Extensive Biodegradation of Nigerian Crude Oil ...

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Extensive Biodegradation of Nigerian Crude Oil (Escravos Light) by Newly Characterized Yeast Strains a

a

b

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M. O. Ilori , S. A. Adebusoye , O. S. Obayori , G. O. Oyetibo , c

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O. Ajidahun , C. James & O. O. Amund a

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Microbiology, University of Lagos, Akoka, Lagos, Nigeria

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Department of Microbiology, Lagos State University, Ojo, Lagos, Nigeria c

Department of Chemistry, University of Lagos, Akoka, Lagos, Nigeria Available online: 14 Sep 2011

To cite this article: M. O. Ilori, S. A. Adebusoye, O. S. Obayori, G. O. Oyetibo, O. Ajidahun, C. James & O. O. Amund (2011): Extensive Biodegradation of Nigerian Crude Oil (Escravos Light) by Newly Characterized Yeast Strains, Petroleum Science and Technology, 29:21, 2191-2208 To link to this article: http://dx.doi.org/10.1080/10916461003681620

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Petroleum Science and Technology, 29:2191–2208, 2011 Copyright © Taylor & Francis Group, LLC ISSN: 1091-6466 print/1532-2459 online DOI: 10.1080/10916461003681620

Extensive Biodegradation of Nigerian Crude Oil (Escravos Light) by Newly Characterized Yeast Strains M. O. ILORI,1 S. A. ADEBUSOYE,1 O. S. OBAYORI,2 G. O. OYETIBO,1 O. AJIDAHUN,3 C. JAMES,3 AND O. O. AMUND1 Downloaded by [Ganiyu Oyetibo] at 15:01 06 October 2011

1

Microbiology, University of Lagos, Akoka, Lagos, Nigeria Department of Microbiology, Lagos State University, Ojo, Lagos, Nigeria 3 Department of Chemistry, University of Lagos, Akoka, Lagos, Nigeria 2

Abstract Because microbial degradation is known to be an efficient process in the in situ decontamination of oil-bearing environments, it is believed that development of effective bioremediation strategies will be aided by microbial sourcing of novel and competent hydrocarbon degraders with a broad and unusual substrate spectrum. Thus, in keeping with this objective, two Candida strains (MN1 and MC1) isolated after a repeated batch enrichment technique were tested for their biodegradation potentials on Nigerian crude oil, Escravos light. Axenic cultures of strains MN1 and MC1 grew at a rate of 1.623 and 0.586 d 1 , respectively, in mineral salts medium supplemented with 8.4 g L 1 of crude oil. Whereas strain MN1 degraded aliphatic fractions by 97.6% and the aromatics by 74.61%, the corresponding values obtained for MC1 were 97.2% and 67.29% during the 14-day incubation period. The gas chromatography (GC) fingerprinting of aliphatic fractions showed major degradation of heptadecane (C17), octadecane (C18), nonadecane (C19), eicosane (C20), undodecane (C21), tricosane (C23), hexacosane (C26), octacosane (C28), and nonacosane (C29) in less than 6 days, whereas nearly 100% of these fractions including the isoprenoid molecules was metabolized in 14 days. Among the aromatic fractions that were nearly eliminated during the cultivation period were naphthalene, phenanthrene, fluoranthrene, chrysene, benzo(a)anthracene, benzo(b)fluoranthrene, and benzo(a)pyrene. Interestingly, substrate uptake studies showed that both strains grew very well on petroleum cuts, biphenyl, phenol, xylene, and quite a number of polycyclic aromatic hydrocarbons including pyrene, phenanthrene, and anthracene. Keywords biodegradation, bioremediation, Candida spp., hydrocarbons, pollution

Introduction One of the issues that has remained permanent in public discourse in Nigeria is the unprecedented pollution of the Niger-Delta region of the country, which unfortunately has precipitated massive militancy activities. More than 90% of Nigeria’s oil reserves are found in this region. Unfortunately, activities of oil prospecting and petrochemical and allied industries have contributed immensely to the massive degradation of the NigerDelta environment for more than six decades. The problem is further compounded by Address correspondence to Sunday A. Adebusoye, Ph.D., Faculty of Science, Department of Microbiology, University of Lagos, Akoka, Yaba, Lagos, Nigeria. E-mail: [email protected]

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the lack of appropriate regulatory acts and/or enforcement. Oil spills are not properly monitored and often not reported. Spillage resulting from accidents and sabotage are regular occurrences, and these have resulted in unquantifiable human and economic losses as well as mounting hostilities between oil companies and their host communities. It has become a national priority, therefore, to decommission these sites in order to arrest further ecological consequences and the chaotic relationships between the oil companies and their host communities by developing effective treatment techniques. Although a number of well-established physical and chemical techniques exist for petroleum contamination, stricter environmental regulations, high costs, and low public acceptability is driving the quest for dependable, cost-effective methods for destruction of petroleum pollutants, and biological methods are accepted as the most cost competitive (Diaz et al., 2002; Van Hamme et al., 2003; Hamamura et al., 2006; Gouda et al., 2007). Because effective biological treatment of petroleum pollution relies heavily on a fundamental understanding of how microorganisms grow on hydrocarbons, it is necessary to perform laboratory feasibility tests to determine the microbial potential to degrade the pollutants. Such studies would lead to a better understanding of the growth dynamics of hydrocarbonoclastic organisms, the extent of petroleum metabolism, end product distribution, and the diversity of substrates utilized by the organisms. Degradation of crude petroleum is complicated, because they are made up of a complex mixture of various concentrations of aliphatic (n-alkanes, iso-alkanes, cycloalkanes), phenolics, aromatics, polycyclic aromatics (PAHs), heavy metals, asphaltenes, and resins (Medina-Beliver et al., 2005; Hamamura et al., 2006). This complexity presents a particular challenge to oil-degrading microorganisms. Studies concerning the relationship of the chemical composition of petroleum showed that oil containing a higher concentration of aliphatics was found to be more susceptible to microbial attack (Walker et al., 1976; Amund and Akangbou, 1993; Lal and Khanna, 1996). Among the aliphatics, straight- and branchedchain alkanes are metabolized first by microorganisms, whereas the long-chain alkanes (C28-C32), cycloalkanes, and asphaltenes are very refractory to degradation (Perry, 1984; Leahy and Colwell, 1990). Previously, Amund and Akangbou (1993) reported differential degradation of four Nigerian crude oils that differ substantially in fractional composition. Among the four crudes, the highest degradation (45%) was obtained for Bonny Light, which, interestingly, had the highest proportion of saturates (81.11%) compared with the other three. The abilities of axenic cultures of microorganisms and a consortium of known and unknown microbial composition to metabolize crude petroleum have been demonstrated by many investigators (Atlas and Bartha, 1972; Amund, 1984; Lal and Khanna, 1996; Adebusoye et al., 2007). Most of these studies relied mainly on the disappearance of the aliphatic fractions as a measure of biological degradation with little or no information on the fate of the aromatic components, which, unfortunately, are mostly responsible for the toxicity, carcinogenicity, and refractile properties of crude oil. However, Venkateswaran et al. (1991) reported the degradation of 50% of the total hydrocarbons of weathered crude oil, of which PAHs accounted for 25%. Similarly, Lal and Khanna (1996), using a pure culture of Acinetobacter calcoaceticus S30, observed 50% degradation of Bombay light crude, of which aliphatics were degraded more (74.1%) than the aromatics (54.3%), using a pure culture of Acinetobacter calcoaceticus S30. In the same study, using a different crude oil (Gujarat), the authors reported 37% degradation, with both aliphatics and aromatics degraded equally when Alcaligenes odorans P20 was used as inoculum compared to 29% degradation obtained for strain S30, of which the aliphatics accounted for sixfold higher degradation than the aromatic fractions. In spite of this body of knowledge on

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microbial degradation of petroleum, very few studies (Amund and Nwokoye 1993; April et al., 2000; Clemente et al., 2001; Saraswathy and Hallberg, 2002; Yamada et al., 2002; Mesyami and Baheri, 2003; Husaini et al., 2008) have demonstrated biodegradation capabilities of yeast species. Recently, Husaini et al. (2008) isolated multiple fungi species with the ability to degrade linear alkanes (n-C15 to n-C23 range) present in spent motor oil; no mention was made of the potentials of these organisms on the aromatic fractions of the oil. In the current study, however, we investigated the biodegradability of a Nigerian crude oil (Escravos light) by two yeast strains isolated from a soil with a long history of hydrocarbon contamination. Contrary to previous findings that yeasts are only capable of utilizing the aliphatic fractions of crude oil (West et al., 1984; Okpokwasili and Ibe, 1987), our investigation revealed extensive degradation of both the aliphatic and aromatic fractions, thus effecting extensive degradation of the oil.

Experimental Source of Microorganisms Hydrocarbon-degrading yeasts were developed by an enrichment procedure from soil samples collected from Total Nigeria PLC fuel depot, Apapa, Lagos. The depot receives about 40 million liters of petroleum products monthly from the Apapa jetty through a network of pipelines of 25 km long. The pipeline, although deeply buried, is prone to leakages due to age, pressure from the product vessel, illegal road repairs, and sabotage. Consequently, the Apapa soil is constantly contaminated with hydrocarbons, including spent oil drained from heavy-duty power generators. The soil collected between 0 and 15 cm depth with the aid of a sterile soil auger was used to inoculate a mineral salts (MS) medium previously described by Kästner et al. (1994). The medium was fortified with trace metals solution (Bauchop and Elsden, 1960), made selective by the addition of streptomycin, after which the pH was adjusted to 4.5. The medium was supplemented with 1% (v/v) Escravos crude oil. Incubation was performed at room temperature (29ıC ˙ 1.0ı C) for 14 days. Isolates were purified on Sabouraud dextrose agar. The yeast isolates were subsequently identified based on the taxonomic schemes and descriptions by Smith (1969), Barnett and Pankhurst (1974), and O’Donnell (1979). Physicochemical Analysis of the Soil Sample The soil physicochemistry was evaluated using standard analytical protocols described previously (Nelson and Sommers, 1982; Association of Official Analytical Chemists, 1990; Chopra and Kanwar, 1998; Singh et al., 1999). Heavy metal analyses of the samples were determined using flame atomic absorption spectrophotometry. Growth of Organisms Growth of the isolates was monitored in a 250-mL Erlenmeyer flask containing 50 mL of MS medium with 8.4 g L 1 of Escravos light crude oil as substrate. Organisms were grown overnight on Sabouraud dextrose broth, centrifuged and washed twice with MS medium, and then used as 1% (v/v 1 ) inoculum (106 cfu/mL 1 ). The flasks were set up in three replicates and were incubated at room temperature. Control flasks were inoculated with heat-attenuated cells. A set of replicate flasks was sacrificed at regular

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intervals to evaluate the extent of degradation as well as the cell densities. Cell densities were determined as described recently (Ilori et al., 2008).


Oil Analyses Residual oil was extracted from the culture fluid with n-hexane and quantified gravimetrically (Yveline et al., 1997). The extracted oil was fractionated into saturate and aromatic fractions by silica gel liquid column chromatography (Wang et al., 1994). Fractions were analyzed by a gas chromatograph fitted with flame ionization detector (Varian 3700 GCFID, Varian Inc., Palo Alto, CA) using a 30 m 0.25 mm (internal diameter) 0.25 m (film thickness) glass capillary column (HP-5) and helium as the carrier gas. Gas chromatography–mass spectrometry (GC-MS) analyses were carried out using an ion trap detector coupled to a Varian 3700 GC. The carrier gas flow rate was 0.7 cm3 s 1 . The oven for both GC-FID and GC-MS was maintained at an initial temperature of 50ı C for 5 min, increased by 8ı C min 1 to 160ıC, followed by 15ıC min 1 to 180ı C and 6ı C min 1 to a final temperature of 300ıC, which was maintained for 2 min. The injector and detector temperatures were set at 200ıC and 320ıC, respectively. Degradation was determined as percentage of hydrocarbons relative to the amount of the hydrocarbons of the corresponding control samples. Individual peaks were identified based on a preset library in the system. Chemicals High-purity hydrocarbons as well as Escravos light crude oil were obtained from Chevron Nigerian Limited. All other chemicals were of analytical grade or higher. The Escravos crude was dark brown in color, had a specific gravity of 0.84 and pH 5.24, as well as aromatic and saturate concentrations of 22.05% and 69.74%, respectively (Amund and Akangbou, 1993). Statistical Analyses Mean generation times and specific growth rates were calculated using nonlinear regression of growth curves. Regression, correlation, t-test. and variance analyses were performed using the Prism version 5 software program (GraphPad Software, San Diego, CA).

Results Physicochemical Composition of the Apapa Soil The physicochemistry of the contaminated soil is presented in Table 1. The soil is oily, non-sticky, and dark brown in appearance. The results showed that the sample was heavily contaminated with hydrocarbons (14.28%) and some heavy metals, including lead (8.80 mg kg 1 ) and iron (15.38 mg kg 1 ). In addition to being alkaline, the soil had a salinity of 10.2%. The data also revealed a general lack of mineral nutrients, which are needed in relatively large quantities for proper functioning of microbial cells. For instance, the total sulfate and nitrogen detected were 0.002 and 0.142 mg kg 1 , respectively, and the level of phosphate was 1.12 mg kg 1 . By implication, the biological treatment of this soil would require necessary adjustments of nitrogen, phosphorus, and potassium


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Table 1 Physicochemistry of the polluted soil Parameter

Level detected

Physical appearance pH Moisture (%) PO34 (mg/kg) Sulfur (mg/kg) Total nitrogen (mg/kg) Salinity (%) Cu (mg/kg) Fe (mg/kg) Pb (mg/kg) Zn (mg/kg) K (mg/kg) Mn (mg/kg) Total organic matter (%) Total hydrocarbon (%)

Dark brown, not sticky and oily 8.0 9.73 1.12 0.002 0.142 10.2 1.93 15.38 8.80 3.05 0.01 8.03 25.71 14.28

Values are means of three replicate determinations.

concentrations to stimulate biodegradation of the petroleum contaminant. The proportion of hydrocarbonoclastic yeasts within the heterotrophic community was on the order of 1.5%, which is quite significant compared to less than the 1.0% that would be expected from a pristine soil. The results further exposed the level of pollution that the soil was constantly subjected to. Isolation and Identification of Yeast Strains Several hydrocarbons present in crude oil were used to select for competent organisms. A microbial consortium developed from the enrichment protocol degraded the oil in less than 7 days. The composition of this consortium indicated the presence of only four types of clones on solid medium. Two of the strains exhibiting excellent metabolism of crude oil and diverse hydrocarbon compounds were subsequently selected and used in this study. The isolates were identified using their microscopic morphological, biochemical, and physiological attributes as Candida parapsilosis strain MN1 and Candida sp. strain MC1. These organisms were medium, oval, budding cells that produce pseudomycelia and ferment and assimilate sugars including glucose, galactose, sucrose, maltose, and raffinose. The temperature optimum for growth was 20ıC–30ıC. Whereas strain MN1 failed to assimilate citrate and nitrate, MC1 was sustainable on the former and capable of reducing the latter. An investigation into the substrate versatility of the yeast isolates revealed broad substrate diversity when grown on pure hydrocarbons as sole sources of carbon and energy. Among the substrates that supported growth as depicted in Table 2 were anthracene, pyrene, phenol, benzene, phenanthrene, biphyenyl, benzoate, toluene, xylene, and petroleum cuts. However, benzene and cyclohexane were not utilized.

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M. O. Ilori et al. Table 2 Substrate spectral of yeast strains


Substrate Benzene Biphenyl Phenanthrene Pyrene Anthracene Xylene Toluene Cyclohexane Phenol Crude oil Kerosene Diesel Spent motor oil Engine oil

MN1

MC1

C C C CC C CC

CC CC C C C CC

CC CCC CC CCC CCC CC

CC CCC CC CCC CCC CC

C, Poor growth; CC, good growth; CCC, luxuriant growth. Liquid and solid substrates were supplied at 1% (v/v) and 100 ppm concentrations.

Growth Characteristics of Strain MN1 and MC1 Escravos light was tested for its biodegradability using the two yeast species. The cultures were sampled at different time points, at which time the concentrations of n-alkanes, branched alkanes, and PAHs remaining in the culture fluids were determined by GC-FID. At each time point the population densities of the organisms were evaluated by a standard plate count procedure concomitant with residual crude oil concentration by a simple weight loss technique. In this study, losses due to nonbiological factors were eliminated because the data obtained from experimental flasks were resolved with reference to heatinactivated controls. The growth curves as illustrated in Figure 1 revealed that none of the strains grew with a lag. The fungal populations increased by 6–7 orders of magnitude with generation times of 0.427 and 1.196 days (t D 0–10 days), respectively, for MN1 and MC1 (Table 3). This growth increase was accompanied by substrate-level depletion as well as reduction in the pH of the culture media from 4.72 to 3.45 for MN1 and from 4.61 to 3.69 for MC1 (Figure 1). According to Figure 2, at the beginning of the experiment (within 6 days) the oil was rapidly depleted at least down to concentrations lower than 30%. Overall, strain MN1 degraded nearly 92% of the oil in 14 days, whereas approximately 90% was obtained for MC1. During this period, amounts of aliphatics depleted by MN1 were 97.63% and aromatics 74.61%. The corresponding values obtained for MC1 were 97.19% and 67.29%, respectively, for aliphatics and aromatics (Figure 2, Table 3). It is noteworthy that despite the fact that cell population assumed a decreasing trend by day 10, metabolism of the oil continued, although at a much slower pace. The rate of utilization of the oil and aliphatic fractions was fastest (nearly twice the rate observed for the next 8 days) within the first 6 days of incubation and was faster in MN1 that MC1. For instance, the rate of consumption of the aliphatic fractions obtained during the first 6 days of propagation in flasks inoculated with strain MN1 was 12.40%/d



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Figure 1. Growth curves of yeast strains on Nigerian Escravos light crude oil. Data values represent averages of three replicate determinations.

, MN1; #, MC1.

compared to 2.91% d 1 recorded 8 days after, thus, giving an overall consumption rate of 6.97%/d. The trend observed for the aromatic fractions contrasted sharply with the aliphatic components. The degradation of the former was slower at the onset but increased subsequently (see Table 3). According to the kinetic data summarized in Table 3, one is apt to conclude that strain MN1 was more efficient in detoxification of the oil components. Table 3 Growth kinetics of hydrocarbon-utilizing yeast strains Degradation rate (%d % Degradation Isolate

Tg (d) (d

MN1 MC1

0.427 1.196

1

1.623 0.586

)

Day 6

1)

Day 14

Overall

Crude

Aliph

Arom

Crude

Aliph

Arom

Crude

Aliph

Arom

Crude

Aliph

Arom

91.87 89.71

97.6 97.2

74.61 67.29

10.44 8.53

12.40 9.87

4.58 4.52

3.65 4.75

2.91 4.75

5.90 5.02

6.52 6.41

6.97 6.94

5.33 4.81

Tg, mean generation time (d); , specific growth rate (d 1); Crude, crude oil; Aliph, aliphatic; Arom, aromatic. Percentage degradation values were evaluated with reference to the amount recovered from control flasks at day 14. Crude oil was supplied at 8.4 g/L concentration. All values are means for triplicate determinations.


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Figure 2. Time course degradation of aliphatic () and aromatic () fractions of Escravos light crude by strains MC1 and MN1. Crude oil degradation ( ) was determined by gravimetric analysis and fractions were evaluated by net decrease (FID area counts) in experimental cultures compared with that of the heat-attenuated controls. Initial substrate concentration was 8.4 g/L. The oil was steadily emulsified and depleted completely with visual disappearance of the slick from the liquid surface. Data values represent averages of three replicate determinations.

However, comparison of the degradation competence of both strains yielded lack of statistical significance at a 0.05% confidence limit, thus suggesting relatively similar catabolic functions. Changes in Profile of Aliphatic and Aromatic Fractions of the Oil Scrutiny of the GC fingerprints can be useful to gain more insight into microbial metabolism of hydrocarbon mixtures. As a general rule, the fingerprints of crude petroleum are constituted by a continuum, built up by a number of fused peaks belonging to the compounds present in relatively low concentrations superimposed to this, some single peaks are generally present belonging to compounds with relatively higher concentrations. Among the latter are all n-alkanes, which can be easily recognized because they are generally more intense and equispaced in the chromatogram. Figures 3 and 4, panel A, show the GC-FID traces of the Nigerian Eseravos crude in control flasks; panels B and C illustrate the fingerprints of the residual oil extracted from the culture

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Figure 3. Gas chromatographic profiles for Escravos crude oil recovered from the culture fluid of MN1 at intervals during a 14-day aerated batch cultivation. Undegraded oil (control), A; B and C are respective degraded oil samples after 6 and 14 days of incubation. The oil substrate was supplied at a concentration of 8.4 g/L. Note the complete disappearance of hydrocarbon peaks in panel C.

M. O. Ilori et al.


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Figure 4. Gas chromatographic profiles for Escravos crude oil recovered from the culture fluid of MC1 at intervals during a 14-day aerated batch cultivation. Undegraded oil (control), A; B and C are respective degraded oil samples after 6 and 14 days of incubation. The oil substrate was supplied at a concentration of 8.4 g/L. Note the complete disappearance of hydrocarbon peaks in panel C.



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fluids of MN1 and MC1 after incubation periods of 6 and 14 days. The biodegradation of the crude was readily apparent in the GC charts, which showed that n-alkanes and other aliphatics have been removed. An analysis of the fingerprints summarized in Figures 5–7 showed that all saturate components of the complex mixture were consumed, although to a different extent and with varying depletion rates that decreased with increasing number of carbon atoms. This observation is especially true for extracts recovered from MN1 incubations, although not without some exceptions. Nearly all of the lighter and heavy fractions ranging from C12 to C29 with the exception of docosane (C22), tetracosane (C24), hexacosane (C26), and octacosane (C28) were degraded by this strain within 6 days (Figure 5). In the case of strain MC1, though significant reduction (65–83%) in the n-alkane fractions was observed in 6 days with the exception of C28 (15%), total consumption of all of the representative peaks occurred by day 14. An interesting observation in the MC1 incubations is the fact that increasing carbon length appeared to have little or no influence on the degradation dynamics of the organism. For instance, C16, C23, C26, and C29 were completely utilized within the first 6 days of cultivation, whereas it took additional 8 days to achieve the same fit with other saturates of the oil (Figures 4 and 5). In spite of the near disappearance

Figure 5. Analysis of the aliphatic components of crude oil recovered in culture fluids at day 6 ( ) and day 14 () during growth of yeast strains MN1 and MC1. Percentage degradation represents the net decrease (in FID area counts) in experimental cultures, compared with that of the heat-attenuated controls. Values presented are averages of three replicate determinations. The crude oil substrate was supplied at a concentration of 8.4 g/L.


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Figure 6. Percentage reduction in nC17/phytane and nC18/phytane ratios during growth of strains MN1 and MC1 on Escravos crude oil. and represent percentage reduction in the ratios at days 6 and 14 of incubation.

of the saturate fractions observed in both incubations, statistically (t-test), the catabolic functions of the organisms were significantly different. The nC17/phytane and nC18/phytane ratios were measured to provide an estimation of the degree of degradation of the aliphatic fractions of the hydrocarbons. Changes in the relative abundance of the C19 and C20 isoprenoidal alkanes, pristane and phytane, to their association with nC17 and nC18 n-alkanes, heptadecane and octadecane, are often used to assess the degree of microbial degradation that an oil has undergone. Bacteria degrade n-alkanes quite readily, whereas the isoprenoids are relatively resistant to microbial degradation, resulting in a decrease in the ratios with increasing levels of degradation. Changes in nC17/pristane and nC18/phytane ratios as well as the percent degradation of the isoprenoids recovered from the culture fluids are presented in Figure 6. As depicted in this figure, pristane was degraded at a faster rate than phytane by both strains. In fact, twice as much phytane utilized was obtained for pristane in 6 days. During this period, significant reduction in the ratios was observed for strain MN1, whereas an insignificant decrease in nC17/pristane ratio (from 2.718 to 2.628; 3.10%) was obtained



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Figure 7. Analysis of the aromatic components of crude oil recovered in culture fluids at day 6 ( ) and day 14 () during growth of yeast strains MN1 and MC1. Percentage degradation represents the net decrease (in FID area counts) in experimental cultures, compared with that of the heat-attenuated controls. Values presented are averages of three replicate determinations. The crude oil substrate was supplied at a concentration of 8.4 g/L. Naph, naphthalene; Aceph, acephthene; Fluor, fluorene; Phen, phenanthrene; Anth, anthracene; Fluora, fluoranthrene; Pyre, pyrene; Benza, benzo(a)anthracene; Chrys, chrysene; Benzf, benzo(b)fluoranthrene; Benzp, benzo(a)pyrene; Indeno, indeno(1,2,3cd)pyrene; Benzk, benzo(k)fluoranthrene; Benzg, benzo(g,h,i)fluoranthrene.

in MC1 incubations. In both cases, however, there was a total disappearance of the isoprenoid peaks by the end of the experiment. The dynamics of degradation of the aromatic components was slightly different from the aliphatics. Both Candida spp. exhibited a similar attack on the aromatics as shown in Figure 7. The profiles of the aromatics during the first 6 days indicate a recovery of significant concentrations of these fractions (with some exceptions) in the culture fluids of the organisms. With the exception of acephthene (15.1–25.1%) and fluorene (12.3– 37.38%), which was sparingly attacked, significant degradation was observed for all of the aromatic components of the oil. Although the compositions of benzo(b)fluoranthrene and benzo(a)pyrene were lowest, all of these compounds were completely depleted within 6 days of incubation. When the concentrations of individual aromatics recovered from

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the yeast’s culture fluids at the end of the study were fitted into statistical analysis, in sharp contrast to aliphatic analysis, the results did not differ significantly, indicating that relatively similar aromatic catabolic functions exist in both strains.


Discussion Hydrocarbon degradation, particularly of crude oil by microorganisms, has been extensively reported (Leahy and Colwell, 1990; Amund and Akangbou, 1993; Lal and Khanna, 1996; Head et al., 2003; Hamamura et al., 2006; Adebusoye et al., 2007). These studies have also shown that membrane-bound, group-specific oxygenases and mechanisms for optimizing contact between the microorganism and the water-insoluble hydrocarbon are the two essential characteristics that define the hydrocarbon-oxidizing microorganisms. The fact that the oxygenases are group specific means that individual organisms could metabolize only a limited range of hydrocarbon substrates (Britton, 1984) and that only mixture of different organisms could efficiently degrade crude oil and petroleum fractions. However, it appears that our isolates are exceptional in that axenic cultures of both Candida strains MN1 and MC1 exhibited extensive metabolism of the aliphatic and aromatic components of the oil. The proportions of hydrocarbon-utilizing yeasts relative to heterotrophic ones was quite high (Table 1) and suggestive of heavy petroleum contamination of the soil; previous studies have demonstrated that undisturbed soils usually have less than a 1.0% hydrocarbonoclastic population (Atlas, 1992, 1995). This extensive pollution may have paved the way for selective pressure for the evolution of new catabolic pathways and competent petroleum degraders such as the yeast strains obtained in the present study. It is generally known that polluted sites exert environmental pressure on fundamental ecological parameters such as abundance, diversity, nutrient recycling, and the food chain. In these niches, therefore, pathways and metabolic networks have been developed as adaptive responses to harsh conditions. It is therefore not surprising that most xenobiotic degraders have been isolated from sites contaminated with target pollutants. The biomass concentrations increased considerably during the first 10 days as illustrated in Figure 1. The growth rate was higher in MN1 than MC1, though not significantly .p < 0:05/. Subsequently, the cell populations decreased. Quite interestingly, consumption of the oil substrate continued in spite of changes in the growth curves from the exponential to decline phase. It is, therefore, obvious that this trend cannot be ascribed to a decrease in oil concentration alone or a decline in the available carbon source; rather, it could be due to the accumulation of toxic metabolites and/or a decrease in pH of the culture media. A more logical explanation would be that further utilization of the oil at this stage of growth did not result in growth or the carbon was not incorporated into the cellular architecture. Qualitative analysis of the crude petroleum before and after biodegradation showed that the n-alkanes fractions are more easily degraded than aromatic fractions. These results are in close agreement with those obtained previously by several workers (Wang et al., 1998; Nocentini et al., 2000; Diaz et al., 2002). Similar findings were published recently by Gouda et al. (2007) while working on the ability of pure bacterial cultures to degrade kerosene. Overall, our study revealed that nearly 92% of the initial hydrocarbons contained in the crude oil were utilized by both strains MN1 and MC1. It is obvious from Figures 3 and 4 that nearly all of the linear alkanes were significantly metabolized within the first 6 days of the experiment, with near disappearance in MN1 incubation. The isoprenoids resisted for a while but were eventually completely assimilated (Sugiura



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et al., 1997; Chaineau et al., 2005; Adebusoye et al., 2007). It is noteworthy that linear alkanes were degraded at relatively similar rates (especially in flasks inoculated with MN1) regardless of the number of C atoms of the linear hydrocarbon, contrary to earlier reports (Gouda et al., 2007; Husaini et al., 2008). The ratios of nC17/pristane and nC18/phytane were remarkably different during the first 6 days of cultivation (Figure 6). The decrease in these ratios is evidence that the destruction of nC17 and nC18 is preferential to the isoprenoids. According to April et al. (2000), the reduction in peak heights of pristane and phytane is indicative of microbial degradation and susceptibility of other isoprenoidal alkanes to attack. Many investigators have used this observation to assess the degree of oil degradation in natural systems following bioremediation (Diaz et al., 2002; Adebusoye et al., 2007). The disappearance of pristane and phytane from both MN1 and MC1 incubations in less than 14 days shows that these hydrocarbons are biodegradable and their use as biodegradation index may only be valuable in the early stages of biodegradation. Therefore, their use as biomarkers should be approached with caution. Pritchard et al. (1992) observed that indigenous microorganisms in Prince William Sound biodegraded pristane and phytane almost as fast as their corresponding readily biodegradable straight-chain C17 and C18 analogues. The use of more recalcitrant isporenoids should be advocated for in order to quantify biodegradation more rigorously, even after pristane and phytane have been substantially degraded. Between 67 and 75% of the aromatic fractions of the crude oil was degraded in the 14-day incubation period. Most of these ring compounds were utilized between days 7 and 14. Therefore, the hydrocarbon residue left in the culture flask was most likely to contain mostly asphaltenes and aromatics. Analysis of the individual aromatics making up the oil showed that all of the PAHs were substantially metabolized in 14 days with the exception of acephthene and fluorene (Figure 4). It is not surprising that both yeast strains were able to grow on all aromatics tested with the exception of benzene as sole sources of carbon and energy (Table 2). Previously, Fedorak and Westlake (1981) reported a more rapid attack of aromatic hydrocarbons during the degradation of crude oil by marine microbial populations from a pristine site and a commercial harbor. In a parallel study, Horowitz and Atlas (1977), using an in situ continuous flow system, and Bertrand et al. (1983), using a continuous culture fermentor, observed degradation of all fractions of crude oil at similar rates, in marked contrast to results obtained from other investigations. A possible explanation for the observed degradation of the aromatic fractions, which were previously considered relatively recalcitrant to biodegradation (Walker et al., 1975), can be ascribed to cometabolism in which the PAHs (nongrowth hydrocarbons) are oxidized in the presence of hydrocarbons, which can serve as growth substrates (Perry, 1979) and/or relaxed specificity of the requisite enzymes. Evidence exists that naphthalene dioxygenase, for instance, is now known to be a versatile enzyme, able to catalyze a wide variety of reactions (van Hamme et al., 2003). Similarly, there is molecular and biochemical evidence that the naphthalene plasmid degradative enzyme system could mineralize other PAHs, such as phenanthrene and anthracene (Sanseverino et al., 1993). The present study has shown that Candida strains were able to grow effectively on crude oil, metabolizing both the aliphatic and aromatic components. Problems associated with the use of fungi in hydrocarbon biodegradation bothered on slow growth and concentration of pollutant utilized. Though bacteria can grow rapidly on hydrocarbons at elevated concentrations, such features are rare in fungi. For instance, Husaini et al. (2008) recently demonstrated the ability of Penicillium sp. P1, Trichodermal asperellum TUB F-756, and T. asperellum Tr48 to grow on spent motor oil at 1% (v/v 1 ) concentration.

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However, these strains were propagated for over 2 months before extensive degradation of the oil fractions was observed. It is noteworthy that our isolates extensively degraded similar concentrations of crude oil in less than 14 days. Overall, the broad PAH and aliphatic-degrading capabilities in our organisms may be attributed to relaxed initial enzyme specificity for PAHs, the presence of multiple oxygenases, and the presence of multiple metabolic pathways or multiple genes for isofunctional pathways. Although as reviewed by van Hamme et al. (2003), the presence of both alkane- and aromatic hydrocarbon-degrading genes in single strains appear to be common, it is relatively a rare phenomenon in yeasts; to our knowledge, none has been demonstrated to process the catabolic qualities observed in the yeasts reported in this study.


Acknowledgments This article is dedicated to the memory of Christy James, who passed on shortly before its publication. May her gentle soul rest in peace.

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