Bioremediation of Spent Engine Oil Contaminated Soil by Using Fungus, Penicillium sp.

Abstract

This study investigated the ability of Penicillium sp. to bio-remediate spent engine oil contaminated soil both in vitro and in vivo. In the in vitro assay, mycelium of a seven day old culture of Penicillium sp. grown on Sabouraud Dextrose Agar (SDA) was punched out using a 0.5mm Cork borer and inoculated on the centre of Petri dishes containing the spent and unspent engine oil and incubated for seven days and daily reading of the mycelia growth obtained using a metre rule. For the in vivo assay, soil received 0 (control), 20/180, 40/360, 60/540, 80/720 and 100ml/900mm concentrations/treatments (inoculation with mycelium of Penicillium sp.). Seeds of Telfeira occidentalis was sown on the soil and assessed for growth performance (plant height, leaf area (using a metre rule) and leaf count (number of leaves) for 7, 14, 21 and 28 Days after Planting (DAP). Results of the in vitro assay showed a significant increase (p<0.05) in the growth diameter of Penicillium sp. relative to control. Results of the in vivo assay showed that spent engine oil had no significant effect (p<0.05) on the growth performance of T. occidentalis at 7, 14, 21 and 28 DAP and on fresh and dry weight (g) 28 DAP relative to control. After 28 days of plant growth, the added spent engine oil was no longer detected. The plant began producing pods 61 DAP. This study showed that Penicillium sp. can biodegrade hydrocarbons present in spent engine oil and as such is a good tool for bioremediation.

Full text

Bioremediation of Spent Engine Oil Contaminated Soil
by Using Fungus, Penicillium sp.
Umana, Etim Johnson1, *Akwaji, Patrick Ishoro1
and Markson, Aniedi-Abasi Akpan1
1Department of Botany, University of Calabar, Calabar, Cross River State, Nigeria.
* akwajiisnever@yahoo.com
Keywords: Bioremediation, spent engine oil, Penicillium sp, soil.
Abstract. This study investigated the ability of Penicillium sp. to bio-remediate spent engine oil
contaminated soil both in vitro and in vivo. In the in vitro assay, mycelium of a seven day old
culture of Penicillium sp. grown on Sabouraud Dextrose Agar (SDA) was punched out using a
0.5mm Cork borer and inoculated on the centre of Petri dishes containing the spent and unspent
engine oil and incubated for seven days and daily reading of the mycelia growth obtained using a
metre rule. For the in vivo assay, soil received 0 (control), 20/180, 40/360, 60/540, 80/720 and
100ml/900mm concentrations/treatments (inoculation with mycelium of Penicillium sp.). Seeds of
Telfeira occidentalis was sown on the soil and assessed for growth performance (plant height, leaf
area (using a metre rule) and leaf count (number of leaves) for 7, 14, 21 and 28 Days after Planting
(DAP). Results of the in vitro assay showed a significant increase (p<0.05) in the growth diameter
of Penicillium sp. relative to control. Results of the in vivo assay showed that spent engine oil had
no significant effect (p<0.05) on the growth performance of T. occidentalis at 7, 14, 21 and 28 DAP
and on fresh and dry weight (g) 28 DAP relative to control. After 28 days of plant growth, the
added spent engine oil was no longer detected. The plant began producing pods 61 DAP. This study
showed that Penicillium sp. can biodegrade hydrocarbons present in spent engine oil and as such is
a good tool for bioremediation.
Introduction
Bioremediation is a process that uses microorganisms such as fungi (mycoremediation) and
green plants to remove contaminants such as oil from the environment, which could be in-situ or ex-
situ [1]. Mycoremediation refers to the use of fungi to clean contaminated soil [2]. According to
[3], to achieve a successful mycoremediation process, fungi must grow and survive in soils
contaminated with oil. In oil contaminated sites, mycoremediation can be applied as a final clean up
measure to further breakdown residual hydrocarbons as well as to improve soil quality [4].
Mycoremediation is a viable method that can be used to bio-remediate areas contaminated with
pollutants because it is affordable and environmentally friendly [5].
According to Achuba et al. [6] and Wang et al. [7] spent engine oil is a brown-to-black liquid
and a mixture of various chemicals such as aliphatic hydrocarbons, aromatic hydrocarbons,
polychlorinated biphenyls, chlorodibenzofurans, lubricative additives, decomposition products and
heavy metal contaminants that come from engine parts as they wear out.
Similarly, Anoliefo et al. [8] reported that there are large amounts of hydrocarbons present in
spent engine oil such as the highly toxic polycyclic aromatic hydrocarbon (PAH). Furthermore, Wei
et al. [9] reported that spent engine oil in soil creates conditions that are unhealthy for plant growth
such as heavy metal toxicity to poor aeration of soil. Odjegba and Sadiq [10] reported that
contamination from spent engine oil is a major environmental challenge and is more widespread
than crude oil pollution. According to Meinz [11] spent engine oil as a petroleum product contains
potentially hazardous chemicals, especially the polycyclic aromatic hydrocarbons (PAHs), heavy
metals and chemicals additives such as amines, phenol and benzenes while Ikhajiagbe and Anoliefo
[12] reported that spent engine oil pollution can affect a vast area when they are carried by run-off
during rainfall to nearby farms and Fetzer [13] reported that chemicals found in oil contaminated
International Letters of Natural Sciences Submitted: 2016-07-20
ISSN: 2300-9675, Vol. 59, pp 82-91 Revised: 2016-10-04
doi:10.18052/www.scipress.com/ILNS.59.82 Accepted: 2016-10-04
© 2016 SciPress Ltd., Switzerland Online: 2016-10-07
SciPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/

soil can cause a reduction in the level of available plant nutrients and a rise to a toxic level of
elements such as manganese.
Over the years, automobiles repairs and maintenance activities have been carried out by auto
mechanics at the Uyo Mechanic Village located at Afa Ofot in Uyo Metropolis of Akwa Ibom
State, Nigeria. The site is well known as a farming area where crops consumed around Uyo
Metropolis, Ediene-Abak, Abak and Ikot Ekpene are harvested. In recent times, complaints by
farmers have been received concerning loss in produce due to land pollution as a result of spent
engine oil released by the auto-mechanics. In view of this and based on the menace caused by oil
pollution on plants, a research on how to reclaim the farmland ex-situ was carried out using
mycelium of Penicillium sp, a fungus also found within these areas. The objective of the present
study therefore, was to test Penicillium sp. for its ability to bio-remediate spent engine oil
contaminated soils both in vitro and in vivo.
Materials and Methods
Sources of Materials
Matured dry fluted pumpkin (Telfeira occidentalis) seeds were obtained in Uyo main market
in Uyo Metropolis, while spent engine oil was obtained from the Uyo Mechanic Village, Afa Ofot
in Uyo Metropolis, both in Akwa Ibom State, Nigeria. Soil sample (Sandy-loam) was obtained from
Afa Ofot, Abak Road in Uyo Metropolis, Akwa Ibom State, Nigeria for soil analysis. The study was
carried out in the Laboratory and Green House of the Department of Botany, University of Calabar,
Calabar, Cross River State, Nigeria.
Source of fungus and morphological identification
The fungus used in this research work was isolated from spent engine oil contaminated soil
collected from the Uyo Mechanic Village, Uyo Metropolis, Akwa Ibom State, Nigeria using Direct
Plate Method (DPM). Approximately 2g of the spent engine oil contaminated soil was placed on
Sabouraud Dextrose Agar (SDA) in Petri dishes. Four (4) sections were inoculated per Petri dish.
The plates were incubated at 28 ± 1°C until fungus growth was noticed. After 5 days, the isolate
was sub-cultured on freshly prepared SDA to obtain pure culture. Isolated fungus was
microscopically (Olympus optical, Phillipines) identified as far as possible using the identification
guides of the International Mycological Institute, Kew [14].
In vitro assay
2ml of spent and unspent engine oil was first poured into different Petri dishes (90mm) using
sterile syringe, and with a sterilized No.2 cork borer of 5.5mm in diameter, a disc of the matured
culture was punched out and inoculated at the centre of plates and incubated at room temperature of
(28±1oC) for 7days. As a control, the fungus was inoculated on Potato Dextrose Agar instead of
spent and unspent engine oil. Three (3) control plates were prepared for each sample. Measurement
of the mycelium growth diameter was obtained daily for seven days using a calliper and metre rule
[15].
In vivo assay
Soil analysis
Sandy-loam soil was collected and analysed at the Research Laboratory of the Department of
Soil Science, University of Calabar, Calabar, Cross River State, Nigeria for percentage moisture,
pH, total Nitrogen N (determined using Kjedahl’s method followed by spectrophotometry
procedure), organic carbon (determined by oxidation with K2Cr2O7 [16], Available phosphorus P,
calcium Ca, and magnesium Mg (determined using the method of [17], Potassium K (determined
using flame photometry).
International Letters of Natural Sciences Vol. 59 83

Soil sterilization
Soil sterilization was conducted in the Department of Botany green house, University of
Calabar, Nigeria under mean temperature of 27˚C. The top soil collected at 0-45cm depth were heat
sterilized in a cut covered metal drum using firewood at 100˚C for 20 minutes and allowed to cool.
The sterilized soil was dispensed into polyethylene bags.
Soil treatment/Soil inoculation
Polyethylene bags were filled with about 5 kilogram (5kg) of the sandy-loam soil treated with
20, 40, 60, 80 and 100ml concentration of spent engine oil. The treatments were replicated thrice
and laid out (Experimental Design) in a Complete Randomized Design (CRD). Soil inoculation was
carried out using the methods of [18]. 180mm (2 Petri dishes) containing seven day old mycelium
of Penicillium sp. grown on PDA was dissolved in 100ml of distilled water and inoculated into the
soil treated with 20ml of spent engine oil, while 360mm (4 Petri dishes) was dissolved in 100ml of
distilled water and inoculated into the soil treated with 40ml of spent engine oil. Soil treated with
60ml of spent engine oil was inoculated with 540mm (6 Petri dishes) dissolved in 100ml of distilled
water, while 80ml was inoculated with 720mm (8 Petri dishes) dissolved in 100ml of distilled water
and 100ml treatment was inoculated with 900mm (10 Petri dishes) of seven day old mycelium of
Penicillium sp. dissolved in 100ml of distilled water. Soil, treatment (spent engine oil) and
mycelium of Penicillium sp. were thoroughly mixed before planting with Telfairia occidentalis
seeds.
Planting of T. occidentalis
Three to four seeds of T. occidentalis were sown in polyethylene bags containing spent engine
oil polluted soil and inoculated with mycelium of Penicillium sp. After seed emergence, the plant
was reduced to two stands per bag. As the plants grew, growth parameters such as plant height
(PH), leaf area (LA), and number of leaves (NL) was collected at 7 Days after Planting (DAP), 14
(DAP), 21 (DAP) and 28 (DAP). Fresh weight (FW) and Dry weight (DW) were collected at 28
(DAP) in three replicates. Frequency of watering was morning and evening.
Statistical analysis
Data obtained in this research work were analysed by one way analysis of variance (ANOVA)
using IBM SPSS ver. 21 and sample means were compared using Least Significant Difference
(LSD) and Duncan multiple range test to obtain significant data.
Results
Isolated fungus
Penicillium sp. (Figure 1) was isolated from spent engine oil contaminated soil and used in this
study.
In vitro bioremediation assay
Results of in vitro bioremediation potentials of Penicillium sp. grown on spent and unspent engine
oil carried out in this study is presented in (Table 1). Results show that the growth diameter of
Penicillium sp. inoculated on spent and unspent engine oil was 1.12 ± 0.04cm, 3.46 ± 0.02cm,
3.57 ± 0.05cm, 3.67± 0.01cm, 3.69 ± 0.03cm, 4.08 ± 0.02cm, 4.50 ± 0.04cm and 0.81± 0.01cm,
2.14 ± 0.04cm, 2.47 ± 0.02cm, 3.31± 0.03cm, 4.07 ± 0.01cm, 4.50 ± 0.02cm and 4.50 ± 0.03cm on
the first to seventh day of incubation respectively while that of the control (Untreated) was
1.29 ± 0.01cm, 2.46 ± 0.03cm, 3.37 ± 0.02cm, 3.49 ± 0.04cm, 3.53 ± 0.02cm, 3.63 ± 0.01cm and
3.73 ± 0.02cm on the first to seventh day of incubation respectively. Results therefore, showed that
Penicillium sp. had a significant effect (p≤0.05) in degrading the hydrocarbons present in the spent
and unspent engine oil relative to control after seven days observation period.
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In vivo bioremediation assay
Soil analysis
Soil analysis revealed the presence of reasonable level of sand (20.2%), silt (51.2%), and Clay
(22.4%) as well as macronutrients Potassium (K) 139mg/kg, Phosphorus (P) 63mg/kg. Nitrogen
(N), organic carbon (C), Magnesium (Mg) and Calcium (Ca) was 38mg/kg, 1.90mg/kg, 136mg/kg
and 107mg/kg respectively. The soil pH was 7.2 as presented in (Table 2).
Fig. 1: Photomicrograph of Penicillium sp. × 40
Table 1: Growth diameter of Penicillium sp. grown on spent and unspent engine oil
for seven days (cm)
Treatments
Days of incubation and mycelium growth (cm)
1
2
3
4
5
6
7
Spent
engine oil
1.12±0.04
3.46±0.02
3.57±0.05
3.67±0.01
3.69±0.03
4.08±0.02
4.50±0.04
Unspent
engine oil
0.81±0.01
2.14±0.04
2.47±0.02
3.31±0.03
4.07±0.01
4.50±0.02
4.50±0.03
Control
1.29±0.01
2.46±0.03
3.37±0.02
3.49±0.04
3.53±0.02
3.63±0.01
3.73±0.02
Least
Significant
Difference
0.98*
Note: n = 3; Mean ± Standard Deviation
Table 2: Soil analysis
Soil constituents
content
Texture (%)
Sand
20.2
Silt
51.2
Clay
22.4
Soil pH
pH
7.2
Nutrients (mg/kg)
Potassium (K)
139
Phosphorus (P)
63
Nitrogen (N)
38
Organic carbon (C)
1.90
Magnesium (Mg)
136
Calcium (Ca)
107
International Letters of Natural Sciences Vol. 59 85

Bioremediation effect of Penicillium sp. on growth performance of T. occidentalis grown in
spent engine oil contaminated soil at the different concentrations
Growth performance of T. occidentalis on spent engine oil contaminated soil at the different
concentrations 0ml (control), 20, 40, 60, 80, and 100mls and treatment levels with mycelium of
Penicillium sp. at 20ml/180mm, 40ml/360mm, 60ml/540mm, 80ml/720mm and 100ml/900mm for
7, 14, 21 and 28 (DAP) is given below.
Plant height (cm)
Results of the mean plant height of T. occidentalis grown on spent engine oil contaminated
soil at the different concentrations inoculated with mycelium of Penicillium sp. is presented in
(Table 3). Results showed that the mean plant height of the control (untreated) 0ml (12.2 ± 0.5) 7
DAP was not significantly greater (p<0.05) than those means for plant grown in spent engine oil
contaminated soil at 20/180 (13.8 ± 0.04), 40/360 (13.7 ±0.03), 60/540 (14.7 ± 0.03), 80/720
(13.3 ± 0.01) and 100ml/900mm (14.5 ± 0.02) concentrations/treatments as presented in (Table 3).
Results therefore, showed that there was no progressive reduction in plant height of T. occidentalis
as the concentration of the spent engine oil increased from 20 to 100ml. At 14 DAP; the mean plant
height of the control 0ml (17.6 ± 0.02) was also not significantly greater than those means for plant
grown in soil at 20/180, 40/360, 60/520 and 80ml/720mm concentrations/treatments except at
100ml/900mm (15.5 ± 0.02) concentration/treatment. However, retardation of growth was not
observed at this treatment level. At 21 DAP, it was observed that the mean plant height of the
control 0ml (20.7 ±0.1) was also not significantly greater (p<0.05) than those plant grown in 20 to
80ml concentrations except at100ml/900mm (13.5 ± 0.02) concentration/treatment. At 28 DAP, it
was observed that the soil became very hard and no trace of spent engine oil was observed on the
surface of the soil. At 28 DAP, the growth of T. occidentalis initially retarded at 100ml
concentration 21 DAP was progressive. This shows that the Penicillium sp. was able biodegrade the
hydrocarbons which initially created a hydrophobic environment limiting water absorption through
the roots. Also at 28 DAP, there was no toxic effect observed on the T. occidentalis as a result of
treatment with spent engine oil, rather the T. occidentalis showed reasonable growth level at the
different concentration/treatment levels as compared with the control. The plant began producing
pods 61 DAP.
Table 3: Mean plant height of T. occidentalis grown in spent engine oil contaminated soil
inoculated with mycelium of Penicillium sp.
Concentration
(ml)/treatment
(mm)
0(control)
20/180
40/360
60 /540
80/720
100/900
Plant height (cm)
7 DAP
12.2 ± 0.5a
13.8± 0.04a
13.1± 0.04a
14.7± 0.03a
13.5± 0.01a
14.5± 0.02a
14 DAP
17.6 ± 0.02b
15.7± 0.03a
15.6± 0.02a
17.4 ± 0.2b
14.1± 0.02a
15.5± 0.02b
21 DAP
20.7 ± 0.1c
19.1± 0.02b
19.1± 0.04b
18.2± 0.03c
18. 8 ± 0.1b
13.5± 0.02a
28 DAP
22.8 ± 0.2d
23.2 ± 0.1c
21.2 ± 0.2c
20.1± 0.01d
19.1± 0.04c
18.2 ± 0.5c
Note: (a, b, c and d are subscript), Values with same subscript in the same column are not
significantly different, but values with different subscript in the same column are significantly
different at (p<0.05)
Leaf area (cm)
Results of the mean leaf area of T. occidentalis grown on spent engine oil contaminated soil at
the different concentrations inoculated with mycelium of Penicillium sp. is presented in (Table 4).
Results showed that at 7 DAP, the mean leaf area of the T. occidentalis grown in spent engine oil
contaminated soil at 20/180 (8.5 ± 0.02), 40/ 360 (9.3 ± 0.02), 60/ 540 (7.7 ± 0.02), 80/720 (8.3 ±
0.03) and 100ml/900mm (8.4 ± 0.001) concentrations/treatments was significantly greater (p<0.05)
than the mean leaf area of the control 0ml (5.1 ± 0.2). At 7 DAP, The leaf area of T. occidentalis
86 Volume 59

was observed to increase tremendously as compared to the control (Table 4). At 14 DAP; the mean
leaf area of the control 0ml (10.6 ± 0.1) was not significantly greater (p<0.05) than the mean leaf
area of those plant grown on 20/180 (10.7 ± 0.01), 40/360 (11.7 ± 0.03), 60/540 (10.3 ± 0.01),
80/720 (10.1 ± 0.02) and 100ml/900mm (10.8 ± 0.03) concentrations/treatment of spent engine oil
contaminated soil. At 21 DAP; there was also no significant difference observed between the mean
leaf area of the control and those plants grown on spent engine contaminated soil at the different
concentrations/treatments. Leaf curl was however observed on some of the plant at higher
concentrations of 80 and 100mls. At 28 DAP, the leaf curl initially observed at 21 DAP was no
longer visible. There was also no significant difference (p<0.05) between the control 0ml (20.1 ±
0.02) and the plant grown on 20/180 (19.2 ± 0.1), 40/360 (18.2 ± 0.05), 60/520 (18.1 ± 0.02),
80/720 (18.2 ± 0.03) and 100ml/900mm (17.9 ± 0.02) concentrations/treatments. Results therefore,
showed that there was a reasonable increase in the mean leaf area of T. occidentalis as compared to
the control.
Table 4: Mean leaf area of T. occidentalis grown in spent engine oil contaminated soil inoculated
with mycelium of Penicillium sp.
Concentration
(ml)/treatment
(mm)
0(control)
20/180
40/360
60 /540
80/720
100/900
Leaf area (cm)
7 DAP
5.1 ± 0.2a
8.5 ± 0.02a
9.3 ± 0.02a
7.7 ± 0.02a
8.3 ± 0.03a
8.4 ± 0.01a
14 DAP
10.6 ± 0.1b
10.7± 0.01a
11.7± 0.03b
17.4 ± 0.2b
10.1 ± 0.02a
15.5± 0.02b
21 DAP
15.0 ± 0.03c
15.8± 0.03b
19.1± 0.04c
14.1± 0.03c
13. 6 ± 0.02b
13.5± 0.02c
28 DAP
20.1 ± 0.02d
19.2 ± 0.1c
21.2 ± 0.2d
18.2± 0.01d
18.2 ± 0.03c
17.9 ± 0.5d
Note: (a, b, c and d are subscript), Values with same subscript in the same column are not
significantly different, but values with different subscript in the same column are significantly
different at (p<0.05)
Leaf count (Number of leaves)
Results of the mean leaf count of T. occidentalis grown in spent engine oil contaminated soil
at the different concentrations inoculated with mycelium of Penicillium sp. is presented in (Table
5). Results showed that at 7 DAP, the mean leaf count of the T. occidentalis grown in spent engine
oil contaminated soil at 20/180 (6.0 ± 0.03), 40/360 (6.0 ± 0.02), 60/540 (6.0 ± 0.02) and
80ml/720mm (7.0 ± 0.02) concentrations/treatments was significantly greater (p<0.05) than the
mean leaf count of the control 0ml (5.1 ± 0.2) except at 100ml/900mm 4.0 ± 0.02
concentration/treatment. At 14 DAP; the mean leaf count of the control 0ml (11.0 ± 0.02) was not
significantly higher than those grown in 20/180 (10.7 ± 005), 40/360 (10.5 ± 0.1), 60/540 (10.1 ±
0.01) and 80ml/720mm (10.2 ± 0.02) except at 100ml/900mm (8.2 ± 0.03) concentration/treatment.
However, at 21 DAP number of mean leaf count of the control 0ml (16.5 ± 0.1) was not
significantly higher (p<0.05) than those plant grown in spent engine oil contaminated soil at
20ml/180mm (16.0 ± 0.2), 40ml/360mm (15.0 ± 0.03), 60ml/520mm (15.0 ± 0.5), 80ml/720mm
(15.3 ± 0.02) and 100ml/900mm (15.0 ± 0.01) concentrations/treatments. At 28 DAP, number of
mean leaf count of the control 0ml (18.2 ± 0.1) was not significantly higher than those grown in
20/180 (18.1 ± 0.03), 40/360 (17.1 ±0.02), 60/520 (17.7 ± 0.2), and 80ml/720mm (17.2 ± 0.1)
concentrations/treatments except at 100ml/900mm (15.0 ± 0.01) concentration/treatment level. It is
noteworthy to state that at 28 DAP, the leaves of T. occidentalis were observed to be evergreen, this
confirms that Penicillium sp. was able to biodegrade the hydrocarbons present in the spent engine
oil contaminated soil and as such T. occidentalis was able to combat stomata and transpiration
problems.
International Letters of Natural Sciences Vol. 59 87

Table 5: Mean leaf count of T. occidentalis grown in spent engine oil contaminated soil inoculated
with mycelium of Penicillium sp.
Concentration
(ml)/treatment
(mm)
0(control)
20/180
40/360
60 /540
80/720
100/900
Leaf count (Number of leaves)
7 DAP
5.0 ± 0.2a
6.0 ± 0.03a
6.0 ± 0.02a
6.0 ± 0.02a
7.0 ± 0.02a
4.0 ± 0.02a
14 DAP
11.9 ± 0.02b
10.7 ± 0.05b
10.5 ± 0.1b
10.0 ± 0.01b
10.2 ± 0.02b
8.2 ± 0.03b
21 DAP
16.5 ± 0.1c
16.0 ± 0.2c
15.0 ± 0.03c
15.0 ± 0.5c
14. 3 ± 0.02c
15.0± 0.01c
28 DAP
18.2 ± 0.1d
18.1 ± 0.03d
17.1 ± 0.02d
17.7 ± 0.2d
17.2 ± 0.1c
15.0± 0.01d
Note: (a, b, c and d are subscript), Values with same subscript in the same column are not
significantly different, but values with different subscript in the same column are significantly
different at (p<0.05)
Fresh weight (g) and Dry weight (g)
Results of the mean fresh weight (g) and dry weight (g) of T. occidentalis grown in spent
engine contaminated soil inoculated with mycelium of Penicillium sp. obtained 28 DAP is
presented in (Table 6). At 28 DAP, mean Fresh weight (FW) (g) of the control 0ml (3.60 ± 0.01)
was not significantly higher (p<0.05) than those that were grown in spent engine contaminated soil
and treated with mycelium of Penicillium sp. at 20/180 (3.56 ± 0.01), 40/360 (3.44 ± 0.01) and
60ml/540mm (3.43 ± 0.0)1 concentrations/treatments except at 80ml/720mm (3.30 ± 0.01) and
100ml/900mm (3.21 ± 0.01) concentration/treatment. There was however, not much progressive
reduction in plant fresh weight as the concentrations/treatments of the spent engine oil increased
from 20 to 100ml and 180mm to 900mm respectively. At 28 DAP, mean Dry weight (DW) (g) of
the control 0ml (2.1 ± 0.01) was also not significantly higher (p<0.05) than those plant grown on
spent engine oil contaminated soil and treated with mycelium of Penicillium sp. at 20/180 (2.1 ±
0.01), 40/360 (2.1 ± 0.01), 60/540 (1.9 ± 0.01) and 80ml/720mm (1.9 ± 0.01)
concentrations/treatments except at 100ml/900mm (1.5 ± 0.01) concentration/treatment. Increase in
the concentration of the spent engine oil slightly reduced the fresh and dry weight of T. occidentalis
at the higher concentrations/treatments but showed no significant difference as compared with the
control.
Table 6: Mean fresh and dry weight (g) of T. occidentalis grown in spent engine oil contaminated
soil inoculated with mycelium of Penicillium sp.
Concentration
(ml)/treatment (mm)
FW (g)
DW (g)
0 (control)
3.60 ± 0.01a
2.1 ± 0.01a
20/180
3.56 ± 0.01a
2.1 ± 0.01a
40/360
3.44 ± 0.01a
2.1 ± 0.01a
60/540
3.43 ± 0.01a
1.9 ± 0.01a
80/720
3.30 ± 0.01b
1.9 ± 0.01a
100/900
3.21 ± 0.01c
1.5 ± 0.01b
Note: (a, b, c and d are subscript), Values with same subscript in the same column are not
significantly different, but values with different subscript in the same column are significantly
different at (p<0.05)
Discussion
Spent engine oil contaminated soil is a major factor limiting the growth and yield of crops and
as such effective management is critical for the profitable production of crops. In this study
Penicillium sp. isolated from spent engine oil contaminated soil obtained at the Uyo Mechanic
village in Afa ofot in Uyo Metropolis of Akwa Ibom State, Nigeria was studied for its ability to bio-
remediate spent engine oil contaminated soil at different concentrations both in vitro and in vivo.
88 Volume 59

Authors like Mandri and Lin [19], Quinones-Aquilar et al. [20] and Bouchez et al. [21] have
reported on fungi that are able to degrade various pollutants while Yateem et al. [22], Juhasz and
Naidu [23], Saraswathy and Hallberg [24], Adekunle et al. [25], Atagana et al. [26], Husaini et al.
[27], Gesinde et al. [28], Obire and Anyanwu [29], and Hadibarata and Tachibana [30] studied the
biodegradation of petroleum products by fungi which is in conformity with this study. Soil borne
fungi such as Penicillium sp. has been reported to produce extracellular enzymes which breakdown
complex carbohydrates and as such make possible the degradation of various pollutants. Romero et
al. [31] reported the ability of Penicillium sp. to remediate pollutants in the presence of salt which is
a useful biological treatment without damage to the physically sensitive ecosystem. Penicillium sp.
was used in this study to test its ability to bio-remediate spent engine oil polluted soil both in vitro
and in vivo. Results of the in vitro bioremediation assay showed a significant increase (p<0.05) in
the mycelia growth of Penicillium sp. relative to the control (Table 2) when inoculated on spent and
unspent engine oil and incubated for seven days. This finding is in conformity with that of
Vanishree et al. [32] who reported on the biodegradation of petrol using Penicillium sp. The
increase rates of mycelia growth of Penicillium sp. fungus on spent and unspent engine oil in this
study might have been due to the fact that the fungus utilized spent engine oil as a medium for its
growth using extracellular enzymes which agrees with the work of Bartha and Atlas [33].
Researchers like Singh [34] listed some genera of fungi that were isolated from an oil polluted
environment which had been demonstrated to contain members that can degrade petroleum
hydrocarbons. Juhasz and Naidu [23], also mentioned some soil borne fungi such as Aspergillus and
Penicillium which were found to be potential degraders of crude oil hydrocarbons while researchers
like Ryan et al. [35] and Srivastava and Thakur [36] reported Fusarium solani, Fusarium
oxysporum, Trichoderma viride and Aspergillus niger grown in acidic medium which also showed
good growth respectively.
In this study, results of the in vivo bioremediation assay using Penicillium sp. mycelium
treated spent engine oil contaminated soil at different concentrations/treatment levels of 20/180,
40/360, 60/540, 80/720 and 100ml/900mm showed that the spent engine oil had no significant
effect (p<0.05) on the growth performance (plant height, leaf area, leaf count (number of leaves) of
T. occidentalis at 7, 14, 21 and 28 DAP and on fresh and dry weight 28 DAP when compared with
the control (0ml) (Tables 3-6). The observed effect of Penicillium sp. treated spent engine oil
contaminated soil on the growth performance of T. occidentalis agrees with the findings of other
researchers like Adekunle et al. [25] that strains of the genus Penicillium are good hydrocarbon
assimilators and that there have the ability to transform xenobiotics compounds like phenol into less
mutagenic products. Workers like Pedro et al. [37] and Abdusalam et al. [38] also reported that
Penicillium sp has the ability to degrade monocyclic aromatic hydro carbons such as benzene,
toluene, ethyl benzene and xylene; BTEX), phenol compounds and heavy metals like lead, nickel
and iron using mono-oxygenases, forming a trans-diol.
Conclusion
This study showed that Penicillium sp. a soil borne fungus can biodegrade hydrocarbons
present in spent engine oil and as such is a good tool for bioremediation.
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