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Hydrocarbon degraders from tropical marine environments
Marine Pollution Bulletin 44 (2002) 118–121 www.elsevier.com/locate/marpolbul
Hydrocarbon degraders from tropical marine environments S.S. Zinjarde a,b, A.A. Pant a,* a
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India b Department of Biotechnology, University of Pune, Pune 411007, India
Abstract Analysis of 20 samples of marine mud and water around Mumbai resulted in the isolation of 17 bacteria and yeasts all of which were able to degrade more than 10% of the supplied crude oil. The yeasts strains were important degraders of the aliphatic fraction of crude. All the isolated yeasts belonged to the genus Candida. Using biochemical tests these were identified as Candida parapsilosis, C. albicans, C. guilliermondii, Yarrowia lipolytica, C. tropicalis and C. intermedia. Y. lipolytica was the best degrader utilizing 78% of the aliphatic fraction of Bombay High crude oil. None of these isolates degraded the aromatic or ashphaltene fractions. All the isolates required aeration, nitrogen and phosphate supplementation for optimal degradation. Four out of the six yeasts are human pathogens. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Candida species; Crude oil degradation; Tropical marine environments
1. Introduction Chronic inputs of oil account for a major fraction of the oil entering ports such as those in Mumbai. Transport, pumping of ballast waters, effluent from dry docking, servicing of oil tankers and ships are the major factors contributing towards oil pollution which also affect beaches contiguous to the ports. Chronic oil pollution results in sizable populations of hydrocarbon degraders (Braddock et al., 1995; MacNaughton et al., 1999). Ahrean et al. (1971) and Bhonsale and Mavinkurve (1984) have reported the natural occurrence of marine yeasts in oil-polluted coastal regions. The present paper reports investigations on hydrocarbon degraders in the Mumbai region and identification of yeast isolates responsible for maximum degradation of crude oil.
Artificial seawater contained per litre of deionized water, NaCl, 24.5 g; CaCl2 2H2 O, 1.54 g; KBr, 0.1 g; NaF, 0.003 g; KCl, 0.7 g; H3 BO3 , 0.03 g; Na2 SO4 , 4.09 g; NaHCO3 , 0.2 g; SrCl2 6H2 O, 0.017 g; MgCl2 6H2 O, 11.1 g, pH 8.0 adjusted with 1 N NaOH. The enrichment medium contained ammonium sulphate, 0.5% (76 mM Nitrogen); dipotassium hydrogen phosphate, 0.001% and Bombay High crude oil, 1% as the sole carbon source in artificial seawater at pH 8.0. Environmental samples were incubated at 30 °C for five days. An aliquot of this culture was transferred to fresh medium and the procedure was repeated thrice. The crude oil degrading isolates were maintained on Nutrient Agar and MGYP agar (Malt extract, 0.3%; Yeast extract, 0.3%; peptone, 0.5%; glucose 1%; agar, 2.5%) prepared in seawater. The number of yeast colonies in various samples was determined by total viable counts on seawater MGYP agar plates.
2. Materials and methods 2.2. Oil degradation studies 2.1. Samples and media Samples of ship scrapings, oil contaminated water, sediments and floating debris were obtained from Mumbai harbour and suspended in artificial seawater. *
Corresponding author. Tel.: +91-30-589-3300; fax: +91-20-5893761. E-mail address: [email protected] (A.A. Pant).
After a series of preliminary gravimetric analysis, all degradation data was analyzed on a Shimadzu gas chromatograph. Organisms were grown in 250 ml flasks with 50 ml medium. The test results were compared with uninoculated controls incubated under similar conditions. Residual oil was extracted in 50 ml dichloromethane, concentrated to 5 ml, fractionated on a silica gel column into aliphatic, aromatic and ashphaltene
0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 1 ) 0 0 1 8 5 - 0
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
fractions according to the method of Walker and Colwell (1974). The aliphatic fraction was concentrated to 2 ml and 1 ll was loaded on to a SE 30 column on a GC R1A with nitrogen as carrier gas. Peaks were determined with a flame ionization detector. Temperature was programmed from 60 °C to 250 °C with a 6 °C rise per minute. The results were quantified by comparing the integrated areas under the peaks of test samples with uninoculated controls (Dibble and Bartha, 1976). All results are averages of three observations in duplicate. Proportions of aliphatic, aromatic and ashphaltenes fractions in three different Indian crudes (Bombay High, Assam and Gujarat crude) were determined after fractionation by the method of Walker and Colwell (1974).
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Table 1 Degradation of crude oil by the isolated organisms Isolate no. (bacteria)
% degradation of crude
Isolate no. (yeasts)
% degradation of crude
1, 2, 10 3 4,11 5 6 7 8 9
15 10 12 11 14 13 21 20
1 2 3 4 5 6
34 34 35 45 38 30
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at 200 rpm. % degradation of crude oil (Gravimetry).
2.3. Identification of yeast cultures 3.1. Identification of the yeasts isolates Yeasts were identified in accordance with the methods published by Meyer et al. (1984) and van der Walt and Yarrow (1984) using morphological and biochemical characteristics. Fatty acid methyl ester patterns were determined as described earlier (Zinjarde et al., 1998). 2.4. Standardization of conditions for oil degradation Ammonium sulphate, urea and sodium nitrate were used at concentrations calculated to provide 70 mM nitrogen. Paraffinized ammonium sulphate and urea were used in separate combination with dipotassium hydrogen phosphate (6.5 mM phosphorus). Paraffinized magnesium ammonium phosphate at concentrations providing 70 mM nitrogen and 6.5 mM phosphorus was also checked. Oil degradation in seawater medium was compared with that in distilled water medium. The effect of temperature was determined by incubating the cultures at 15 °C, 30 °C, 40 °C and 50 °C for 5 days. The effect of aeration was determined by varying the agitation rates of the shaker from 0 to 200 rpm. Crude oil was provided in concentrations varying from 0.5% to 2.5%.
3. Results and discussion Enrichment of samples from 20 different sites in the Mumbai coastal region yielded 50 isolates. Among these, seventeen degraded more than 10% of the provided crude (Table 1). Out of these 17, 11 were bacterial cultures and six were yeasts. The yeasts were dominant in populations from dockyards polluted with crude oil. All dockyard samples yielded yeast isolates and in some cases these were the only microbial flora. Total viable counts here were in the order of 107 –108 as compared to 103 –104 per ml from locales outside the dockyards.
None of the yeasts produced a red colour with diazonium blue nor did they produce basidiospores characteristic of basidiomycetous yeasts. On ascosporeinducing media none produced ascospores. Thus they were members of the deuteromycetous group which includes 17 genera. The yeasts produced cream-coloured, non-mucoid colonies without any characteristic aroma, they were unable to form conidia, lacked ballistospores and did not show agglomerates characteristic of Sarcinosporon. The cells of all these yeasts were oval. Oval cells are produced by Candida and Malassezia. Optimum temperature for growth was 30 °C suggesting that the yeasts belonged to the genus Candida. Fig. 1 shows the slide cultures of the isolates. Morphological typing is supported by evidence from fatty acid methyl ester fingerprinting on the Microbial Identification System which confirmed that these yeasts belonged to the genus Candida. The six yeast isolates were identified to the species level using biochemical assimilation and fermentation tests. Meyer et al. (1984) have classified the genus Candida into 10 groups. Isolate No. 4 showed characters of Group V, No. 1 and No. 5 of Group VI and Nos. 2, 3 and 6 of Group VII. The isolate No. 4 belonging to Group V was separated from other species of this group on the basis of its ability to grow at 25 °C, inability to assimilate maltose, cellobiose, its inability to ferment glucose and was identified as Candida (Yarrowia) lipolytica. Isolate No. 1 belonging to group VI was identified as C. guilliermondii on the basis of assimilation of galactose, mannitol, cellobiose, melezitose, melibiose, L -arabinose, ability to grow at 37 °C and glucose fermentation. On the basis of assimilation of galactose, mannitol, cellobiose, melezitose, lactose, ability to ferment glucose but not lactose, isolate No. 5 was identified as C. intermedia. Isolate No. 2 of group VII assimilated galactose, starch, did not assimilate lactose, and fermented
120
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
Fig. 1. Slide cultures of the yeast cultures on Corn Meal agar incubated at 30 °C for 96 h. (a) C. guilliermondii, (b) C. albicans, (c) C. parapsilosis, (d) Y. lipolytica, (e) C. intermedia, (f) C. tropicalis.
maltose but not sucrose. These biochemical traits identified the isolate as C. albicans. Isolate No. 6 showed a similar pattern but fermented sucrose which is a characteristic of C. tropicalis. Yeast No. 3 assimilated galactose and arabinose but not cellobiose or starch. Since it fermented glucose it was classified as C. parapsilosis. Thus isolate No. 1–6 were identified as C. guilliermondii, C. albicans, C. parapsilosis, Y. lipolytica, C. intermedia and C. tropicalis, respectively. The strain of Y. lipolytica was found to be the best degrader (Table 1). Y. liploytica has often been isolated from oil-polluted environments (Ahrean et al., 1971; Heslot, 1990). Four of these isolates namely, C. tropicalis, C.albicans, C.intermedia, and C. guilliermondii are known to be human pathogens. 3.2. Studies on crude oil degradation The yeast isolates showed better degradation of Bombay High crude oil which had the highest percentage of aliphatics (Table 2) in comparison to the other two crudes and this was used for all further studies. Oils containing a high percentage of saturates are known to be degraded easily (Jobson et al., 1972; Walker et al., 1976) by most organisms and the Candida group is Table 2 Composition of the three crude oil samples Crude oil
Aliphatics
Aromatics
Ashphaltenes
Bombay High Assam crude Gujarat crude
82 69 55
17 22 37
1 9 8
All values are expressed as percentages.
known to be efficient in degradation of alkanes and alkenes (Klug and Markovetz, 1967). Although there are reports of yeasts transforming polycyclic aromatic hydrocarbons (MacGillivary and Shiaris, 1993), the present yeasts were unable to attack the aromatic and ashphaltenes fraction of crude oil. The yeast cultures degraded the aliphatic fraction of crude oil in the presence of ammonium salts and urea (Table 3). C. guilliermondii and C. albicans used ammonium sulphate and urea equally well but all other isolates used ammonium sulphate as the preferred source. Nitrogen concentration above 70 mM nitrogen did not increase oil degradation. Magnesium ammonium phosphate was found to enhance the oil degradation capability of the yeast isolates by about 10–15%. Oleophilic fertilizers and oil degraders associate themselves at oil–water interfaces. Therefore, such fertilizers are specifically available to oil degraders rather than the entire heterotrophic population. Paraffinized fertilizers have been effective in treatment of oil-
Table 3 Degradation of the aliphatic fraction of Bombay High crude oil in the presence of soluble nitrogen sources Yeast isolate
Ammonium sulphate
Sodium nitrate
Urea
C. C. C. Y. C. C.
62 2:7 58 2:8 60 2:2 78 2:5 65 2:3 52 2:9
9 2:4 10 2:9 7 2:2 6:0 2:8 8 2:7 5 2:3
58 3:1 55 2:8 45 1:8 57 2:2 55 2:3 43 1:9
guilliermondii albicans parapsilosis lipolytica intermedia tropicalis
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, the respective nitrogen sources providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at 200 rpm.
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
spills (Prince, 1997; Santas et al., 1999). Olivieri et al. (1976) first described the use of this water-insoluble, non-toxic, oleophilic fertilizer providing both nitrogen and phosphorus and such products have been successfully used in oil-spill remediation (Prince, 1997). The yeasts were able to degrade oil both in seawater and freshwater media though degradation was about 10% lesser in freshwater. The yeasts appear to have adapted to the marine environment from which they were isolated. This suggests that these organisms can be used for bioaugmentation studies in most aquatic environments. The optimum temperature for oil degradation was found to be 30 °C. Increased rates of agitation improved degradation. For example, with Y. lipolytica increased rates of agitation from 0 to 200 rpm resulted in an increase in degradation from 0% to 78% (Table 4). An increase in crude oil concentration resulted in increased degradation upto 2 g of aliphatic fraction followed by a decrease in degradation (Table 5). In this respect, Sikkema et al. (1995) have reviewed the details of hydro carbon toxicity. Thus yeasts appear to be important microflora of oilcontaminated tropical marine environments. There are
Table 4 Effect of agitation rates on degradation of aliphatic fraction of crude oil by Y. lipolytica NCIM 3589 Rate of agitation (rpm)
Percentage degradation of aliphatic fraction of crude oil
0 50 100 150 200
ND 46 2:5 57 2:4 73 2:6 78 2:5
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at varying agitation rates. ND: Not detected.
Table 5 Effect of crude oil concentrations on degradation of the aliphatic fraction of Bombay High crude oil by Y. lipolytica NCIM 3589 Concentration Weight of ali% degradation Average weight of crude oil (%) phatic fraction in of aliphatic loss in aliphatic initial medium (g) fraction fraction (g)a 0.5 1.0 1.5 2.0 2.5 3.0
0.41 0.82 1.23 1.64 2.05 2.46
92 1:5 78 2:5 60 2:4 56 2:5 55 2:7 15 2:6
0.37 0.63 0.74 0.91 1.12 0.37
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and varying concentrations of crude oil incubated at 30 °C for 5 days at 200 rpm. a Initial weight of aliphatic fraction minus remnant weight of aliphatic fraction.
121
significantly lower yeast viable counts away from centers of high oil-pollution. The yeasts seem to have been adapted to the marine environment and are not true marine forms. Selected strains can degrade upto 80% of the crude under optimal conditions in culture although their efficacy in the environment may be constrained by availability of oxygen and nutrients. Since some of these species are known pathogens there is cause for concern in their occurrence where people are exposed to them. References Ahrean, D.G., Meyer, S.P., Standard, P.G., 1971. The role of yeasts in the decomposition of oils in marine environments. In: Murray, E.D. (Ed.), Developments in Industrial Microbiology, vol. 12. American Institute of Biological Sciences, Washington, DC, pp. 126–134. Bhonsale, N.B., Mavinkurve, S., 1984. Effects of dispersants on microbial growth and biodegradation of crude oils. Mahasagar 17, 233–236. Braddock, J.F., Lindstrom, J.E., Brown, E.J., 1995. Distribution of hydrocarbon degrading microorganisms in sediments from Prince William Sound Alaska, following the Exxon Valdez oil spill. Mar. Pollut. Bull. 30, 125–132. Dibble, J.T., Bartha, R., 1976. Effect of iron on the biodegradation of petroleum in seawater. Appl. Environ. Microbiol. 31, 544–550. Heslot, H., 1990. Genetic anad genetic engineering of the industrial yeast Yarrowia lipolytica. In: Fiechter, A. (Ed.), Advances in Biochemistry Engineering, vol. 43. Springer, Berlin, pp. 43–73. Jobson, A., Cook, F.D., Westlake, D.W.S., 1972. Microbial utilization of crude oil. Appl. Microbiol. 23, 1082–1089. Klug, M.J., Markovetz, A.J., 1967. Degradation of hydrocarbons by members of the genus Candida. J. Bacteriol. 93, 1847–1852. MacGillivary, A.R., Shiaris, M.P., 1993. Biotransformation of polycyclic aromatic hydrocarbons by yeasts isolated from coastal sediments. Appl. Environ. Microbiol. 59, 1613–1618. MacNaughton, S.J., Stephen, J.R., Venosa, A.D., Davis, G.A., Chang, Y.J., White, D.C., 1999. Microbial population changes during bioremediation of an experimental oil spill. Appl. Environ. Microbiol. 65, 3566–3574. Meyer, S.A., Ahrean, D.G., Yarrow, D., 1984. Candida. In: Kregervanrij, N.J.W. (Ed.), Yeasts: A Taxonomic Study. Elsevier, Amsterdam, pp. 585–844. Olivieri, R., Bacchin, P., Robertiello, A., Oddo, N., Degen, L., Tonolo, A., 1976. Microbial degradation of oil spills enhanced by a slow release fertilizer. Appl. Environ. Microbiol. 31, 629–634. Prince, R.C., 1997. Bioremediation of marine oil spills. Trends Biotechnol. 15, 158–160. Santas, R., Korda, A., Tenete, A., Buchholz, K., Santas, Ph., 1999. Mesocosm assays of oil spill bioremediation with oleophilic fertilizers Inipol, F1 or both. Mar. Pollut. Bull. 38, 44–48. Sikkema, J., de Bont, J.A., Poolman, B., 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev. 59, 201–222. van der Walt, Yarrow, J.P., 1984. Methods for the isolation, maintenance, classification and identification of yeasts. In: Kreger van rij, N.J.W. (Ed.), Yeasts: A Taxonomic Study. Elsevier, Amsterdam, pp. 45–104. Walker, J.D., Colwell, R.R., 1974. Microbial petroleum degradation: uses of mixed hydrocarbon substrates. Appl. Microbiol. 27, 1053– 1060. Walker, J.D., Colwell, R.R., Petrakis, L., 1976. Biodegradation rates of components of petroleum. Can. J. Microbiol. 22, 1209–1213. Zinjarde, S.S., Pant, A., Deshpande, M.V., 1998. Dimorphic transition in Yarrowia lipolytica isolated from oil-polluted seawater. Mycolog. Res. 102, 553–558.
Hydrocarbon degraders from tropical marine environments S.S. Zinjarde a,b, A.A. Pant a,* a
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India b Department of Biotechnology, University of Pune, Pune 411007, India
Abstract Analysis of 20 samples of marine mud and water around Mumbai resulted in the isolation of 17 bacteria and yeasts all of which were able to degrade more than 10% of the supplied crude oil. The yeasts strains were important degraders of the aliphatic fraction of crude. All the isolated yeasts belonged to the genus Candida. Using biochemical tests these were identified as Candida parapsilosis, C. albicans, C. guilliermondii, Yarrowia lipolytica, C. tropicalis and C. intermedia. Y. lipolytica was the best degrader utilizing 78% of the aliphatic fraction of Bombay High crude oil. None of these isolates degraded the aromatic or ashphaltene fractions. All the isolates required aeration, nitrogen and phosphate supplementation for optimal degradation. Four out of the six yeasts are human pathogens. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Candida species; Crude oil degradation; Tropical marine environments
1. Introduction Chronic inputs of oil account for a major fraction of the oil entering ports such as those in Mumbai. Transport, pumping of ballast waters, effluent from dry docking, servicing of oil tankers and ships are the major factors contributing towards oil pollution which also affect beaches contiguous to the ports. Chronic oil pollution results in sizable populations of hydrocarbon degraders (Braddock et al., 1995; MacNaughton et al., 1999). Ahrean et al. (1971) and Bhonsale and Mavinkurve (1984) have reported the natural occurrence of marine yeasts in oil-polluted coastal regions. The present paper reports investigations on hydrocarbon degraders in the Mumbai region and identification of yeast isolates responsible for maximum degradation of crude oil.
Artificial seawater contained per litre of deionized water, NaCl, 24.5 g; CaCl2 2H2 O, 1.54 g; KBr, 0.1 g; NaF, 0.003 g; KCl, 0.7 g; H3 BO3 , 0.03 g; Na2 SO4 , 4.09 g; NaHCO3 , 0.2 g; SrCl2 6H2 O, 0.017 g; MgCl2 6H2 O, 11.1 g, pH 8.0 adjusted with 1 N NaOH. The enrichment medium contained ammonium sulphate, 0.5% (76 mM Nitrogen); dipotassium hydrogen phosphate, 0.001% and Bombay High crude oil, 1% as the sole carbon source in artificial seawater at pH 8.0. Environmental samples were incubated at 30 °C for five days. An aliquot of this culture was transferred to fresh medium and the procedure was repeated thrice. The crude oil degrading isolates were maintained on Nutrient Agar and MGYP agar (Malt extract, 0.3%; Yeast extract, 0.3%; peptone, 0.5%; glucose 1%; agar, 2.5%) prepared in seawater. The number of yeast colonies in various samples was determined by total viable counts on seawater MGYP agar plates.
2. Materials and methods 2.2. Oil degradation studies 2.1. Samples and media Samples of ship scrapings, oil contaminated water, sediments and floating debris were obtained from Mumbai harbour and suspended in artificial seawater. *
Corresponding author. Tel.: +91-30-589-3300; fax: +91-20-5893761. E-mail address: [email protected] (A.A. Pant).
After a series of preliminary gravimetric analysis, all degradation data was analyzed on a Shimadzu gas chromatograph. Organisms were grown in 250 ml flasks with 50 ml medium. The test results were compared with uninoculated controls incubated under similar conditions. Residual oil was extracted in 50 ml dichloromethane, concentrated to 5 ml, fractionated on a silica gel column into aliphatic, aromatic and ashphaltene
0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 1 ) 0 0 1 8 5 - 0
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
fractions according to the method of Walker and Colwell (1974). The aliphatic fraction was concentrated to 2 ml and 1 ll was loaded on to a SE 30 column on a GC R1A with nitrogen as carrier gas. Peaks were determined with a flame ionization detector. Temperature was programmed from 60 °C to 250 °C with a 6 °C rise per minute. The results were quantified by comparing the integrated areas under the peaks of test samples with uninoculated controls (Dibble and Bartha, 1976). All results are averages of three observations in duplicate. Proportions of aliphatic, aromatic and ashphaltenes fractions in three different Indian crudes (Bombay High, Assam and Gujarat crude) were determined after fractionation by the method of Walker and Colwell (1974).
119
Table 1 Degradation of crude oil by the isolated organisms Isolate no. (bacteria)
% degradation of crude
Isolate no. (yeasts)
% degradation of crude
1, 2, 10 3 4,11 5 6 7 8 9
15 10 12 11 14 13 21 20
1 2 3 4 5 6
34 34 35 45 38 30
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at 200 rpm. % degradation of crude oil (Gravimetry).
2.3. Identification of yeast cultures 3.1. Identification of the yeasts isolates Yeasts were identified in accordance with the methods published by Meyer et al. (1984) and van der Walt and Yarrow (1984) using morphological and biochemical characteristics. Fatty acid methyl ester patterns were determined as described earlier (Zinjarde et al., 1998). 2.4. Standardization of conditions for oil degradation Ammonium sulphate, urea and sodium nitrate were used at concentrations calculated to provide 70 mM nitrogen. Paraffinized ammonium sulphate and urea were used in separate combination with dipotassium hydrogen phosphate (6.5 mM phosphorus). Paraffinized magnesium ammonium phosphate at concentrations providing 70 mM nitrogen and 6.5 mM phosphorus was also checked. Oil degradation in seawater medium was compared with that in distilled water medium. The effect of temperature was determined by incubating the cultures at 15 °C, 30 °C, 40 °C and 50 °C for 5 days. The effect of aeration was determined by varying the agitation rates of the shaker from 0 to 200 rpm. Crude oil was provided in concentrations varying from 0.5% to 2.5%.
3. Results and discussion Enrichment of samples from 20 different sites in the Mumbai coastal region yielded 50 isolates. Among these, seventeen degraded more than 10% of the provided crude (Table 1). Out of these 17, 11 were bacterial cultures and six were yeasts. The yeasts were dominant in populations from dockyards polluted with crude oil. All dockyard samples yielded yeast isolates and in some cases these were the only microbial flora. Total viable counts here were in the order of 107 –108 as compared to 103 –104 per ml from locales outside the dockyards.
None of the yeasts produced a red colour with diazonium blue nor did they produce basidiospores characteristic of basidiomycetous yeasts. On ascosporeinducing media none produced ascospores. Thus they were members of the deuteromycetous group which includes 17 genera. The yeasts produced cream-coloured, non-mucoid colonies without any characteristic aroma, they were unable to form conidia, lacked ballistospores and did not show agglomerates characteristic of Sarcinosporon. The cells of all these yeasts were oval. Oval cells are produced by Candida and Malassezia. Optimum temperature for growth was 30 °C suggesting that the yeasts belonged to the genus Candida. Fig. 1 shows the slide cultures of the isolates. Morphological typing is supported by evidence from fatty acid methyl ester fingerprinting on the Microbial Identification System which confirmed that these yeasts belonged to the genus Candida. The six yeast isolates were identified to the species level using biochemical assimilation and fermentation tests. Meyer et al. (1984) have classified the genus Candida into 10 groups. Isolate No. 4 showed characters of Group V, No. 1 and No. 5 of Group VI and Nos. 2, 3 and 6 of Group VII. The isolate No. 4 belonging to Group V was separated from other species of this group on the basis of its ability to grow at 25 °C, inability to assimilate maltose, cellobiose, its inability to ferment glucose and was identified as Candida (Yarrowia) lipolytica. Isolate No. 1 belonging to group VI was identified as C. guilliermondii on the basis of assimilation of galactose, mannitol, cellobiose, melezitose, melibiose, L -arabinose, ability to grow at 37 °C and glucose fermentation. On the basis of assimilation of galactose, mannitol, cellobiose, melezitose, lactose, ability to ferment glucose but not lactose, isolate No. 5 was identified as C. intermedia. Isolate No. 2 of group VII assimilated galactose, starch, did not assimilate lactose, and fermented
120
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
Fig. 1. Slide cultures of the yeast cultures on Corn Meal agar incubated at 30 °C for 96 h. (a) C. guilliermondii, (b) C. albicans, (c) C. parapsilosis, (d) Y. lipolytica, (e) C. intermedia, (f) C. tropicalis.
maltose but not sucrose. These biochemical traits identified the isolate as C. albicans. Isolate No. 6 showed a similar pattern but fermented sucrose which is a characteristic of C. tropicalis. Yeast No. 3 assimilated galactose and arabinose but not cellobiose or starch. Since it fermented glucose it was classified as C. parapsilosis. Thus isolate No. 1–6 were identified as C. guilliermondii, C. albicans, C. parapsilosis, Y. lipolytica, C. intermedia and C. tropicalis, respectively. The strain of Y. lipolytica was found to be the best degrader (Table 1). Y. liploytica has often been isolated from oil-polluted environments (Ahrean et al., 1971; Heslot, 1990). Four of these isolates namely, C. tropicalis, C.albicans, C.intermedia, and C. guilliermondii are known to be human pathogens. 3.2. Studies on crude oil degradation The yeast isolates showed better degradation of Bombay High crude oil which had the highest percentage of aliphatics (Table 2) in comparison to the other two crudes and this was used for all further studies. Oils containing a high percentage of saturates are known to be degraded easily (Jobson et al., 1972; Walker et al., 1976) by most organisms and the Candida group is Table 2 Composition of the three crude oil samples Crude oil
Aliphatics
Aromatics
Ashphaltenes
Bombay High Assam crude Gujarat crude
82 69 55
17 22 37
1 9 8
All values are expressed as percentages.
known to be efficient in degradation of alkanes and alkenes (Klug and Markovetz, 1967). Although there are reports of yeasts transforming polycyclic aromatic hydrocarbons (MacGillivary and Shiaris, 1993), the present yeasts were unable to attack the aromatic and ashphaltenes fraction of crude oil. The yeast cultures degraded the aliphatic fraction of crude oil in the presence of ammonium salts and urea (Table 3). C. guilliermondii and C. albicans used ammonium sulphate and urea equally well but all other isolates used ammonium sulphate as the preferred source. Nitrogen concentration above 70 mM nitrogen did not increase oil degradation. Magnesium ammonium phosphate was found to enhance the oil degradation capability of the yeast isolates by about 10–15%. Oleophilic fertilizers and oil degraders associate themselves at oil–water interfaces. Therefore, such fertilizers are specifically available to oil degraders rather than the entire heterotrophic population. Paraffinized fertilizers have been effective in treatment of oil-
Table 3 Degradation of the aliphatic fraction of Bombay High crude oil in the presence of soluble nitrogen sources Yeast isolate
Ammonium sulphate
Sodium nitrate
Urea
C. C. C. Y. C. C.
62 2:7 58 2:8 60 2:2 78 2:5 65 2:3 52 2:9
9 2:4 10 2:9 7 2:2 6:0 2:8 8 2:7 5 2:3
58 3:1 55 2:8 45 1:8 57 2:2 55 2:3 43 1:9
guilliermondii albicans parapsilosis lipolytica intermedia tropicalis
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, the respective nitrogen sources providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at 200 rpm.
S.S. Zinjarde, A.A. Pant / Marine Pollution Bulletin 44 (2002) 118–121
spills (Prince, 1997; Santas et al., 1999). Olivieri et al. (1976) first described the use of this water-insoluble, non-toxic, oleophilic fertilizer providing both nitrogen and phosphorus and such products have been successfully used in oil-spill remediation (Prince, 1997). The yeasts were able to degrade oil both in seawater and freshwater media though degradation was about 10% lesser in freshwater. The yeasts appear to have adapted to the marine environment from which they were isolated. This suggests that these organisms can be used for bioaugmentation studies in most aquatic environments. The optimum temperature for oil degradation was found to be 30 °C. Increased rates of agitation improved degradation. For example, with Y. lipolytica increased rates of agitation from 0 to 200 rpm resulted in an increase in degradation from 0% to 78% (Table 4). An increase in crude oil concentration resulted in increased degradation upto 2 g of aliphatic fraction followed by a decrease in degradation (Table 5). In this respect, Sikkema et al. (1995) have reviewed the details of hydro carbon toxicity. Thus yeasts appear to be important microflora of oilcontaminated tropical marine environments. There are
Table 4 Effect of agitation rates on degradation of aliphatic fraction of crude oil by Y. lipolytica NCIM 3589 Rate of agitation (rpm)
Percentage degradation of aliphatic fraction of crude oil
0 50 100 150 200
ND 46 2:5 57 2:4 73 2:6 78 2:5
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and 1% crude oil incubated at 30 °C for 5 days at varying agitation rates. ND: Not detected.
Table 5 Effect of crude oil concentrations on degradation of the aliphatic fraction of Bombay High crude oil by Y. lipolytica NCIM 3589 Concentration Weight of ali% degradation Average weight of crude oil (%) phatic fraction in of aliphatic loss in aliphatic initial medium (g) fraction fraction (g)a 0.5 1.0 1.5 2.0 2.5 3.0
0.41 0.82 1.23 1.64 2.05 2.46
92 1:5 78 2:5 60 2:4 56 2:5 55 2:7 15 2:6
0.37 0.63 0.74 0.91 1.12 0.37
Medium: Artificial seawater containing 0.001% dipotassium hydrogen phosphate, ammonium sulphate providing 70 mM nitrogen and varying concentrations of crude oil incubated at 30 °C for 5 days at 200 rpm. a Initial weight of aliphatic fraction minus remnant weight of aliphatic fraction.
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