecting the
rate of biodegradation of PAHS.
International Journal of Pure and Applied
Bioscience. 2014;2(3):185-202.
4. Ijah UJJ, Ndana M. Stimulated
biodegradation of crude oil in soil amended
with periwinkle shells. The
Environmentalist. 2003;23:249-254.
5. Orji FA, Ibiene AA, Dike EN. Laboratory
scale bioremediation of petroleum
hydrocarbons polluted mangrove swamps
in the Niger Delta using cow dung.
Malaysian Journal of Microbial. 2012;8(4):
219-228.
6. Orji FA, Ibiene AA, Okerentugba PO.
Bioremediation of petroleum hydrocarbon
polluted mangrove swamps in the Niger
Delta using nutrient formula from water
hyacinth. American Journal of
Environmental Science. 2013;9(4):348-
366.
7. Walkley A, Black IA. An examination of the
Degtjareff method for determining organic
carbon in soils: Effect of variations in
digestion conditions and of inorganic soil
constituents. Soil Science. 1934;63:251-
263.
8. American Public Health Association
(APHA): Standard methods for the
examination of water and waste water.
16
th
edition, American Public health
association. Washington, D.C.; 1985.
9. Chikere CB, Okpokwasili GS. Monitoring of
microbial hydrocarbon remediation in the
soil. African Journal of Biotechnology.
2011;1(3):117-138.
10. Ibiene AA, Orji FA, Ezidi CO, Ngwobia CL.
Bioremediation of hydrocarbon
contaminated soil in the Niger Delta using
spent mushroom compost and other
organic wastes. Nigerian Journal of
0
2000
4000
6000
8000
10000
12000
14000
0
14
28
42
TPH Concentration (mg/kg)
Time (Days)
Set-Up A
Set-Up B
Set-Up C
Stanley et al.; AJARR, 2(1): 1-7, 2018; Article no.AJARR.44326
7
Agricultural Food and Environment. 2011;
7(3):1-7.
11. Adenipekun CO, Ogunjobi AA.
Bioremediation of cutting fluids
contaminated soil by pleurotus tuber-
region. The Environmentalist. 2011;32:11-
18.
12. Stanley HO, Offorbuike OM, Stanley CN.
Bioremediation of crude oil contaminated
soil using Pleurotus pulmonarius, a white-
rot fungus. IOSR Journal of Environment
Science, Toxicology and Food Technology.
2017;11(4):122-128.
13. Okerentugba PO, Orji FA, Ibiene AA,
Elemo GN. Spent mushroom compost for
bioremediation of petroleum hydrocarbon
polluted soil: A review. Global Advanced
Research Journal of Environmental
Science and Toxicology. 2015;4(1):001-
007.
_________________________________________________________________________________
© 2018 Stanley 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/26510
