J. Basic Microbiol. 42 (2002) 1, 67 – 73
(Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India)
Emulsifier from a tropical marine yeast, Yarrowia lipolytica NCIM 3589 SMITA S. ZINJARDE and ADITI PANT*) (Received 30 May 2001/Accepted 28 September 2001) A tropical marine strain of Yarrowia lipolytica, NCIM 3589 produced emulsifier in the presence of alkanes or crude oil. The mode of alkane uptake in this organism was by attachment to large droplets. An emulsifier (lipid-carbohydrate-protein) complex was associated with the cell wall. This emulsifier increased the hydrophobicity of the cells during the growth phase. In the stationary phase, the organism produced the emulsifier extracellularly under conditions of carbon excess and nitrogen limitation. Other requirements for extracellular emulsifier production included an initial pH of 8.0 and the presence of sodium chloride at a concentration of 2 to 3 % (342 to 513 mM). The cell-associated and extracellular emulsifier was shown to have similar properties.
Emulsifiers play an important physiological role in hydrocarbon degrading organisms. Hydrocarbons are used as growth substrates by a large variety of microbes including bacteria, filamentous fungi and yeasts. The insoluble nature of hydrocarbons is one of the properties that makes their utilisation rather difficult. Hydrocarbon degraders show different adaptations for utilising these water insoluble substrates. Some produce growth-associated extracellular emulsifiers. These emulsifiers emulsify hydrocarbons to sub-micron droplets which are then utilised by the microorganisms (IGUCHI et al. 1969, RAPP et al. 1979, ITOH and INOUE 1982). Other microorganisms produce emulsifiers that make the cell surfaces hydrophobic and enhance hydrocarbon uptake (KAPPELI and FIECHTER 1977, KAPPELI et al. 1977). We report here, cell-associated and extracellular emulsifier production by a tropical marine yeast isolate, Yarrowia lipolytica NCIM 3589.
Materials and methods Microorganism: The oil degrading Y. lipolytica strain was isolated from a sample of sea water near Mumbai, India (ZINJARDE et al. 1998). Media and growth conditions: Cells were grown in a pre-inoculum medium containing per litre of artificial sea water, peptone, 5.0 g; yeast extract, 3.0 g, pH 8.0 for 24 h. After growth, the medium was centrifuged, cells washed and 2 × 109 cells/ml were inoculated in the emulsifier production medium which contained per litre of sea water, ammonium sulphate, 5.0 g; dipotassium hydrogen phosphate 0.01 g and n-hexadecane, 10.0 g, pH 8.0. All experiments were carried out in 250 ml flasks with 50 ml medium. Experimental cultures were incubated at 30 °C for 6 days at 200 rpm. Growth was determined in terms of dry weight after centrifugation at 5000 g for 10 min, washing and drying to a constant weight. Cell attachment to alkanes was checked under a LEITZ microscope. Emulsification assay, purification and characterisation was carried out as described earlier (ZINJARDE et al. 1997). *) Corresponding author Dr. ADITI PANT; e-mail:
[email protected]
WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2002 0233-111X/02/0103-0067 $ 17.50+.50/0
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Determination of cell-associated emulsifier and cell hydrophobicity: Cells were homogenised in a BRAUNS Homogenizer for 2 min. (4 cycles of 15 sec) and emulsification activity in the homogenate was determined after treatment with pronase and the emulsifier was isolated. Hydrophobicity of cells grown on glucose or n-hexadecane was compared according to the method of KAPPELI and FIECHTER (1976). Cells were harvested, washed in citrate phosphate buffer (50 mM) pH 6.0 and 20 mg dry weight suspended in the same buffer containing 0.005 mM KCN. These cells were incubated with 0.2 ml of freshly emulsified n-hexadecane for 2 min and centrifuged at 3000 g for 10 min. The unbound alkane was removed with acetone. The cells were resuspended in buffer and the cell-bound alkane was extracted with 10 ml diethyl ether. This was concentrated to 2 ml and 1 µl loaded on SE30 column fitted onto a GC (SHIMADZU R 1 A) with nitrogen as carrier gas using FID under isothermal conditions at 150 °C. To determine the significance of the emulsified alkane for growth and alkane utilisation, Tween 80, or the isolated emulsifier was added into the emulsifier production medium at a concentration of 3 µg/ml. The organism was inoculated and growth and alkane utilisation monitored. Effect of culture conditions on growth and extracellular emulsifier production: Glucose, sodium acetate, glycerol, n-alkanes (C10 – C18) and crude oil were added into the growth medium as carbon sources at a concentration of 1% (w/v). Nitrogen in the form of (NH4)2SO4, NH4Cl, NH4NO3 or urea were supplied to provide 70 mM nitrogen in the growth medium with n-hexadecane as sole carbon source. Concentration of ammonium sulphate, one of the best used nitrogen sources was varied to provide 17, 35, 53, 70 and 105 mM N. Initial pH of the medium was varied from 3 to 10. pH after growth was also checked. In a medium containing all components of sea water except NaCl, 70 mM N as ammonium sulphate and 0.01 g ⋅ l–1 dipotassium hydrogen phosphate, concentration of sodium chloride was varied from 0 to 2.56 M. Time course of emulsifier production was studied under optimal conditions for a period of 7 days and compared with growth and emulsifier production in fresh water medium.
Results and discussion Yeasts may take up water insoluble alkanes either by releasing emulsifiers into the medium (IGUCHI et al. 1969, ITOH and INOUE 1982) or they may attach onto large droplets of alkane and form flocs (BLANCH and EISNELE 1973, MALLE and BLANCH 1977). Microscopic observations of the present strain of Y. lipolytica showed large droplets of alkanes with attached yeast cells which are characteristic of floc formation (Fig. 1). Such observations were also reported by MIURA et al. (1977) in case of cells of Candida intermedia. Addition of the isolated emulsifier produced by this yeast itself or Tween 80 did not enhance growth or alkane utilisation. Substrate emulsification is not a pre-requisite for growth of this organism on alkanes. Candida guilliermondii and Candida tropicalis on the other hand utilise emulsified alkanes (MIURA 1977).
Fig. 1 Microscopic observations of Yarrowia lipolytica NCIM 3589 grown on n-hexadecane after incubation for 48 h (at 400 X magnification)
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Yarrowia lipolytica emulsifier
Emulsifiers are known to be present as an integral part of the cell wall where they increase the alkane binding capacity and enhance alkane uptake. KAPPELI et al. (1977) isolated a cell wall associated fatty acid-mannan complex from alkane grown Candida tropicalis. This emulsifier increases the alkane binding capacity of the cells. Glucose grown cells lacked this complex and exhibited lower hydrophobicity. In the present study, the hydrophobicity of Y. lipolytica cells grown on alkanes was 2.5 times greater than those grown on glucose. Homogenised alkane grown cells showed the presence of an emulsifier even on the first day after inoculation into fresh alkane containing medium although cell-free broth did not show any emulsification activity. Glucose grown cells had neither cell-associated nor extracellular emulsification activity. The cell associated emulsifier present only in alkane grown cells presumably makes the cell surface hydrophobic, enhances floc formation and consequently alkane uptake in a manner similar to that observed in C. tropicalis (KAPPELLI and FIECHTER 1977, KAPPELLI et al. 1977). Their strain of C. tropicalis did not liberate the emulsifier into the medium. On the other hand, NCIM 3589 produces an emulsifier which remains associated with the cells until the stationary phase of growth when it liberates the emulsifier extracellularly. Conditions for extracellular emulsifier production Effect of carbon sources on growth and emulsifier production showed that carbon sources such as glucose, alcohol, sodium acetate or glycerol did not result in emulsifier production. With crude oil or alkanes (C10 –C18), NCIM 3589 showed emulsifier production. This is in agreement with the results of PAREILLEUX (1979), KIM and REHM (1982) and CIRIGLIANO and CARMAN (1984), who have all reported emulsifier production in the presence of water insoluble substrates. n-Hexadecane at a concentration of 1% w/v was used for all studies although, emulsifier production was similar on alkanes of different carbon chain lengths. Nitrogen sources such as ammonium sulphate and ammonium chloride supported maximum emulsifier production. (Table 1). The concentration of ammonium sulphate was varied and 70 mM were found to yield a maximum of 3.0 units/ml emulsification activity (Table 2). Table 1 Effect of nitrogen sources on growth and emulsifier production by Yarrowia lipolytica NCIM 3589 Nitrogen source
Emulsification activity (units/ml)
Biomass (mg/ml)
Ammonium sulphate Ammonium chloride Ammonium nitrate Urea Sodium nitrate
3.0 3.0 1.7 1.0 ND*
3.0 3.0 1.8 0.9 0.12
* ND Not detected. Table 2 Effect of varying concentrations of ammonium sulphate on growth and emulsifier production by Yarrowia lipolytica NCIM 3589 Ammonium sulphate mM Nitrogen
Emulsification activity (units/ml)
Biomass (mg/ml)
17 35 53 70 89 105
1.6 2.0 2.5 3.0 0.07 0.06
1.7 2.1 2.6 3.0 3.8 3.8
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At this concentration, ammonium ions were exhausted after 72 h (Fig. 3 a) creating nitrogen limitation but, alkane was incompletely utilised as estimated by GC. With initial nitrogen concentrations higher than the optimum for example, 105 mM and the same concentration of alkane, ammonium ions were detected in the medium even upto 8 days, but alkane was below detectable limits. No extracellular emulsification activity was produced although, cell biomass was higher (3.8 mg/ml). Such observations were also made in Rhodococcus erythropolis by SINGER and FINNERTY (1990). At concentration less than optimum, for example with 35 mM nitrogen ammonium ions were exhausted after 48 h and extracellular emulsifier was detected within the next 8 h. The emulsifier was thus produced extracellularly under conditions of nitrogen limitation. In a similar manner, Pseudomonas species, Candida antarctica and R. erythropolis produce emulsifiers under conditions of nitrogen limitation (SKYDATK and WAGNER 1987, KITAMATO et al. 1989, SINGER and FINNERTY 1990). BOULTON and RATLEDGE (1987) suggested that under conditions of nutrient limitation and carbon excess, growth does not occur but, carbon is transported into the cell where it maybe utilised for biosynthesis of lipids, polysaccharides or secondary metabolites such as antibiotics. Effect of initial pH on growth and emulsifier production is shown in Table 3. This Y. lipolytica strain isolated from sea water grows best and produces maximum emulsifier at an initial pH of 8.0 which is that of natural sea water. Table 3 Effect of initial pH on growth and emulsifier production by Yarrowia lipolytica NCIM 3589 Initial pH
Emulsification activity (units/ml)
Biomass (mg/ml)
2.0 4.0 6.0 8.0 10.0
ND* 1.5 2.5 3.0 ND*
0.06 2.0 2.7 3.0 0.06
* ND Not detected
Fig. 2 Effect of sodium chloride concentration on growth and emulsifier production by Yarrowia lipolytica NCIM 3589
Yarrowia lipolytica emulsifier
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Fig. 3 a. Time course of emulsifier production by Yarrowia lipolytica NCIM 3589 grown in sea water medium b. Time course of emulsifier production by Yarrowia lipolytica NCIM 3589 grown in fresh water medium
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GOMA et al. (1973), PAREILLEUX (1979) and CIRIGLIANO and CARMAN (1984) have cultivated different emulsifier producing strains of Y. lipolytica in the acidic pH range (3.7 to 5.0). The final pH of the medium at the end of 6 days was 3.0. VELDKAMP (1970) suggested two reasons for a drop in the pH namely, the use of ammonium sulphate in the medium and production of organic acids. In the present study, organic acids were produced only in trace amounts (data not presented) and this drop in pH may be mainly associated with the use of ammonium sulphate in the medium. The effect of sodium chloride on growth and emulsifier production is shown in Fig. 2. NaCl at concentrations ranging between 342 to 513 mM (2 to 3%) either in the presence or absence of other sea water salts, enhanced emulsifier production. Increased emulsifier production in the presence of NaCl has not been reported earlier though NaCl is known to increase the production of a non-emulsifying biopolymer in the cyanobacterium Westiellopsis prolifa (SAXENA and KAUSHIK 1992). The emulsifier production was 3 times higher in sea water (3 units/ml) compared to 1 unit/ml in fresh water (Fig. 3 a and b). In sea water, 0.72 mg/ml of emulsifier and in fresh water 0.24 mg/ml was produced. The final biomass of the organism was the same in sea water and in fresh water. COMBS et al. (1968) showed that NaCl reduces growth yields of the yeast Candida albicans but this is not expected from NCIM 3589 which has been isolated from a marine environment. This yeast utilised the supplied alkane more proficiently in sea water than in fresh water (Fig. 3 a and b). In fresh water, 20% of the initial alkane could be measured after 6 days of growth but in sea water, residual alkane was not detected under the same experimental conditions. Time course of emulsifier production in the optimised medium showed that the emulsifier was a stationary phase metabolite produced extracellularly in 72 h (Fig. 3a). Maximum emulsifier production was obtained after 6 days. CIRIGLIANO and CARMAN (1984) have also reported the production of a carbohydrate protein complex in a Y. lipolytica strain during the stationary phase. SARUBBO et al. (1999) have recently reported an extracellular emulsifier by another strain of Y. lipolytica (IA 1055) using babassu oil as sole carbon source after 60 h of growth. We have described here a strain of Y. lipolytica producing cell-associated emulsifier in the earlier stages of growth that makes the cell surface hydrophobic enhancing attachment to large alkane droplets. The organism displays extracellular emulsifier activity in the stationary phase under conditions of nitrogen limitation in the presence of NaCl at a concentration of 2–3%. The cell-associated and extracellular emulsifier could be isolated in the pure form by the method described by us earlier (ZINJARDE et al. 1997). Both the emulsifiers showed similar characteristics with respect to composition and other physical properties.
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[email protected] Dr. S. S. ZINJARDE, Department of Biotechnology, University of Pune, Pune, 411007, India e-mail:
[email protected]
