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

. 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 exsitu [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 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.

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].

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 K 2 Cr 2 O 7 [16], Available phosphorus P, calcium Ca, and magnesium Mg (determined using the method of [17], Potassium K (determined using flame photometry).

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.

Isolated fungus
Penicillium sp. (Figure 1) was isolated from spent engine oil contaminated soil and used in this study.

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In vivo bioremediation assay

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.

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was observed to increase tremendously as compared to the control (

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.

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 bioremediate spent engine oil contaminated soil at different concentrations both in vitro and in vivo.

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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.