Biodegradation of Crude-oil by Fungi Isolated from Cow Dung- contaminated soils

This study assayed oil-degrading potential of fungi isolated from cow dung-contaminated soils. The samples were collected aseptically from four different cow dung dumping sites with no history of crude-oil pollution in Akure metropolis. These samples were analyzed for fungal loads and oil-degrading fungi using potato dextrose agar and mineral salt medium respectively. The biodegradation of crude oil was observed spectrophotometrically using the broth culture of the fungal isolates for a period of 15 days on mineral salt medium. The fungi were identified based on the microscopic and macroscopic features of the hyphal mass, nature of the fruiting bodies and the morphology of cells and spores. The sixteen (16) fungi identified from the contaminated soils include; Bdellospora helicoides, Aspergillus fumigatus, Gonadobotricum apiculata, Aspergillus niger, Trichoderma viridae, Pleurothecium recurvatum, Streptothrix atra, Thysarophora longispora, Candida albicans, Aspergillus flavus, Helminthosporium velutinum, Botrytis cinerea, Zoophage nitospora, Varicosporium elodeae, Articulospora inflata and Neurospora crassa. All fungi showed degradation of the crude oil, with Trichoderma viridae, Aspergillus flavus and Varicosporium elodeae demonstrating best degradation ability. Trichoderma viridae exhibited highest degradation (66.2%) while Varicosporium elodeae exhibited least degradation (40%). The measurement of the rate of biodegradation of crude oil by the three fungi was further confirmed using Gas Chromatography and Mass Spectrophotometer (GC-MS). The GC-MS analysis showed that the fungi degraded the hydrocarbon compounds when compared to that of the control. The result obtained revealed that oil-degrading fungi can be isolated from cow dung dumping sites and they are competent mycoflora for the biodegradation of crude oil polluted soils. They can be used as a better approach to restoring oil contaminated environments through bioremediation process.


Introduction
The common problems associated with petroleum industry are accidental and deliberate discharge of oil spills.Oil spillage is known to be a major environmental problem in Nigeria, most especially in the Niger-Delta region, attracting considerable attention in the recent years.Many approaches (physical, chemical and biological methods) have been employed in order to reduce or eliminate the effect of oil spillage on the environment and living organisms.However, most of these efforts have limitations in their applications, either as being too expensive or posing threats to the ecosystem.The most promising of many researches carried out to deal with large-scale oil spills is the use of microorganisms to provide an effective alternative (Singh et al., 2001).This approach is referred to as 'bioremediation', and it is one of the most rapidly growing areas of environmental microbiology, which has been used for cleaning up pollutants.This is because of its low cost, safety and its public acceptability (Grazyna et al., 2001).Microbial degradation represents the major route responsible for the ecological recovery of oil spills (Johnsen, et al., 2005).
Many species of bacteria and fungi isolated from oil spilled sites have been shown in recent years to have the abilities to use petroleum hydrocarbon as sole source of carbon and energy (Olukunle, et al., 2012;Boboye et al., 2010 andOjo, 2006).Fungi have been found to be better degraders of petroleum than traditional bioremediation techniques including bacteria (Batelle, 2000).Although, hydrocarbon degraders may be expected to be readily isolated from a petroleum oil-associated environment, the same degree of expectation may be anticipated for microorganisms isolated from a totally unrelated environment (Ojo, 2006).
The source of fungi for the biodegradation of petroleum oil becomes the issue.In most previous researches, the source of fungi is either selected from crude oil contaminated soils or water (their natural environments), rhizoplanes, phyloplanes, food or crops and root tubers.Some other fungal source for biodegradation of petroleum oil, are the experimental use of mycorrhizal fungi and wood rotting fungi in the biodegradation of petroleum.In this study, attention is paid to source for fungi from cow dung-contaminated soils.Bioremediation techniques involves biostimulation or bioaugmentation i.e. addition of nutrients or addition of oil-degrading microorganisms respectively (Samantha, 2002).The objective of this study therefore was to isolate fungi from cow dung-contaminated soils and assay for their degrading activities on crude oil using turbidity growth and GC-MS with the view to providing them for bioaugumentation so that soil could be enhanced for productivity.

Materials and Methods
The soil samples used were collected at cow dung-contaminated sites at a depth of about 10-15cm in various locations in Akure, Ondo State for the cultivation of fungal isolates while the petroleum sample was a Bonnylight crude oil collected from the Shell Petroleum Development Corporation refinery in Port Harcourt, Rivers State.
Pour plate technique was used for the microbiological analysis of samples collected from cow dung-contaminated sites (Song and Bartha, 1990).One gram of the soil and 1 ml of the water sample was mixed with 9ml of normal saline.One millilitre of the soil and water each was diluted from 10 -1 to 10 -10 .An aliquot of (1ml) dilution 10 -5 was seeded into potato dextrose agar (PDA).The inoculated plates were incubated at 28 o C for 72 hr.The fungal spore forming units (Sfu/g) were then counted.Repeated subculturing was done for each isolate to get pure culture of the fungi for easy identification.
Pure Fungal cultures were observed while still on plates and the isolates were characterized using cultural characteristics such as the colour of aerial and substrate hyphae, type of hyphae, shape and kind of asexual spore, presence of foot cell, sporangiophore or conidiophores and the characteristics of spore head using microscopic and macroscopic methods.The identities of the organisms were determined by comparing their characteristics with those of known taxa as described by Domsch and Gams (1970).
The screening of fungal isolates for the ability to utilize petroleum was done according to the method of Ijah and Antai (2005) by growing the isolates in potato dextrose broth for 3 days before they were transferred to oil broth (1.2g K 2 HPO 4 , 1.8g K 2 PO 4 , 4.0g NH 4 Cl, 0.2g MgSO 4 .7H 2 O, 0.1g NaCl, 0.01g FeSO 4 .7H 2 O, in 1000ml distilled water, 1percent crude oil) and incubated at room temperature.After every 3 days, 1ml of the oil broth was extracted to know the extent of oil degradation by measuring the turbidity on a spectrophotometer at wavelength of 540nm and the best and most efficient oil degraders were selected for further studies using Gas Chromatography and Mass spectrophotometry (GC-MS).The best and most efficient oil degraders were later grown in 5mls of Potato dextrose broth in test tubes for three days.At the end of 3 days of incubation, the fungi were introduced into 7mls of sterile oil broth in McCartney bottles and incubated at room temperature.This was done in duplicates for each of the fungal isolates.Negative control was also prepared without adding any fungal isolates.After every 4 days, residual crude oil was extracted using diethyl ether.The diethyl ether was then allowed to evaporate overnight after which the remnant crude oil was taken for Gas Chromatography and Mass Spectrophotometry analysis.The analysis was conducted at the Central Science Laboratory, Obafemi Awolowo University, Ile-Ife, Nigeria.Four microlitres (4µl) of the extractable crude oil was diluted with 4µl of n-hexane and analyzed on a 19091J-413HP-5 capillary column installed in a Gas Chromatograph (Agilent Technologies, 7890 GC system) equipped with a Mass spectrophotometer (5975 series MSD).Split injection was used with helium as carrier gas.The oven temperature was initially set at 35°c for 2 minutes and increased at a rate of 5°c/min to 280°c and ran for 60 minutes.
The turbidity growth of the fungi species are shown in Fig. 1.Trichoderma viridae, Aspergillus flavus and Varicosporium elodeae had the best growth in the mineral salt broth after 15 days of incubation.From the Gas Chromatography and Mass Spectrophotometric analysis on the three isolates, Fig. 2 shows the gas chromatogram of undegraded (incubated control) crude oil.Fig. 3 shows gas chromatographic patterns of crude oil degraded by Aspergillus flavus after 4, 8, 12 and 16 days of incubation, while Fig. 4 and Fig. 5 show the gas chromatographic patterns of crude oil degraded by Varicosporium eloedea and Trichoderma viridae respectively after 4, 8, 12 and 16 days of incubation.
Light chain alkanes were probably lost by volatilization during the 16 days of incubation.Comparing the chromatograms in Fig. 3, Fig. 4 and Fig. 5 with those of the control (Fig. 2), it was observed that the fungi were slow in attacking or degrading the crude oil on the 4 th , 8 th and 12 th day of incubation but were later able to degrade the oil effectively.This is due to the fact that the fungi are slow growers, so it took them longer time to start attacking the crude oil.It was also observed that after 4, 8 and 12 days of incubation the organisms did not show any significant degradation but after the 16 th day of incubation, it was observed that Trichoderma viridae degraded the crude oil best while Varicosporium eloedea was the least in degrading it.This may be due to the differences in competence of degradative enzymes and pathways of degradation used by the organisms.It was also observed that there were reductions in the quality of some compounds in the crude oil.Therefore, according to the results obtained, Trichoderma viridae and Aspergillus flavus should be preferred to Varicosporium eloedea when microbial seeding of oil spills in tropical soils is intended.

White mycelium growth
The hyphae is aerial, slender and non-septate; a short haustoria, branched, conidia hyaline and elongate as well as catenulate

2.
Brown mycelia growth An upright conidiophores that terminates in a davate swelling bearing phialides at the apex or radiating from the entire surface; conidia are 1-celled and globose

3.
Black mycelia growth and fully extended in the growth medium An upright conidiophores that terminates in a davate swelling bearing phialides at the apex or radiating from the entire surface; conidia are 1-celled and globose

7.
Cotton-like mycelia at 24 hours turning dirty with development of black spores on mycelium Non-septate hyphae, thin sporagiophore with a sporangium in club-like form Thysarophora longispora 8.

Greenish mould
Mycelium are not extensive, conidia are 1-celled, ovoid to fusoid, forming short chains by budding which are produced on mycelium epically or laterally.

9.
Yellow mycelia growth An upright conidiophores that terminates in a davate swelling bearing phialides at the apex or radiating from the entire surface; conidia are 1-celled and globose

11.
Grey/Green mould Long conidiophores which are septate, conidia are borne in clusters on branched sterigmata.

White mycelium when young but turn yellow at maturity
Branched septate conidiophore, produced golden yellow conidia in masses Neurospora crassa

16.
Cotton-like mycelia at 24 hours turning dirty with development of black spores on mycelium Non-septate hyphae, thin sporagiophore with a sporangium in club-like form Gonadobotricum apiculata

Discussions
Various fungal genera have been isolated from non-hydrocarbon contaminated soils, playing important ecological role in removal of hydrocarbons (Van Beilen et al., 2006;Challan et al., 2004).The high microbial count from the various cow dung polluted sites indicated that cow dung polluted soil is a very rich source of mycoflora, (coprophilous fungi) as shown in Fig. 1.This supports the work of Richardson ( 2001) that the animal faeces provide an environment rich in nitrogenous material suitable for the proliferation of microorganisms.It is also in line with the work of Obire, et al (2008) that the presence of cow dung in the soil resulted in an increase in the population of total saprophytic fungi and an increase in the population of petroleum-utilizing fungi in soil.
Results (Fig. 2 -5) showed that the mycoflora of cow dung possess the ability to utilize crude oil.All the fungal isolates showed varying degree of biodegradation of crude oil in Mineral salt medium indicating that they all utilize crude oil as their main source of carbon.An interesting demonstration generated in this work shows an increase in rates of fungal growth in the media containing crude oil which might be due to the fact that the fungi used crude oil as a substrate for their growth using extra cellular enzymes to break down the recalcitrant hydrocarbon molecules, by dismantling the long chains of hydrogen and carbon, thereby, converting crude oil into simpler forms or products that can be absorbed for the growth and nutrition of the fungi as shown by Adekunle and Adebambo, (2007).It also agreed with the work of Akinde and Obire (2008), which revealed the presence of potential hydrocarbon degraders in cow dung.
From the biodegradation study, it was observed that Aspergillus flavus, Varicosporium elodeae and Trichoderma viridae had the best growth in the oil broth.This is in line with the work of other researchers such as Vidali, (2001) and Bhattacharya et al., (2003), who reported similar findings which made their application for bioremediation efforts suitable.This study also revealed that crude oil-degrading fungi are not restricted to oil contaminated soil as Aspergillus flavus, Varicosporium elodeae and Trichoderma viridae were isolated from cow dungcontaminated soil with no history of oil pollution.This finding supports the report of Odu (1978) that crude oil degrading microorganisms are widely distributed in the Nigerian environment (Ijah, 1998).The efficient crude oil degrading ability of the organisms may be due to the competent degradative enzyme system of the organisms.
The high degradation ability of these organisms especially Aspergillus flavus and Trichoderma viridae may be due to the higher production of extracellular enzymes and organic acids that enable the organisms to utilize the hydrocarbon faster, (Damisa et al., 2013).This agrees with the findings of Stamets (1999), that mycelial mats are used for bioremediation because they produce extracellular enzymes and acids that break and dismantle the long chains of hydrocarbon, the base structure common to oils, petroleum products and many other pollutants.The fact that the species of Aspergillus, Trichoderma and Varicosporium could degrade crude oil efficiently point to their potential usefulness in cleaning oil spills in tropical soils.
This study showed that there were reductions in the quality of some compounds in the crude oil according to the GMSC analysis.By comparing the GMSC profiles for the different days, it was observed that there was a significant decrease in the peaks corresponding to the hydrocarbon fractions of the oil.According to April et al., (2000), the reduction in peak heights of the different hydrocarbon fractions of the crude oil indicates the degradation of the components.This is well observed in all of the profiles which showed that the fungal isolates are capable of degrading the crude oil.This study supports the findings by April et al., (2000) that reported 22 species of Penicillium and 5 species of Aspergillus isolated from the flare pit soils in Northern and Southern Canada.These isolates showed the ability to degrade hydrocarbons on solid medium amended with crude oil.It was also observed that after 4, 8 and 12 days of incubation the organisms did not show any significant degradation but after the 16 th day of incubation it was observed that Trichoderma viridae degraded the crude oil best while Varicosporium eloedea was the least in degrading it.This may be due to the differences in competence of degradative enzymes and pathways of degradation used by the organisms.This could also be as a result of the fact that hydrocarbons differ in their susceptibility to microbial attack, (Ulrici et al., 2000).
It was observed from Table 2 that there was a significant degradation of some of the selected compounds from the GCMS result.There was complete degradation of 1-Methyl-4-(1-methylethyl)-3-[1-methyl-1-(4-methylpentyl)-5-methylheptyl] Cyclohexene, with retention time of 49.857 by Trichoderma viride.There is scarce information about the genus Trichoderma under petroleum hydrocarbon-contaminated systems in order to make comparisons about the tolerance of Trichoderma to either crude oil or Polycyclic Aromatic Hydrocarbon.Although there has been some research about the utilization of PAHs by this fungus (Li, et al., 2008;Atagana, 2006).Elad et al., (1981) also referred to the utilization of a PAH as a selective medium for the growth of Trichoderma viride.Nevertheless, the present study demonstrates that Trichoderma viride is capable of degrading PAHs and can be seeded for the bioremediation of some recalcitrant hydrocarbons.
It is widely believed that microorganisms isolated from crude oil contaminated environment degrade crude oil more efficiently than their counterparts from uncontaminated environments.It is also generally upheld that bacteria are better crude oil degraders than fungi, (Ijah, 1998).These should not be held as absolute rules since the present study has revealed that Aspergillus flavus and Trichoderma viridae isolated from crude oil free soil caused a high extensive degradation of crude oil.The information provided by this study in regards to the rate at which oil is metabolized by fungi from crude oil unpolluted tropical soil is crucial especially now that the emphasis is on finding efficient hydrocarbon degrading microorganisms for seeding oil contaminated environments.It also contributes to the limited information in literature on the potentials of fungi in degrading oil pollutant in soil when used in microbial seeding process, (Ijah and George, 2003).This study also showed that the cow dung contaminated soil has relatively high populations of crude oil-degrading fungi.This however means that enhanced biodegradation of crude oil in soil through microbial seeding should not be ignored.It is essential for speedy recovery of oil spills in the environment.
The results obtained in this study indicate that Trichoderma viride was the best candidate in degrading the crude oil which means that it should be preferred to either Aspergillus flavus or Varicosporium eloedea when microbial seeding of oil spills in tropical soils is intended.Moreover, the mixed culture of petroleum degrading fungi present in cow dung can be harnessed by researchers in the search for mixed culture of microorganisms with naturally enhanced oil degrading capabilities or which could be genetically engineered for enhanced bioremediation of contaminated sites, (Obire et al., 2008).

Conclusion
It can be concluded from this study that the rate of biodegradation of petroleum hydrocarbon mixtures in soil could be enhanced by the addition of cow dung in soil media.The fungi isolated and tested for biodegradation were very effective and since they were isolated from cow dung-contaminated soils, the use of cow dung to stimulate petroleum hydrocarbon mixtures degradation in the soil could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil and the organisms Aspergillus spp, Trichoderma spp and Varicosporium spp may be pointed out as potential fungi to degrade petroleum hydrocarbons, especially those spilled out over tropical soil.

Table 1 : Suspected fungi from cow dung contaminated soils
with retention time of 49.857 by Trichoderma viride.

Table 2 :
The percentage depletion of some selected compounds by the fungal isolates