Comparative Studies on Biodegradative Abilities of Pleurotus ostreatus and P . pulmonarius in Soils Contaminated with Crude and Used Engine Oils

Crude and used engine oil degrading ability of two white rot fungi Pleurotus ostreatus and P. pulmonarius were investigated for six months. One hundred grams of sterilized soil moistened with 75% distilled water (w/v) were weighed into 9 × 9 × 4 cm (350 cm3) jam bottles and mixed thoroughly with bonny light crude oil and used engine oil at different concentrations (0%, 5%, 10%, 15%, 25% and 30%), separately. Each bottle was then inoculated with two agar plugs of a vigorously grown mycelium of P. ostreatus and P. pulmonarius using a sterile cork borer. The bottles were incubated at room temperature for 6 months. The mycelia-ramified waste was separated from soils and analysed for physicochemical parameters such as organic matter, carbon, nitrogen, phosphorus, potassium, pH and total hydrocarbon content, (THC) after drying. The organic carbon, nitrogen and phosphorus contents in contaminated and inoculated soils were increased after six months. However, decrease in potassium, pH and THC occurred in these soils after the period of investigation. P. ostreatus reduced the initial THC to 8% and 9% in soils contaminated with 20% of crude and engine oils, respectively, which was lower than that of P. pulmonarius. The two white rot fungi could be exploited in bioremediation of soils contaminated with bonny light crude and used engine oils.


Introduction
Crude oil is a major contaminant of soil and water in oil producing countries as a result of extraction and proc-essing of the oil [1].Crude oil spills from pipelines and refineries cause damage to the environment.Improper disposal and handling of oily sludge contaminate soil and may pose a serious threat to groundwater.The contamination changes the physicochemical and biological properties of the soil because oil may be toxic to some soil microorganisms and plants [2].
Augmentation of contaminated soils with appropriate [1] inocula of microorganisms is a promising technique to enhance the biodegradation of hydrocarbons [3] [4].White rot fungi (WRF) are increasingly being investigated and used in bioremediation, because of their ability to degrade an extremely diverse range of very persistent or toxic environmental pollutants [3].Adenipekun and Isikhuemhen [5] reported that engine oil contaminated soil incubated with Lentinus squarrosulus resulted in an increase in nutrient contents and a high percentage degradation of total petroleum hydrocarbon after 90 days of incubation.The use of fungi is expected to be relatively economical as they can be grown on a number of inexpensive agricultural or forest wastes such as corncobs and sawdust.Moreover, their utilization is a gentle and non-aggressive approach.The application of bioremediation capabilities of indigenous organisms to clean up pollutants is viable and has economic values [6].The present investigation was carried out to determine the biodegrading potentials of P. ostreatus and P. pulmonarius on bonny light crude oil and used engine oil contaminated soils.

Collection of Materials
Mushroom spawns of P. ostreatus and P. pulmonarius were obtained from the Department of Microbiology, University of Ibadan, Nigeria while bonny light crude oil and used engine oil were collected from Port Harcourt and a mechanic workshop along Federal University of Technology, Akure (FUTA) respectively.Soil samples were obtained from FUTA research farm while rice bran was purchased at Oba market, Akure.

Cultivation of P. ostreatus and P. pulmonarius
Twenty grams of pretreated rice bran were laid on the contaminated soil in each bottle, covered with aluminum foil and autoclaved at 121˚C for 15 minutes.The culture conditions were according to the method of Baldrian et al. [7] and modified as follows.A 100 g of sterilized soil moistened with 75% distilled water (w/v) were weighed into 9 × 9 × 4 cm (350 cm 3 ) jam bottles and then mixed thoroughly with bonny light crude oil and used engine oil concentrations (1%, 5%, 10%, 15%, 20%, 25% and 30%) separately.Each bottle was then inoculated with two agar plugs of a vigorously growing mycelium of P. ostreatus and P. pulmonarius using a 7 mm sterile cork borer.The bottles were incubated at room temperature for 3 -6 months.For the control experiment, crude oil and used engine oil were not added to the soils but inoculated with the fungi.At 3 and 6 months after incubation, the mycelia-ramified waste was separated from the soils and analyzed for physicochemical parameters after air-drying.A completely randomized design experiment was used for biodegradation studies and each replicated three times.

Determination of Organic Carbon, Nitrogen, Phosphorus, Potassium and pH Bonny Light Crude Oil and Engine Oil Contaminated Soils after 3 and 6 Months
Organic matter, organic carbon, percentage nitrogen, phosphorus and potassium contents were determined following the method of the Association of Official Agricultural Chemists [8].The soil pH was determined by mixing each soil sample with water in ratio 1:1 and the pH value determined with Jenway 3015 pH meter according to the procedure of Bates [9].

Determination of Total Petroleum Hydrocarbon Content of the Soil
Total petroleum hydrocarbon content of the soil before and after degradation was determined using a Fourier transform infrared spectrometry at the Global Environmental Consultants Laboratory Warri, Nigeria.

Effect of Pleurotus ostreatus and P. pulmonarius on Nutrient Analysis of Bonny Light Crude Oil and Engine Oil Contaminated Soils after 3 and 6 Months
Generally, the organic matter of soil contaminated with crude oil and inoculated with P. ostreatus was higher except at concentration 30% after three months (Figure 1(a)).It was also observed that the organic matter of soil contaminated with from zero to 20% concentration used engine oil and inoculated with P. ostreatus was higher at six months.Also, there was a progressive increase in the organic matter when P. pulmonarius was inoculated after six months (Figure 1

(b)).
As the percentage of crude oil increased, there was increase in percentage carbon (% C) in soils inoculated with P. ostreatus after 3 and 6 months.Percentage carbon also increased in soil contaminated with engine oil and inoculated with P. pulmonarius after 6 months.However, there was slight decrease in % C of all the soils with P. pulmonarius when compared with P. ostreatus after 3 and 6 months of contamination with crude and engine oils (Figure 2 A general decrease was observed in percentage nitrogen (% N) as the concentration of crude oil applied increased in soils treated with P. ostreatus and P. pulmonarius after 3 months.The % N of soils treated with crude oil and inoculated with P. ostreatus was higher than that inoculated with P. pulmonarius after 3 and 6 months (Figure 3(a)).There was a progressive decrease in nitrogen content of soil with increase in concentration of used engine oil and inoculated with P. ostreatus whereas that of P. pulmonarius increased after three months from concentration 5 to 20.Also, the nitrogen content of soil inoculated with P. pulmonarius was higher than that of P. ostreatus after six months (Figure 3(b)).
Generally, there was increase in phosphorus content of soil samples contaminated with crude oil and inoculated with P. ostreatus and P. pulmonarius after the third and sixth months from concentrations 5% to 20% although the phosphorus content of P. ostreatus inoculated soil was higher (Figure 4(a)).Similar trend was ob- served in soil treated with engine oil and inoculated with P. ostreatus and P. pulmonarius after 6 months (Figure 4(b)).
The potassium content of soils contaminated with crude oil and inoculated with P. pulmonarius was higher than that of P. ostreatus up to concentration 25% after the third month.However, potassium content of crude oil contaminated soil was higher when P. ostreatus was inoculated after six months although there was decrease in potassium content with increase in concentration of crude oil (Figure 5(a)).The potassium content of soil contaminated with used engine oil increased when P. ostreatus was inoculated after 3 months (Figure 5

(b)).
There was slight increase in pH values of almost all the soils contaminated with crude oil after inoculation with both Pleurotus species at three months although the pH values of P. ostreatus inoculated soils were higher.However, the pH values of P. pulmonarius inoculated soils were higher than that of P. ostreatus from 25% to 30% of crude oil after six months (Figure 6(a)).The pH values of P. ostreatus inoculated soils was higher than that of P. pulmonarius from 10% to 20% of used engine oil as shown in Figure 6(b).
In soils treated with 20% crude oil and inoculated with P. ostreatus, the total petroleum content (TPC) reduced from 165,724 to 14,581 during 0 to 6 months.Similar reduction of TPC from 74,816 to 6972 (about 9% of initial TPC) was observed after treatment with 20% engine oil and inoculation with P. ostreatus (Table 1).

Discussion
Bioremediation is a cheap and easy method to reduce oily sludge contamination.This approach does not require the soil to be moved from its site, thus reducing any chance of secondary contamination.Fungi show tremend-  ous diversity and adaptability in utilization of different organic molecule as a carbon source; however their abilities to degrade a specific hydrocarbon as a source of energy and/or biomass may differ [4].Many studies have reported the use of Pleurotus species in bioremediation exercises [7].Ishikuehmen et al. [3] showed that bioremediation of soil contaminated with crude oil was possible, especially when the fungus had been allowed to es- In this study P. ostreatus inoculated on crude oil and used engine oil contaminated soil showed higher nutrient distribution of organic matter, carbon and phosphorus compared to the control with increase in crude oil concentration after 3 and 6 months.A lower value of phosphorus and potassium was observed with increase in crude oil concentration.Aislabie et al. [10] reported a low value of nitrogen, potassium, phosphorus reserves in petroleum hydrocarbon contamination.From this study, a reduction in nutrient contents of contaminated soils after introduction of the white-rot fungi was observed at higher levels of 10% -20% crude oil concentration compared to lower levels of contaminated oil.This is similar to the findings of Isikhuemhen et al. [3].Leahy and Colwell [11] also reported that very high concentrations of hydrocarbon will inhibit biodegradation by nutrient or oxygen limitation.Adenipekun and Fasidi [12] also reported the ability of Lentinus subnudus to mineralize soil contaminated with various concentrations of crude oil.They found that nutrient contents were generating higher after 6 months of incubation except potassium levels which were not significantly different from the control.Leahy and Colwell [11] stated that pH is a predominant factor in determining biodegradation in soil.In this work the pH of contaminated soils reduced after the introduction of the white-rot fungi from 6.81 to 6.01 and finally to 5.05 after 3 and 6 months of incubation, respectively.This is similar to the findings of [13] where they observed a decrease from 7.55 to 7.11 for cement polluted soil after 2 months of incubation.This is also in agreement with the report of Benneth et al. [14].
This study revealed that the total petroleum hydrocarbon values were higher on contaminated soils compared to the control suggesting the presence of more petroleum hydrocarbon.Ogbo and Okhuoya [15] reported similar effect in soil in mangrove and rain forest soils polluted with crude oil.The best rate of biodegradation of crude oil by P. ostreatus was after 3 months as similar to the findings of Pointing [16] who reported that incubation for biodegradation takes several months.A decrease in TPH was recorded from 3 to 6 months and this agrees with the observation of Sorkoh et al. [17].The high rate of hydrocarbon degradation by Pleurotus species could have emanated from their massive growth and enzyme production responses during their growth phases as observed by George-Okafor et al. [18] while working with filamentous fungi.This could also be supported by the reports of Bogan and Lamar [19] which showed that extracellular lignolytic enzymes of white rot fungi are produced in response to their growth phases.

Conclusion and Recommendation
Pleurotus ostreatus and P. pulmonarius can be employed in decontaminating environment polluted with crude oil and used engine oil.Further research is, however, advocated in investigating the toxicity of the Pleurotus ostreatus and P. pulmonarius cultivated on bonnylight crude and engine oil contaminated soils before consumption.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .
Figure 1.(a) Percentage organic matter content of soil contaminated with crude oil and inoculated with Pleurotus species; (b) Percentage organic matter content of soil contaminated with engine oil and inoculated with Pleurotus species.A: Soil inoculated with P. ostreatus after 3 months.B: Soil inoculated with P. pulmonarius after 3 months.C: Soil inoculated with P. ostreatus after 6 months.D: Soil inoculated with P. pulmonarius after 6 months.

Figure 5 .Figure 6 .
Figure 5. (a) Potassium content (meq 100 g) of soils contaminated with oils and inoculated with Pleurotus species; (b) Potassium content (meq 100 g) of soils contaminated with used engine oil and inoculated with Pleurotus species.A: Soil inoculated with P. ostreatus after 3 months.B: Soil inoculated with P. pulmonarius after 3 months.C: Soil inoculated with P. ostreatus after 6 months.D: Soil inoculated with P. pulmonarius after 6 months.
colonize the substrate mixed with the soil.

Table 1 .
Total petroleum contents of soils contaminated with oils and inoculated with Pleurotus species.