Physicochemical characteristics and mycoremediation of Ejamah-Ebubu oil spill site located at Eleme Local Government Area in Rivers State, Nigeria

Mycoremediation is the application of fungi isolate to contaminated sites. The mycological content of Ejamah- Ebubu oil polluted site was carried out. Using composite sampling technique, five sets of samples were collected; At 15 , Bt 15 , Ct 15 , Dt 15 , Et 15 at depth (0 - 15cm) and Ab 30 , Bb 30 , Cb 30 , Db 30 , Eb 30 at depth (15 - 30cm). The parameters analyzed include; pH, conductivity, nitrate, phosphate, sulphate, total heterotrophic fungal count (THF) and total hydrocarbon utilizing fungal count (HUF). The total heterotrophic fungi and hydrocarbon utilizing fungal count for A15 ranges between 5.0 x 10 3 - 1.5x10 4 cfu/g and 1.1 x10 3 - 2.3x10 3 cfu/g while A30 ranges between 4.0 x 10 3 - 1.3x10 4 cfu/g and 3.0 x 10 3 - 1.3x10 3 cfu/g. A total of nine fungal isolate were obtained and identified to belong to the genera: Aspergillus (44.44%), Microsporum (11.11%), Fusarium (11.11%), Penicillium (22, 22%) Acremonium (11.11%). The frequency of occurrence of the isolates have Aspergillus > Penicillium while Microsporium , Fusarium and Acremonium are the same. The unique ability of these isolates to adapt to such conditions of petroleum hydrocarbon content in soil can be effectively used in bioremediation of oil impacted areas in the Niger Delta. Keywords: Mycoremediation, Hydrocarbon, Aspergillus , bioremediation, Impacted, Delta

Mycoremediation is a form of bioremediation in which contaminated sites are converted into less contaminated ones by the use of fungal mycelium (Bennet et al., 2002).It is a complex and technical area of bioremediation.For the last two decades, Mycologists has employed fungal species in the degradation of organic compounds.Mycoremediation involved the mixing of the vegetative part of a fungus (mycelium) into contaminated soil; placing mycelial mats over toxic sites or/and combination of both.Pollution has significantly affected the ecosystem.Over the past few years the soil is getting more and more polluted due to advancement in technology.Remediation of these polluted soils is not an easy job.Mycoremediation technique has been applied to oil spill contaminated and polluted soil, industrial chemicals, contaminated water and even farm waste (Bennet et al., 2002).The cleanup of a contaminated site requires a consortium of microorganisms; bacteria as well as fungi.Fungi have some intrinsic feature that enable them carry out bioremediation.They secret extracellular enzymes, they also have the ability to grow under stress (low nutrient, pH and water capacity) (Obire andAnyanwu, 2009, George et al., 2009).Fungi secretion of extracellular substance during biodegradation initiate primary attack of more complex and recalcitrance pollutants thereby facilitating secondary attack by bacteria.Fungi mycelia penetrate oil, increasing surface area for biodegradation and bacteria attack (Chaillanet al., 2004).This presence study is aim at determining the physiochemical properties of soil sample and identifying the total heterotrophic and hydrocarbon utilizing fungi from Ejamah-Ebubu oil spill site located at Eleme Local Government Area in Rivers state Nigeria

MATERIAL AND METHOD
Sample Collection: Using composite sampling techniques soil samples were collected from Ejamah-Ebubu oil spill site located at Eleme Local Government Area in Rivers state Nigeria.The soil samples were collected by means of a manually driven clean auger at five (5) sampling points from (0 -15) cm depth: At15, Bt15, Ct15, Dt15, Et15 and depth (15 -30) cm Ab30, Bb30, Cb30, Db30, Eb30.Samples were transferred aseptically into sterile flasks and transported to the laboratory for analysis.
Total Heterotrophic Fungal Count: This was done using spread plate technique.About 0.1 ml of the 10 -3 and 10 -4 dilution of each sample was spread on the surface of Potato dextrose agar (PDA) into which 0.1 ml of lactic acid was added and incubated at 28 0 C for 5 days.Distinct fungi colonies were counted as cfu/g and sub-cultured into freshly prepared PDA for further identification.
Total Hydrocarbon Utilizing Fungal Count: About 0.1 of 10 -3 and 10 -4 dilution were spread onto the surface of freshly prepared acidified mineral salt medium which contain in g/l ( 0.4g of MgSO47H2O, 0.29g KCl, 1.25g Physicochemical Characteristics and Mycoremediation of 4 Akomah, ON; Abu, GO KHPO4 , 0.83 K2HPO4, 0.442g NH4NO3, 10g NaCl, 15g Agar).A filter paper dabbed with crude oil was inserted under the cover of the Petri plate and incubated at 28 0 C for 5 days.Distinct fungi colonies were counted as cfu/g and sub-cultured into freshly prepared PDA for further identification.
Characterization, spore staining and identification of Hydrocarbon utilizing Fungi: The method of Bennet (2002) was adapted for characterization and identification.This include macroscopic examination and microscopic examination.Spore staining procedure was used to confirm the presence of spores.A loop of distinct colonies was emulsified in lacto-phenol cotton blue reagent in a clean glass slid and covered with a cover slip.It was then viewed under the microscope using the X40 objective lens.
Preparation of Fungi Innoculum and Biodegradation of crude oil by fungi isolate: Fungi isolates were innoculated into 100 ml of Potato dextrose broth into which 0.1 ml of lactic acid is added.The setup was incubated at 28 0 C for 48 hours.About 5 ml of each fungi isolate were innoculated into 95 ml of potato dextrose broth into which 0.1 ml lactic acid was added.About 1% crude oil was added to the setup and allowed to form a thin layer over the medium surface.The stopper were placed on the flask and each was inverted several times allowing the microorganisms to mix with the oil.The flasks were incubated at 28 0 C.Each flask was observed and inverted every 24hours for 5 days and the samples were then taken for estimation of total petroleum hydrocarbon degradation and each observation was recorded in the appropriate table .pH and Conductivity Measurement: The pH of the sample was determined using the pH meter (Jenway model 015) while the conductivity was determined using conductivity meter (SC-300).
Salts Content: Nutritive salts (nitrate, phosphate and sulphate) were determined by method outlined in APHA (1995).Nitrate was measured using brucine method, phosphate the ascorbic acid method while sulphate turbidometric method.The procedure had been described in earlier research work (Akomah and Abu, 2015).

Total Petroleum Hydrocarbon and Polyaromatic
Hydrocarbon: Residual total petroleum hydrocarbons (TPH) and polyaromatic hydrocarbons (PAHs) were extracted from soil sample and quantified using gas chromatograph-FID.Procedure had been described in previous research work (Akomah and Abu, 2015).

RESULTS AND DISCUSSION
pH and Conductivity: The result of the analysis of physico-chemical properties of the sediment sample are shown in Figures 1 and 2. The pH of the various sampling points is neutral, ranges from 7.21 -7.82.(At -Et)15 recorded a higher pH, indicating decrease in pH as sampling depth increases.The conductivity of sampling points also increases as sampling depth increases indicating the present of ions.

TPH and PAHs Content:
The TPH concentration of sampling points range between (15.65 -31.65) mg/kg while PAHs ranges between (0.059 -0.117) mg/kg.The concentration of TPH at most sampling point is below/close to the permissible limit (30 mg/kg) as indicated in table 1.

Estimation of Total Petroleum Hydrocarbon degradation (oil and grease):
The concentration of total petroleum hydrocarbon decreases as the experiment process, indicating loss of oil.
The present study show that the nutritive salts are moderate when compared with the concentration of total petroleum hydrocarbon present.Nitrate concentration for top soil (At-Et)15 ranges from 10.5 -12.6 mg/kg while bottom soil (Ab-Eb)30 ranges from 12.6-14 mg/kg.Phosphate concentration for (At-Et)15 ranges from 3.7 -4.4 mg/kg while (Ab -Eb)30 ranges from 2.56 -2.90 mg/kg.
The total petroleum hydrocarbon concentration ranges from 15.57 -32.21 mg/kg.The chromatogram shows carbon chain C9 -C40 including pristane and phytane.Most of the sample points have TPH concentration lower than the permissible limit for TPH (30 mg/kg).

Conclusion:
The study revealed presence of hydrocarbon utilizing fungi in the soil sample of Ejamah-Ebubu oil spill site.It is interesting that fungi species are presence at the site because they are considered as primary catalyses for correcting contaminated ecosystem and controlling the flow of nutrients.

Table 1 :
Physicochemical properties, Salts, TPH and PAHs of various sampling points

Table 4 :
Concentration of Total Petroleum hydrocarbon (oil and grease)