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ASSAY OF OIL-DEGRADING POTENTIAL OF FUNGI ISOLATES ON DIESEL, KEROSENE AND PETROL USING ENRICHMENT METHOD. BOBOYE B. , *OLUKUNLE O. F. , ADETUYI F. C. AND ADEBIYI G. A ABSTRACT A study was carried out to assay for oil-degrading potential of fungi isolates on diesel, kerosene and petrol. Water samples were collected aseptically and analyzed microbiologically using standard techniques. The fungi isolated from the water samples were: Trichoderma viridae, Aspergillus niger, Aspergillus fumigatus and Stochyborys atra.
The confirmatory test for oil utilization potential of the isolated fungi was carried out using the enrichment method, minimal salt broth (MSB). There were variations in the growth patterns of each of the fungal species with respect to the different oil (diesel, kerosene, petrol) used. This also signified that fungal species varied in their degradative abilities. The length of incubation for the fungi ranged from the 5th day to the 20th day. The fungi with the highest hydrocarbon utilization potentials were: Aspergillus fumigatus and Aspergillus niger while stachyborys atra had the least hydrocarbon utilization potential.
These hydrocarbon degraders are capable of utilizing oil polluted river and could therefore be employed in bioremediation process. *Corresponding Author: Institute of Microbial Biotechnology and Metagenomics, University of the Western Cape, South Africa KEYWORDS: oil???degrading potential, microorganism, polluted land, hydrocarbon degraders, enrichment medium INTRODUCTION The huge demand for crude oil causes enormous quantities to be moved from production areas to different destinations where the oil will be used.
The greatest environmental problem connected with crude oil exploration in Nigeria is oil spill both on-shore and off-shore (Okpokwasili, 1996). Crude oil, because of its characteristics, is one of the most significant pollutants in the environment as it is capable of causing serious damages to humans and the ecosystem, resulting in the contamination of drinking water, killing the fishes and poisoning aquatic life, thereby, placing hardship on those who make their living by fishing.
Regardless of the source of contamination, some oil or decomposition products may reach groundwater reserves, lakes and other water bodies, polluting water for domestic and industrial use, thus posing serious threat to public health and causing socio-economic hazards to the populace using the water (Okpokwasili, 2003). When oil enters the ocean, it quickly begins to spread and disperse with the aid of the winds and waves while some will evaporate due to its high volatility, some will form into tar balls and sink to the bottom where they may remain for a long time, slowly releasing hydrocarbons into the water.
Though oil is toxic, it becomes less so with time. Winds and waves help spread and disperse the oil. Some oil will evaporate. Microorganisms in the water attack and digest the oil. If people act quickly after the spill, they can scoop up some of the oil and stop it from causing worse damage to the environment (Swannell and Richard, 2003). Many microorganisms are found to posses the ability to actively decompose specified fractions of hydrocarbon (Bartha and Atlas, 1997). Since not all components of petroleum???derived hydrocarbon mixture are decomposed simultaneously, Yuan et al. 2000) suggested the introduction of mixed culture of bacteria and fungi for the degradation of petroleum. However, single cultures of fungi have been found to be better than mixed cultures (Okerentugba and Ezeronye, 2003). The filamentous fungi are known to possess some attributes that enable them as good potential agents of degradation, such as digestion of the hydrocarbons through the secretion of extracellular enzymes and ability to grow under environmental conditions of stress (low pH values, low nutrients and low water activity) (Ojo, 2006).
Different techniques have been employed to clean up the environment polluted with oil. Bioremediation seems to be a good alternative to conventional clean-up technologies because it has proved to be less disruptive to the environment than other techniques. It is found to improve on nature’s own way of degrading oil. Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity. As such, it uses relatively low-cost, low-technology techniques, which generally have a high public acceptance and can often be carried out on site Keeler, 2003).
However, the time scales involved are relatively long, and the residual contaminant levels achievable may not always be appropriate. Bioremediation has been used at a number of sites worldwide, including United States and Europe, with varying degrees of success. Techniques are improving as greater knowledge and experience are gained, and there is no doubt that bioremediation has great potential for dealing with certain types of site contamination (Keeler, 2003). For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products.
Bioremediation can be effective only where environmental conditions permit microbial growth and activity, its application often involves the manipulation of environmental parameters to allow microbial growth and degradation to proceed at a faster rate. Like other technologies, bioremediation has its limitations. Some contaminants, such as chlorinated organic or high aromatic hydrocarbons, are resistant to microbial attack. They are degraded either slowly or not at all, hence it is not easy to predict the rates of clean-up for a bioremediation exercise (Wilson, 2002).
Most bioremediation systems are run under aerobic conditions, but running a system under anaerobic conditions may permit microbial organisms to degrade otherwise recalcitrant molecules (Bonaventura and Johnson, 2005). This study will provide information on the indigenous fungi obtained from oil spilled site and their potentials to utilize petroleum-hydrocarbon. MATERIALS AND METHODS Source and Collection of samples The contaminated water samples were collected from Awoye River in the Southwest of Nigeria where there was an oil spill in 2004.
The water samples were collected aseptically into screw-capped containers from different locations and depths. The depths considered were 0-10 cm, 11-20 cm and 21-30 cm from the water surface. All glasswares used were washed with detergent, rinsed thoroughly with distilled water and allowed to dry. The test tubes containing 9m1 of distilled water and the conical flask containing potato dextrose agar were loaded into the autoclave and checked to ensure it was air tight. It was then operated at 121??C for 15 minutes to ensure complete sterilization. Enumeration of Hydrocarbon-Utilizing Fungi
The minimal salt medium (MSM) of Zajic and Supplison as described by ljah and Abioye (2003) was used. The medium was sterilized by autoclaving at 121??C for 15 min. The sample was serially diluted and 1ml, from l0-l to 10-6 dilutions was seeded in the MSM agar. The medium was supplemented with 1% (v/v) filter sterilized fuel (kerosene, petrol and diesel) to serve as the only source of carbon (Ijah and Abioye, 2003). Lactophenol acid was used to suppress the growth of bacteria in the media. The agar was incubated at 27??C for 3 days. Uninoculated plates were used as control. Isolation of Hydrocarbon-degrading Fungi
Each mycelia colony of fungi from the mixed culture was subcultured on new plates containing already prepared potato dextrose agar with the aid of a sterile inoculating loop. The plates were incubated at 25??C for 72 hrs until pure cultures consisting of only one type of fungus were obtained. Cultural identification of the fungi isolates were carried out by physical examination for the colour, elevation, edge of the fungi colonies and spore formation on the plates and was later confirmed by microscopic examination (Barnett and Hunter, 1972). The spore counts of fungal isolates were estimated using the counting chamber (Weber Scientific Model).
Hydrocarbon Utilization Potential of the Isolated Fungi The enrichment procedure as described by Nwachukwu (2000) was used in the estimation of hydrocarbon utilizers. All the test tubes were then incubated at room temperature (25-28) ??C for 20 days. The test tubes were shaken manually throughout the duration of the experiment to ensure cell phase contact. The growth rate of the organisms in the Mimimal Salt Broth (MSB) was measured every 5 days using spectrophotometer, standardized at wavelength 540nm and using the MSB broth as blank ( Nwachukwu, 2000). RESULTS AND DISCUSSION
Spore counts of fungi isolated from fuel polluted water are shown in Table 1. A total of four fungi isolates were obtained from the water samples based on their cultural and morphological characteristics (Table 2). The fungi isolated from water samples are Trichoderma viridae, Aspergillus niger, Aspergillus fumigatus and Stachybotrys atra. The results of this work also indicate that most of the fungal species isolated from the water are indigenous microorganisms of water because water contains large population of microorganisms due to routine exposure to series of contaminants (Sutherson, 2002).
The results of this work also indicate that many of the fungal species isolated from contaminated water with spilled oil were capable of degrading petroleum hydrocarbons. The growth pattern of fungi isolated from contaminated water in diesel and minimal salt broth is represented in Figure 1. Aspergillus fumigatus had the highest growth peak from 5th to 20th day with optical densities 0. 60, 0. 62, 0. 67 and 0. 70 respectively while Stachybotrys atra had the lowest optical density of 0. 22, 0. 25, 0. 30 and 0. 31 from the 5th to 20th day.
Next to Aspergillus fumigatus is Aspergillus niger with optical density 0. 55 on the 20th day, Trichoderma viridae showed gradual increase in growth peak with optical density 0. 50 on the 20th day. Aspergillus fumigatus had the highest ability to degrade diesel while Stachybotrys atra had the least ability. The growth pattern of fungi isolated from contaminated water in kerosene and minimal salt broth is represented in Figure 2. Aspergillus niger maintained the highest growth peak from 5th to 20th day with optical density 0. 3 on the 20th day while Stachybotrys atra maintained the lowest growth peak with optical density 0. 30 on the 20th day. Aspergillus fumigatus ranked next to Aspergillus niger with optical density of 0. 56 on the 20th day of incubation. Trichoderma viridae maintained a steady increase in growth with optical density of 0. 38 on the 20th day of incubation. Aspergillus niger had the highest ability to degrade kerosene while Stachybotrys atra had the least ability. The growth pattern of fungi isolated from contaminated water in petrol and inimal salt broth is represented on Figure 3. Aspergillus niger had the highest growth peak on the 20th day with optical density 0. 43, on the 5th day, it had a slight difference in optical density on the 10th day with optical density 0. 25 respectively. Stachybotrys atra had a close growth peaks with Trichoderma viridae on the 5th and 10th day but same growth peaks on the 15th day with optical density 0. 25 respectively. On the 20th day, Trichoderma viridae had higher growth peak than Stachybotrys atra with optical densities 0. 4 and 0. 29. Aspergillus niger with optical density 0. 43 on the 20th day had the highest ability to degrade petrol while Stachybotrys atra with optical density 0. 29 on the 20th day had the least ability. Results presented in this study show that the utilization of the different hydrocarbon used, namely diesel, kerosene and petrol vary among the fungi isolated. This was due probably to the difference in growth rates of each fungus with each fungus attaining different maximum growth peaks and the composition of the different oils used.
However, it must be noted that there were also nutrients present in the minimal salt broth though more of it could have been present in the oil which stimulated the growth of each fungus as described by Batelle (2000) who reported that fungi are good degraders and additional nutrients present in the minimal salt broth helped in overcoming nutrient limitation to microbial growth to a certain extent and also helped in creating a favourable environment for the rapid development of the fungi especially at the times when the fungi had not started breaking down the hydrocarbons into simpler molecules.
The fungi feed on the hydrocarbon products (or contaminants using the substrate as food) in order to build up their cells and hence get energy for growth and reproduction. The contaminants are used up and converted into a less harmful form. The composition of the oil (diesel, kerosene and petrol) also enhanced the growth of the microorganisms as described by Francisco and Speight (1999).
The optical density of each fungal isolate was taken for twenty consecutive days which increased gradually from zero to the twentieth day. This suggests that these fungi cells were able to degrade and feed on hydrocarbons for their growth and development. There was variation in the growth patterns of each of the fungal species with respect to the oil used. The highest degraders for contaminated water sample were Aspergillus fumigatus and Aspergillus flavus while the least degrader was Stachybotyrs atra. CONCLUSION
The results show that fungi isolated from contaminated water bodies are responsible for bioremediation and biodegradation of crude oil in water and can be exploited in the biodegradation of oil spills and bioremediation of the environment for improved agricultural productivity. The results of this work indicate a good prospect for monitoring bioremediation of hydrocarbon-polluted sites using the fungi isolates with the highest oil-degrading potential. Table 1: Spore counts of fungi isolated from contaminated water Isolates |Spore counts (sfu/ml) in fuel polluted water| |W1 |4. 68 x 1010 | |W2 |3. 75 x 1010 | |W3 |6. 87 x 1010 | |W4 |1. 87 x 1010 | Table 2: Cultural and morphological characteristics of fungi isolated from contaminated water. Isolates |Colonies on potato dextrose |Microscopy |Probable organisms | | |agar | | | |W1 |Greenish colonies |Conidiophores hyaline, much branched, not | Trichoderma viridae | | | |verticillate, phialides single or in groups, | | | | |borne in small terminal clusters, usually | | | | |recognized by its rapid growth and green patches | | | | |or cushions of conidia. | |W2 |Black colonies |Conidiophores upright, simple, terminating in a |Aspergillus niger | | | |globose or clavate swelling, bearing phialides at| | | | |the apex. | | |W3 |Brown colonies |Conidiophore is smooth walled with flask. Shape |Aspergillus fumigatus | | | |vesicle with flask shape series of sterigmata | | | | |upon which parallel rows of conidia are produced. | |W4 |Dark green colonies |Conidiophores subhyphae to dark simple, bearing |Stachybotrys atra | | | |apex a cluster of thick short phialides, conidia | | | | |(phialospores) dark, moist heads. At the apex of | | | | |the phialides, not catenalate. | | [pic] [pic] [pic] Table 3: Fungi with the highest and least oil-degrading potentials for contaminated water sample Highest |Least |oil | |Aspergillus fumigatus |Stachybotrys atra |Diesel | |Aspergillus niger |Stachybotrys atra |Kerosene | |Aspergillus niger |Stachybotrys atra |Petrol | REFERENCES Barnett, H. L. and Hunter, B.. 1972. Illustrated Genera of Imperfect Fungi (3rd ed. ) Bingess Publishing Company, Minneapolis Minnesota pp. 241. Batelle, C. D. 2000. Filamentous fungi to clean up the environment. Environmental Issues. , 20 (1):10-15 Batha, R. and Atlas, R. M. 1997. Biodegradation of oil in sea water, writing factor and artificial stimulation In: The microbial degradation of oil pollutants. ( D. G. Ahern and S. P. Meyers(eds). Centre for Wetland Resources, Louissiana. Pp 147-152. Bonaventura, C. nd Johnson, F. M. 2005. Healthy environments for healthy people: Bioremediation today and tomorrow”. Environmental Health Perspectives, 105: 5 – 20. Francisco, M. A. and Speight, J. G. 1999. Changes in the nature of chemical constituents during crude oil distillation. Journal of Studies in Petroleum Composition 45 (6): 733 740. Keeler, R. 2003. Bioremediation, healing the environment naturally. Research & Development magazine (2): 34 ??? 40. Nwachukwu, S. C. U. 2000. Enhanced rehabilitation of tropical aquatic environment polluted with crude petroleum using candida utilis. Journal of Environmental Biology 21(3):241-250 Okpokwasili, G. C. 2003.
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