Screening of hydrocarbon degrading fungi in crude oil polluted soil isolated in the Niger Delta

Bioremediation has been argued to be cheaper and more environmental friendly when compared to other remediation technology. Bioremediation make use of the ability of bacteria and fungi to biodegrade organic compound to cleanup hydrocarbon pollution. Many fungi have been shown to biodegrade simple and complex polycyclic aromatic hydrocarbon. In this study, petroleum hydrocarbon degrading fungi have been isolated and identified by microscopic and macroscopic methods in oil polluted soil. The polluted soil samples were collected from an oil polluted site within an oil bearing community in Bayelsa state of Nigeria. Eight different strains of fungi were isolated and identified and they include, Penicillium spp, Candida species, Aspergillus niger, Mucor spp, Rhodotorulla spp, Rhizopus, Trichorderma spp and Cladospermorium spp. Population of hydrocarbon degrading fungi, isolated from the soil collected in three different sampling stations are 5.3x10 4 , 8.3x10 3 and 2.51x10 4


INTRODUCTION
Fungi are group of microorganisms scientifically regarded as non-green plants.They are widely distributed in the environment and mostly found in moist dark places.
While some are harmful as a result of their toxic secretion, some are edible.Apart from being edible some are also beneficial to man in other ways.For instance, *Corresponding author.E-mail: egbomansi@yahoo.com.Tel: 08066677173.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License some help in the breakdown of organic waste that would have otherwise pose threat to humans and the environment.
Like some bacteria and Protists, fungi digest insoluble organic matter by secreting exoenzymes, which absorb the solubilized nutrients (Prescott et al., 2008).Yeast, molds, mushroom, mildews and some other types of fungi are wide spread in the environment and help to breakdown complex organic matter.The ability of fungi and some bacteria to breakdown complex organic and harmful matter into simple and harmless or less toxic compounds which assimilates them as food, has made them useful and find application in the science of environmental restoration (Bioremediation).
Bioremediation is a technology that utilizes the metabolic potentials of microorganisms such as fungi and bacteria to degrade petroleum hydrocarbon spilled in the environment into harmless compounds (Watannabe, 2001).These microbes either degrade, bioaccumulate, bioabsorbes or bioadsorbes the contaminant in sediment, water or soil.(Odokuma and Dickson, 2003).Of all the petroleum pollution cleanup techniques that have been developed, the use of petroleum hydrocarbon degrading microbes has received most attention (Macaulay, 2015).
Petroleum hydrocarbon pollution is hazardous to the ecosystem and public health (IIheoma, et al., 2015).It is because of toxicity of petroleum hydrocarbon that immediate and urgent attention should be given with view to cleaning up the spill.Abi and Nwosu (2009) reported that crude oil negatively affects soil agricultural productivity and soil organisms.Crude oil has also been shown to reduce plant germination and productivity (Agbogidi, et al., 2007).A study by Wokocha, Emeodu and Ihenko (2011) also reported that crude oil pollution increase soil acidity.High soil acidity is not good for plants because it can destroy plants and impotent soil organisms.
Crude oil also kill fishes in concentration level of 4000ppm (Prasad, 1987).Benzene, a volatile compound in crude oil is also reported by Abdel-Shafy and Mansour (2016), to cause Leukemia in humans.Poly aromatic hydrocarbon another component of crude oil is also reported by Selina, (2005) to cause cancer in humans.
Bayelsa as one of the Niger Delta States where oil companies based their operation has experience many oil spillages hence, oil polluted sites are wide spread in the state.Inspite of the numerous oil pollution which have occurred in the state, much research have not been carried out to identify oil degrading fungi and bacteria, as it done in Ogoni land in Rivers State with view to harness them for bioremediation of the numerous oil polluted sites across the state.Bioremediation offers a very feasible alternative for the decontamination of oil spills (Greetha et al., 2013).
This technique is considered as an effective technology for the treatment of oil pollution (Malatova, 2005).

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Bioremediation is an effective, economical and environmentally friendly treatment method in which microbes are used to degrade hydrocarbons (Greetha et al., 2013) Some of these fungi have been previously reported as hydrocarbon degrading.Fungi have proven useful in bioremediation of polluted environment due to their ability to grow under stressed environment, secretion of extracellular enzymes (Chikere and Azubike, 2014).There is also specialty in the components of hydrocarbon degraded by microorganisms.Fungi can degrade certain components of hydrocarbon that bacteria cannot degrade and certain components that fungi may find difficult to degrade can be easily degraded by certain bacteria.Chikere and Azubike (2014) also reported that the ability of fungi to secrete extracellular enzyme which aid in the biodegradation of petroleum hydrocarbon makes these species of microorganisms useful in the science of bioremediation.This study is focus on the isolation and identification of hydrocarbon utilizing (degrading) fungi in oil spilled soil in the Niger Delta region of Nigeria.

MATERIALS AND METHODS
The materials used in for this research include: trowel, shovel, recyclable polythene bags and sack bags for the polluted soil sample collection apparatus which include microscope, glass slides, Petri dishes conical flasks and dropping pipette etc.Crude oil polluted soil samples were collected from three different locations in an oil spill site in Ikaramar community in the Okordia Zarama clan, within Yenagoa Local Government Area of Bayelsa State.This community is host to SPDC and Agip oil facilities within the Niger Delta.The soil samples were collected from a depth of 0 to 30cm of the top layer soil, put into clean and sterile black polythene bag and transported to the laboratory for biological analysis.

Aspergillus niger
Presence of septatehyphae, black long, smooth, erect conidiophores Hyalineichoromously branched vesicle round, radiate head.Brownish-black mycelium with darks pores on the surface.

Aspergilus fumigates
Presence of rough conidiophores, with uni/biseriatephialides whose vesicle is round with radiate head.
Presence of blue-green to yellow coloration from surface.
A yellow-green color with a creamy edge appears on the surface, which appears golden to reddish brown on the reverse side.
Candida species Single clusters of blastoconidia which is round and elongate.Long branched pseudohyphas were also observed.
A creamy to yellowish colonies with smooth, pasty, glistening or dry, wrinkled and dull color.
Penicillium species Presence of red pigment with edges surrounded by whitish margin.Also the conidiophores are branched.Septate and fruity mycelium is observed.
A bluish-green filament is seen which changes to powdery greenish brown.It has brush phialospores arrangement.

Mucor species
Presence of visible spore and short sporangiosphores with non septatehypae.Slimy colonies of texture with dark pigmented spores.

Fusarium species
Presence of dark pigment of micro and macro conidiophores.Presence of sickle-shaped macroconidia that is yellow to purple in colour.

Rhodotorulla
Spherical to elongate budding yeast-like cells or blastoconidia.
Colonies are coral red to salmon-coloured or slightly orange, smooth to wrinkled, highly glossy to semi-glossy.

Rhizopus
Presence of stolon's and pigmented rhizoids, the formation of sporangiosphores, singly or in groups from nodes directly above the rhizoids and apophysate, columellate, multispored, generally globose sporangia.
Presence of zygomycetes spores with dark pigment colonies are fast growing and cover an agar surface with a dense cottony growth that is at first white becoming grey or yellow brown with sporulation.
Presences of green conidia filament that resemble pencilin conidia are mostly green, sometimes.Hypae with smooth or rough walls and are formed in slimy conidia heads (glorospora) clustered at the tips of the phialids.

Cladosporium
Microscopy shows ascending to erect olivaceous-green apically branched, elongate conidiophores producing branched acropetal chains of smooth-walled conidia.Presence of light to grayish surface, gray to black surface blastoconidia.

Isolation, enumeration and identification
The fungi were isolated from the soil sample by culturing them under growth conditions of media (Sabouraud Dextrose Agar and nutrient Agar). 1 g of homogenized and sieved oil polluted soil sample was weighed and dissolved in 9 ml of sterilized distilled water in nine replicates and properly shaken.Each of the resultant solution was made to 10 -1 and the diluents were further serially diluted up to 10 -8 .1ml of each diluent was then inoculated into sterilized petri dish.The prepared and sterilized Sabouraud Dextrose Agar media was poured into each of the Petri dish containing soil water inoculums.The plates were rotated in clockwise and anticlockwise direction to ensure uniform spread of the inoculums.These plates were allowed to set and then incubated upside down position at 37°C for one week.The plates were observed with magnifying hand lens for hydrocarbon degrading fungal growth after 3 to 5 days.
The plate showing between 30 to 300 colonies was recorded.From the counting, the total viable microbe cells in the sample were expressed as colony forming unit (cfu/g) of the sample.

Screening of Fungi hydrocarbon degradation (utility)
A pure growth of each isolate was inoculated into freshly prepared and well sterilized Bushnell Haas Broth Medium incorporated with 1ml sterilized crude oil as a sole source of carbon and energy in a 200 ml conical flask.10 ml of Chloramphenicol was also incorporated into the freshly prepared BHBM as bacteria growth inhibitor.One flask was kept as control and has no organism inside.The flask was then incubated at 28 to 30°C with regular shaking for two weeks.The flasks content were monitored for change in optical density, colour and hydrocarbon concentration for the period of two weeks.Change in TPH concentration was recorded during the period.

Microscopic identification of fungi
A pure culture of hydrocarbon degrading fungi was further subcultured in another prepared Sabuoraud Dextrose agar enriched with nutrient agar for further purification and identification.
Fragment of a well grown pure culture was immersed in 0.1 ml of 95% ethanol on a glass slide.The ethanol was allowed to evaporate and the specimen was stained with 0.1 ml lactophenol blue stain.The stained specimen was then covered with cover slide and viewed microscopically.The resultant morphology was compared using the guide provided by Pepper and Gerba (2005);Elis et al. (2007); Bartnet and Hunter (1972); Benson (2002) (Table 1).

Isolated and identified fungi in biosludge samples
Table 2 shows the isolated and identified fungi biosludge while Table 3 show fungi enumeration for each polluted soil.

DISCUSSION
Bioremediation has been shown to be an effective, economical and environmentally friendly organic pollutant treatment method in which microbes are used to degrade hydrocarbons (Greetha et al., 2013).In this work, hydrocarbon degrading fungi were isolated and screened then identified by microscopic and macroscopic method, in crude oil polluted soil.2009), were eight fungi isolates from petroleum polluted soil were identified to be a hydrocarbon degrading.These include A. Versicolor, A.niger, A. Flavus, Syncephalastrum spp,Tricoderma spp, N. Sitophila, R. arrhizus and Mucor spp. According to April et al. (2000), some of these fungi have been previously reported as hydrocarbon degrading.
This study has further revealed that, oil polluted soil in the Niger Delta is habited by Hydrocarbon degrading fungi, which can be biostimulated to enhance oil pollution cleanup in the area.The result of this study is an indication that many of the oil spilled soil in Bayelsa may be inhabited by potential hydrocarbon degrading fungi which could be exploited for the cleanup of the polluted environment in the state.

Conclusion
In this study, eight different strains of hydrocarbon utilizing fungi have been isolated and screen in polluted soil, obtained from polluted site in Okordia/Zarama land of Bayelsa state.
The result of this study has proven that apart from bacteria that are versatile in the environment and known for hydrocarbon degradation, the oil polluted soil in orkordia/Zarama area of Bayelsa state also contains fungi that can be harnessed and exploited for oil pollution cleanup.

Table 1 .
Microscopic and macroscopic morphology of Fungi isolates.

Table 2 .
Fungi isolates from the three biosludge samples.

Table 3 .
Fungi density in each of the polluted soil samples.
Alternaria alternate and Fusarium spp are strains of fungi that have already been discovered by different scholars, to degrade different components of petroleum.This is affirmed by Uzoamaka et al. (