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International Journal of Advances in Science and Technology, Vol. 3, No.

3, 2011

Studies on the Biodegradation Potentials of Candida tropicalis and Candida utilis on Petroleum Hydrocarbons
Bridget Amechi1
1

Department of Medical Laboratory Services, F.M.C, Umuahia, Nigeria P.O.Box 2378, Umuahia, Abia State, Nigeria. bridget_amechi@yahoo.ie

Ome Achi2 U.N. Ekwenye2


2 Department of Microbiology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

Abstract
Biodegradation studies was performed to investigate the diesel oil and kerosene degrading ability of two strains of yeast identified as Candida tropicalis and C. utilis and isolated from the vicinity of motor mechanic workshops and a railway station. Each treatment contained 1% (v/v) kerosene or diesel as sole carbon and energy source in mineral salts medium. Under aerobic conditions and after 16 days of incubation, C. tropicalis exhibited a marked reduction in the kerosene hydrocarbon components by 74.4% while C. utilis effected a reduction by 60.0%. Similarly, the diesel components were reduced by 69.0% and 48.0% respectively. Statistically, there were no significant differences (p>0.05) in the hydrocarbon utilization by the isolates. The mixed cultures of the organisms reduced kerosene hydrocarbon components by 91.0% and diesel by 88.0%. Gas chromatographic analysis of residual products of the degradation of kerosene and diesel hydrocarbons showed that lower carbon number (C8-C9) alkanes were readily depleted in the medium than C10-C20 carbon numbers. The two organisms showed high biosurfactant production and high adherence to hydrocarbon. A reduced surface tension to values of 28.90.4D/cm and 43.30.3D/cm was observed. The observed reduced surface tension, production of biosurfactants and high cell hydrophobicity are the plausible mechanism by which the isolates degraded the hydrocarbons.

Keywords: Biodegradation, Candida albicans, Candida utilis, Hydrocarbon.

1. Introduction
Technological development, increased industrial activities and oil exploration in Nigeria has necessitated a vast increase in the use of fuel oil. Leaks and accidental spillage is known to be a regular occurrence during the exploration, refining, transport and storage of petroleum and petroleum products (Mukherji et al.,2004). Oil spills caused by blowouts, leakages from tanks and dumping of waste petroleum products, Oil contamination of soil and water is found frequently. Accidents are likely to occur in the form of pipeline leaks, train derailments, ship wreckages, storage tank ruptures, and transport accidents, for example, lead to an elevated loading of petroleum hydrocarbons in the soil. Diesel oil, a distillate fraction of crude oil, is one of the major pollutants of soil and groundwater near petrol stations. This result in significant decline in the quality of soil and make it unfit for use (Shabir et al., 2007).). Crude oil is a complex mixture of aliphatic and aromatic hydrocarbons, including volatile components of gasoline, petrol, kerosene lubricant oil and solid asphaltene residues (Yeung et al., 1997). The kerosene fraction poses the greatest pollution problem (SolanoSerena et al., 2000).

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 Contamination of the environment with petroleum hydrocarbons are on the increase, as a result of the use of large quantities of petroleum products and accidental oil spills. The resultant environmental problems arising from oil spillage is well pronounced in the Niger Delta area of Nigeria and is a cause of serious concern. Between 1986 and 2000, the Nigerian Petroleum Industry experienced three thousand eight hundred and fifty four (3,854) oil spill incidents. These spills resulted in the loss of 437,810 barrels of oil into the Nigerian environment (Adeyemi, 2004). Similarly, hydrocarbon pollution is said to be a common pollution incidence with almost nine incidents per day since 2005. This may account for over 15% of all pollution incidents in England and Whales (Environment Agency, 2006). It has also been estimated that over one million tons of oil are spilled into the UK terrestrial ecosystems every year (Ripley et al., 2002). Among several clean-up technologies available to remove petroleum hydrocarbons from the soil and ground water, bioremediation processes are gaining ground due to their simplicity, higher efficiency and cost effectiveness when compared to other technologies (Alexander, 1994). Bioremediation has become a major method employed in the restoration of oil-polluted environments, and attempts to accelerate the natural hydrocarbon degradation rates by overcoming factors that limit microbial hydrocarbon degrading activities ( Atlas,1991) . Introduction of microbial degraders (bioaugmentation) is widely used in bioremediation, but only few investigations have differentiated the role of the introduced microorganisms from that of the indigenous microorganisms. Petroleum hydrocarbons could be considered extensively biodegradable in soils. Differences in the extent of hydrocarbon degradation have been reported (Huesman, 1997, Leahy and Colwell 1990, Morgan and Watkinson 1989), depending on soil type, concentration of total hydrocarbons and oxygen and nutrient availability. Considerable information on the microbial degradation of refined petroleum hydrocarbons is available in the literature but less is known on the biodegradability of some commercial products (Sugiura et al, 1997) such as kerosene and diesel oil (Wongsa et al. 2004). Yeasts are a heterogeneous group of fungi that superficially appear to be homogeneous. Yeasts grow in a conspicuous unicellular form that reproduces by fission, budding, or a combination of both. True yeasts reproduce sexually, developing ascospores or basidiospores under favorable conditions. Yeast-like fungi (imperfect yeasts) reproduce only by asexual means. The identification of these fungi is based upon a combination of morphological and biochemical criteria. Morphology is primarily used to establish the genera whereas biochemical assimilations are used to differentiate the various species (Hupert et al, 1995). Yeasts are more tolerant of changes in pH, salinity and fluctuating conditions in the environment than bacteria, hence its choice over bacteria in this present work. The primary aim of this work is to study the biodegradation potentials of C. tropicalis and C. utilis, isolated from petroleum hydrocarbons contaminated soils, with the possible application in bioremediation of hydrocarbon contaminated sites.

2. Materials and methods


Top layer (0-10cm) soil samples were collected aseptically from oil-polluted sites in different parts of Umuahia metropolis in Abia State, including mechanic workshops and a railway station. All samples were immediately transported to the laboratory within six hours of collection for immediate inoculation into the appropriate media for the isolation of test organisms.

2.1. Isolation of hydrocarbon-degrading microorganisms


The soil samples were thoroughly mixed and 1g of each was dissolved in 10ml of distilled water. 0.1ml of this was then inoculated onto Sabourauds Dextrose Agar plates fortified with 50mg/L Chloramphenicol to check bacterial growth, (containing crude oil); using the streak plate technique and incubated for seven days at room temperature 2820C. After incubation, clearing of oil droplets around single colonies could be detected, and those discrete colonies (hydrocarbon-solubilizing colonies) that grew within three days were aseptically picked at random. The yeast-like isolates were then maintained in Corn Meal Agar slants and stored at 4oC for identification (Mashreghi and Marialigeti, 2005).

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2.3. Growth on hydrocarbons


The mineral salts medium as specified by Mashreghi and Marialigeti (2005), was used as the basal medium for the screening of growth in the presence of hydrocarbons. The screening was to determine the ability of each isolate to utilize kerosene and diesel oil as sole source of carbon and energy. The cultures were adapted to the presence of the hydrocarbons (kerosene and diesel oil), by adding 1% (v/v) into 10ml of the MSM, as described by Chikere and Okpokwasili, (2003).Thereafter, mineral salts broth was supplemented with 1 % (v/v) substrate/liter as described by Mashreghi and Marialigeti (2005). Erlenmeyer flasks containing 50ml of MSB and hydrocarbon were inoculated with 0.1ml of overnight grown cultures and incubated at room temperature28 2oC. Growth was confirmed by visually observing the increase in cell density in comparison with uninoculated controls with respective hydrocarbon source. When no difference was noticed in the turbidity of the flask and that of the control, it was taken as no growth (-), when slight increase in turbidity was noticed it was taken as poor growth (+). Significant increase in turbidity was taken as good growth (++) while luxuriant growth and increases in turbidity was stated as very good growth (+++).

2.4. Basic tests for identification and characterizations of isolates


The isolates were identified based on the taxonomic schemes of Barnett and Hunter, (1972) and Hupert et al., ( 1995). The isolates were examined macroscopically and microscopically using the needle mounts method. Biochemical characterizations of isolates were carried out, using various biochemical tests, and sugar assimilation tests, as described by Cheesbrough, (1984). Taxonomic identification of isolates was also established using the API 20C identification data base (bioMerieux, Marcy-l'Etoile, France). Upon identification of the yeast isolates, they were kept pure by repeated sub culturing unto Sabauraud agar until pure colonies were obtained.

3. Biosurfactant production and isolation


Three different methods were used to aid the screening for biosurfactant production. These included : (i) Modified drop collapsed methods (Xiao et al. 2006), (ii) Blood haemolysis technique (Carrilo et al. 1996) and (iii) surface tension measurement using the Du Nouy tensiometer (CSC scientific tensiometer) as described by Tadros, (2005). To isolate the biosurfactants, the organisms were grown in 500ml Erlenmeyer flasks, each containing 100ml mineral salts medium and mineral oil (5%, v/v) . The flasks were then incubated at 30oC for 96h with constant shaking. The cells were removed by centrifugation, and the supernatant acidified to pH 2.0 using 6N HCl and kept at 4oC overnight, to allow precipitation. The resulting precipitate was then left to dry as the crude biosurfactant (Emine and Aysun 2009).

3.1 Emulsification Activity


The emulsifying capacity was evaluated by an emulsification index (E24), as described by Bodour et al., (2004). The E24 of culture samples (0.2ml) were mixed with 0.5 ml of TM buffer (20 mm Tris [tris(hydroxymethyl)-aminomethane]/ HCl buffer, pH 7.0 and 10 mm MgSO4) and then 0.2 ml of kerosene or diesel oil was added. The tubes were then vortexed at room temperature and at high speed for 1 min and allowed to stand for 24h. The E24 index is given as percentage of the height of emulsified layer (cm) divided by the total height of the liquid column (cm). The percentage of emulsification index was calculated by using the following equation. E24 = Height of emulsion formed x 100 Total height of solution

3.2. Structural properties of biosurfactant. The structural analyses of the crude biosurfactant were performed with the cell free filtrate, by determining the lipid, protein and carbohydrate contents. Ninhydrin and Biuret tests were used to detect the presence of amino acids and protein contents of biosurfactant, respectively. Ninhydrin reaction was performed as described by Bisswanger, (2004). The presence of carbohydrate was detected using the Molisch method, and quantified by the methods of Dubois

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 et al., (1956). The presence of lipid was established, using the Grease spot test.

3.3. Extracellular glycolipids


Presence of extracellular glycolipids was demonstrated by inoculating the yeast cells onto Cetyl trimethyl ammonium bromide CTAB - methylene blue agar plates, as described by Siegmund and Wagner, (1991). Anionic surfactants form a dark blue, insoluble ion pair with cetyl trimethyl ammonium bromide and methylene blue.

4. Biodegradation experiments 4.1. Determination of hydrocarbon content.


The amount of hydrocarbon left after 16 days incubation was determined by extracting the residual hydrocarbon with 50ml of dichloromethane in a round bottom flask for 4h, using the weight loss method (Nocentini et al., 2000), with slight modifications ( dichloromethane solvent was used instead of chloroform). The solvent was then separated from the residual oil by evaporation at 400C overnight in an oven. The amount of residual oil was obtained by subtracting the final weight from the initial weight. The percentage utilization of hydrocarbon was thus calculated, using the formula as described by Ijah and Ukpe (1992). Weight of initial hydrocarbon- weight of hydrocarbon (after treatment) Weight of initial hydrocarbon x100

4.2. Gas chromatographic (GC) analyses


The GC analyses were done at the onset and at the end of the incubation, to evaluate the overall degradation of the hydrocarbons by the isolates as described by Geerdink et al., (1996). Gas chromatographic analyses were performed using Hewlett Packard GC (HP5890), equipped with a flame ionization detector (FID) on HP1 Cross-linked Methyl Silicone capillary column, 15m long and 0.32mm i.d., film thickness 1.0m. The operating conditions were : temperature from 100-2500C at 20oC/min; injector temperature 250oC; detector temperature 250oC; carrier gas helium, linear flow rate 5.2ml/min; pressure 13 psi; injection volume 4l. Measured quantities of the samples were extracted with 10ml of Hexane in a separating funnel. The extract was passed through a GC column packed with silica gel and the aliphatic components eluted with an acetone Dichloro methane mixture.

4.3. Statistical analysis


Analysis of variance of appropriate data, using the SPSS statistical package version 17 where applicable, was used to establish significant relationships or otherwise of the parameters under investigation. Experiments were carried out in triplicate.

5. Results 5.1. Isolation and identification of hydrocarbon degrading Candida spp.


The microorganisms isolated from the soil samples were morphologically different on solid media. They appeared smooth and creamy, or dry and creamy colonies. The presence of zone of clearing around colonies was an indication of hydrocarbon degradation, while the absence of zone of clearing around colonies was an indication of the absence of hydrocarbon degradation. Several of the isolates (Forty one), formed clear zones on agar plates in varying degrees. They were further screened for hydrocarbon utilization using the mineral salts broth. Several of the isolates (Thirty four) were able to utilize hydrocarbon as measured by the degree of turbidity of the medium. Two of the isolates showed the highest degree of turbidity.

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 Based on the morphological, microscopic appearance and carbohydrates assimilation profile in the API 20C identification system, and based on the results obtained in the hydrocarbon-utilization screening test, the two isolates that showed the highest degree of turbidity were identified as C.tropicalis and C.utilis. They were then chosen for further studies on biodegradation.

5.2. Biosurfactant Production and isolation.


Table 1 shows the biosurfactant properties and the relationship between emulsification activities, surface tension and adherence to hydrocarbon by the isolates. C.tropicalis had 42% adherence to hydrocarbon, while C.utilis adherence to the hydrocarbon was 20%. Emulsifying activity for both C.tropicalis and C. utilis were 53% and 48% respectively. An initial surface tension of 68.40.4 and 59.30.4 were reduced to 28.90.4 and 43.30.3 respectively, after incubation by the isolates.

Table 1. Biosurfactant properties of isolates. Parameter Carbohydrate (mg/l) Protein Lipid Heamolytic Activity (mm) Extracellular glycoprotein Emulsification Activity (%) MATH Test (%) C.tropicalis 1.5 + + 15 + 53 42 C.utilis 0.8 + + 11 + 48 20 59.30.4 43.30.3

Initial 68.40.4 Surface tension (D/cm) Final 28.90.4

+ = Positive reaction - = Negative reaction MATH = Microbial Adherence to Hydrocarbons

5.3. Hydrocarbon hydrolysis by cultures.


The percentage hydrocarbon removal after hydrolysis by the cultures is as shown in Table 2. The results showed the reduction of kerosene by 74.4% by C.tropicalis and 60.0% by C.utilis, while diesel oil was reduced by 69.0% and 48.0% by C.tropicalis and C.utilis respectively. However, kerosene and diesel oil were reduced by 91.0% and 88.0% respectively by the mixed culture.

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 Table 2. Hydrocarbon content of media before and after hydrolysis of substrates by cultures

Incubation time Hydrocarbon (Days) 0 Kerosene 16 % Kerosene removal 0 Diesel oil 16 % Diesel removal

Residual Hydrocarbon Content (mg/l) in culture C.tropicalis 1250.06 320.00 74.40 1250.06 510.00 69.00 C.utilis 1250.06 505.00 60.00 1250.06 650.05 48.00 Mixed culture 1250.06 112.92 91.00 1250.06 150.09 88.00

5.4. Gas chromatographic Analyses of extracted hydrocarbons.


The extent of biodegradation was interpreted based on the variation in the peak areas observed in the GC chromatograms. Figure 1 represents the chromatograms of the peak areas produced on day 0 and day 16 of incubation, during the degradation of diesel oil by C.tropicalis. The results showed the peak areas of n-octane (C8), n -nonatane (C9) and n-dedacane (C10) completely removed, while the other n-alkanes were reduced in varying amount. A new peak n-hexacosane (C26) was produced after degradation as shown (Figure 1). Figure 2 is the peak area produced before incubation and after 16 days degradation of kerosene by C.tropicalis. There was no complete removal of n-alkanes. However, there was a considerable reduction in their peak areas as shown in Figure 2. The peak areas produced during the degradation of diesel oil by C.utilis is as shown in Figure 3. C.utilis completely degraded n-octane (C8) and n- nonatane (C9), while the rest of the n-alkanes present in the diesel oil were reduced in varying amount. The Figure 4 shows the peak area produced during degradation of kerosene by C.utilis. No complete removal of n-alkanes was observed. However, there was a considerable reduction in their peak areas as shown. Figure 5 is the chromatograms showing peak areas of diesel oil degradation by the mixed culture on day 0 and 16 respectively. The n-alkanes n-octane (C8), n-nonacane (C9) and n-Tetracosane (C-24) present in the diesel oil were completely degraded by the mixed culture. The other n-alkanes present were however reduced. Figure 6 is the chromatograms of the peak areas produced during the degradation of kerosene by the mixed culture. The n-octane (C8), n-nonacane (C9), n-pentadecane (C15), n-hexadecane (C16) and n-heptadecane (C17) present in the kerosene were completely degraded by the mixed culture at the end of the incubation period. The figures 7 and 8 are the peak areas of the control kerosene and diesel oil respectively.

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B A

Figure :1. Chromatograms of diesel oil degradation by C.tropicalis.


in Mineral salts medium. A= Day 0; B= Day16.

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Figure :2. Chromatograms of kerosene degradation by C.tropicalis. in Mineral salts medium. A= Day 0; B= Day16

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Figure :3. Chromatograms of diesel oil degradation by C.utilis in Mineral salts medium. A= Day 0; B= Day16

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Figure :4. Chromatograms of kerosene degradation by C.utilis. in Mineral salts medium. A= Day 0; B= Day16

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Figure .5. Chromatograms of diesel oil degradation by Mixed culture in Mineral salts medium. A= Day 0; B= Day16

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Figure :6. Chromatograms of kerosene degradation by Mixed culture. in Mineral salts medium. A= Day 0; B= Day16

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Figure :7. Chromatograms of Control kerosene

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Figure :8. Chromatograms of Control diesel oil.

6. DISCUSSION Microbes are the main degraders of petroleum hydrocarbons in contaminated ecosystem; hence biodegradation of hydrocarbons by native microbial population is the primary mechanism by which hydrocarbon contaminants are removed from the environment. The effectiveness of enhancing hydrocarbon degradation through addition of microbial inocula prepared from non indigenous population has been investigated (Atlas, 1993). In this work, a total of sixty one microorganisms were isolated from soil collected from petroleum hydrocarbon contaminated mechanic workshops and a rail station. They were screened for hydrocarbondegrading capabilities. Of the total number screened, thirty four were able to degrade and utilize hydrocarbons. Candida species have been found at sites polluted by petroleum and petroleum derivatives (Okpokwasili and Nwosu, 1990), thus suggesting this genera effectively metabolize hydrocarbon molecules. Candida species have also been identified as providing excellent degradation of petroleum derivatives (Okpokwasili and Nwosu, 1990, Ijah,1996, Ijah and Akpera,1998). Similarly, Adekunle et. al., 2004 also described the successful use of Saccharomyces cerevisiae in the

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 biodegradation of petroleum products. The preliminary estimation of the isolates degradable activity was used as a criterion in the selection of strains RS3 and MW6 for further studies, identified as Candida tropicalis and Candida utilis. The isolation of these organisms from a contaminated environment gives acclimatization advantages of xenobiotics-degrading microbes over non-acclimatized strains. The substrates kerosene and diesel sustained the growth of the yeast isolates C. utilis and C. tropicalis, but the degree of activities varied. This indicated that the isolates have the potential to utilize kerosene and diesel oil as carbon sources. The growth and hydrocarbon utilizing abilities of C. utilis and C. tropicalis are similar to the findings of Okpokwasili and Nwosu (1990) and Obire and Wemedo (1998) which showed that these organisms were among the best hydrocarbon-degrading species. Accordingly, it can be inferred from this study, that diesel oil was slightly preferred by C.tropicalis, while kerosene was rather preferred by C.utilis. Nevertheless, reports of Ghosh and Banerjee (1984) and Oboh et al., (2006) show a preference for kerosene by C. tropicalis. The results of the hydrocarbon degradation by the isolates, with the range between 71.4% - 91.0%, agrees with the report of Rahman et al. (2003), where they reported the percentage degradation of crude oil range - approximately 100% of C8-C11, 83-98% of C12-C21 80-85% of C22-C31, using bacterial consortium. The optimal removal of short-chain and medium-chain aliphatic compounds compared to longer-chain aliphatic compounds in this work corroborates the works of Mukred et al. (2008), where all the four isolates they used, showed maximum removal of short-chain and medium-chain aliphatic compounds. This may seem logical, since short-chain and medium-chain alkanes are said to be generally more easily degraded due to their lower hydrophobicity (Mukred et al., 2008). Statistically, there was no significant difference (p>0.05) in the degradation competence of the two isolates on the two hydrocarbon substrates. Pre-adaptation to hydrocarbons enhanced the degradative capability of the isolates. This is not surprising considering that oilpolluted environments generally have higher numbers of hydrocarbondegrading microorganisms than pristine sites. The ability of hydrocarbon utilizing yeasts isolated from the study sites to degrade hydrocarbons could be attributed to adaptation to these pollutants in their natural environment since they are continuously exposed. The composition of a microbial consortium is an important factor, which must be made to ensure synergistic enhancement of catabolic activities. Haramaya et al. (1997) reported that a microbial consortium called SM8 exhibited higher activity than an axenic culture of light and heavy crude oils. The use of mixed yeast culture in this study was as a result of reported observations that mixed bacterial cultures performed as much as twice in degradation of hydrocarbons than single cultures, provided non is producing inhibitory substances affecting the other (Wilson and Bouwer,1997).This is in conformity with the results obtained in this study with the mixed yeast culture. This may be due to the fact that only few organisms have the full enzymatic capability to mineralize hydrocarbons to carbon dioxide and water. The catabolisms of aliphatic hydrocarbons follow different metabolic pathways. The production of biosurfactant, measurement of surface tension of media and microbial adherence to hydrocarbons were therefore examined to see whether the yeast used both as energy and carbon source for growth. It is also known that microorganisms capable of petroleum hydrocarbon degradation can sometimes produce biosurfactants, which improves the solubility of hydrophobic pollutants and thus, their biodegradability (Kanga et al., 1997). As a result, the selected strains were examined for their ability to produce biosurfactants. The reduction in surface tension of media by C.tropicalis and C.utilis (initial surface tension of 68.40.4 and 59.30.4 D/cm were reduced to 28.90.4 and 43.30.3D/cm respectively), indicated the production of surface active compounds. Similar results were obtained by Banat (1995) and Tabatabaee et al. (2005). Several bacteria that reduced culture broth surface tension to values below 40 D/cm were isolated by Tabatabaee et al. (2005). Data showed cell surface hydrophobicity of 42% and 20% for C.tropicalis and C.utilis. The chromatographic profile of the kerosene and diesel supplement in inoculated and uninoculated control, showed a comparison of the differences in peak numbers and areas. The peaks in this case represented the concentration of hydrocarbons before and after the incubation period. The reduction in concentration and in some cases appearance of multiple peaks is a clear indication that the components of the hydrocarbons were degraded by the yeast cultures. The decreases in the peaks of a wide range of compounds from nC8 and nC24 suggesting biodegradation of the hydrocarbons, agrees with the results obtained by Sharma and Rehma (2009). The lower carbon number n-alkanes were degraded more readily than the middle chain carbon number n-alkanes. In some cases, the appearance of additional peaks in some of the profiles may be as a result of the production of novel hydrocarbons during

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 biodegradation of the substrates, as seen in this work, and had been reported earlier by Walker and Colwell, (1976). In recent years, much attention has been directed towards biosurfactants due to their broadrange functional properties and the diverse synthetic capabilities of microbes. The most important thing is their environmental acceptability, because they are readily biodegradable and have lower toxicity than synthetic surfactants (Desai and Banat,1997). The outer surfaces of microbial cells contain a variety of chemical compounds which may be involved in the attachment of cells to surfaces. Hydrophobic/hydrophilic interactions play a large role in attachment, leading to the development of the concept of cell surface hydrophobicity as a measure of the tendency of a cell to attach to a surface. The results of this study however showed that growth was not observed in the medium supplemented with kerosene and diesel oil respectively without any inocula, thus kerosene and diesel oil were readily used as carbon and energy sources by the yeasts cells. A positive correlation was observed between emulsification activity, cell adherence to hydrocarbon and biomass growth of the isolates in the hydrocarbon media. The observed reduction in surface tension, presence of biosurfactants and cell hydrophobicity are the most plausible explanation for the mechanism by which the isolates degraded the hydrocarbons.

6.1. Conclussion
It has been established in this study that C.tropicalis is a more efficient degrader of hydrocarbons than C.utilis. It can also be said that the mixed yeast consortium gave better results than the individual isolates. These cultures, especially C.tropicalis with good degradation for a wide range of n-alkanes can be used for bioremediation and treatment of hydrocarbon contaminated environment.

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Authors Profile B.O. Amechi: the corresponding author is a laboratory scientist with the Federal Medical Centre, Umuahia, Abia State, Nigeria. O. K Achi is a Professor of Microbiology, a lecturer in the department of Microbiology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. U.N. Ekwenye is a Doctor of Microbiology, a lecturer in the department of Microbiology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

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