Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture

Bioresource Technology 100 (2009) 6028–6032

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Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture Young-Bum Kim, Hyun Shik Yun, Eun-Ki Kim * National Research Lab. of Bioactive Materials Lab., Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea

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Article history: Received 10 March 2009 Received in revised form 17 June 2009 Accepted 17 June 2009 Available online 16 July 2009 Keywords: Sophorolipid Fed-batch Microbial surfactant Feeding rate control Candida bombicola ATCC 22214

a b s t r a c t To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Glycolipids are non-ionic surfactants composed of a sugar head and a lipid tail (Thanomsub et al., 2006; Van Bogaert et al., 2007). One of these glycolipids, sophorolipid, is produced through the bioconversion of saccharides and/or vegetable oils/hydrocarbons by Candida sp. and secreted extracellularly under suitable growth conditions (Ashby et al., 2005; Sarubbo et al., 2007). Glycolipids and sorphorolipids are produced by the conversion of saccharides, vegetables, and hydrocarbons by Candida sp. They are secreted extracellularly under suitable conditions (Felse et al., 2007). The use of sophorolipid versus the use of chemical surfactants has certainly risen in concerns because of the awareness of potential environmental damage that may be caused by chemical compounds, whereas sophorolipid offer superior environmental compatibility combined with excellent functional properties; therapeutic and surface-active (Owsianiak et al., 2009; Van Bogaert et al., 2007). Typically, there are the two types of sophorolipid produced by Candida sp., lactone form where the carboxylic acid group of the fatty acid is covalently linked to the disaccharide ring and the open-chain form. Due to other variations in the attached groups, sophorolipid are applicable for a wide variety of uses, including but not limited to, cosmetic/personal care applications, detergents, lubricants, improvement of sepsis survival rates, anticancer activity, the curing of the skin diseases, germicidal agents, * Corresponding author. Tel.: +82 32 860 7514; fax: +82 32 872 4046. E-mail address: [email protected] (E.-K. Kim). URL: http://bioactive.inha.ac.kr (E.-K. Kim). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.053

mitigation of harmful algal blooms, and as an oil recovery enhancer (Ashby et al., 2006; Chen et al., 2006; Hardin et al., 2007; Kim et al., 2002; Owsianiak et al., 2009; Sun et al., 2004; Van Bogaert et al., 2007; Yoo et al., 2005). In addition, as an effort to expand their application potentials, many chemical and enzymatic processes that seek to modify the sugar or fatty acid moieties of sophorolipid have been also reported (Rau et al., 2001). The culture conditions were studied thoroughly by many researchers. The optimal growth temperature of Candida bombicola ATCC 2214 was approximately 25 or 30 °C. At 25 °C, cell growth rate was lower, and the glucose consumption rate was higher compared to fermentation at 30 °C (Casas and Garcia-Ochoa, 1999). During the exponential growth phase, pH dropped greatly and maintained at 3.5 by the addition of NaOH for optimal sophorolipid production (Kim et al., 2005). In addition, most of sophorolipid biosynthesis started at stationary phase under nitrogen limitation and does not associate with cell growth (Davila et al., 1992). The lowcost of sophorolipid, accompanied by their large-scale production potential, have been identified to be appropriate for industrial applications (Felse et al., 2007). Variable substrates and culture modes were attempted and employed to increase productivity and expand the application of sophorolipid (Deshpande and Daniels, 1995; Felse et al., 2007; Glenns and Cooper, 2006; Hu and Ju, 2001; Kim et al., 2005; Solaiman et al., 2004). In addition, an unstructured and non-segregated kinetic model for sophorolipid production by C. bombicola was proposed in a kinetic study, with the relationship between growth and sophorolipid production. Also flow cytometry was attempted for the calibration of nucleic acids and proteins during the growth of C. bombicola (Alcon et al., 2004). Although these kinetic models were established to

Y.-B. Kim et al. / Bioresource Technology 100 (2009) 6028–6032

increase the productivity of sophorolipid, but the most of these strategies were found to be difficult to apply to industrial processes because of the many control factors that exist. The purpose of this investigation was to enhance the productivity of sophorolipid on a fed-batch mode. A suitable secondary substrate such as a lipophilic carbon source was selected by productivity of sophorolipid and an optimum concentration of glucose was determined by initial production rate during the early production phase. In addition, a novel control method for maintaining optimum concentrations of the substrates and enhancement of productivity was suggested for simplification of sophorolipid production by pH monitoring. 2. Methods 2.1. Strain and chemicals C. bombicola ATCC 22214 was stored at 70 °C with 50% of glycerol. The inoculum culture was incubated on YM broth (per liter, 3.0 g of yeast extract, 3.0 g of malt extract, 5.0 g of peptone, 10.0 g of glucose) by a shaking incubator (25 °C, 250 rpm, 24 h). The culture broth was then transferred to the production medium and was prepared for cell storage. Glucose was obtained from Sigma–Aldrich to the culture media components were provided by Difco and Merck. All oils were obtained from Shindongbang Co. (Korea). 2.2. Standard medium and culture condition The standard medium for production contained, per liter, 100 g of glucose, 5 g of yeast extract, 1 g of KH2PO4, 0.5 g of MgSO4
7H2O, 0.1 g of CaCl2
2H2O, 0.1 g of NaCl, 0.7 g of peptone and 10% (w/v) of vegetable oil. 5% (v/v) of culture suspension was used as the inoculums for a 2.5 L jar fermenter (Kobiotech, Korea). Culture conditions were as follows: working volume, 1 L; temperature, 30 °C; pH, 3.5; agitation, 550 rpm; aeration rate, 1 vvm and culture time, 3–8 days. The oxygen saturation was controlled at 20% by changing the agitation speed. When necessary, the cultivation pH was adjusted by automatic/manual titration of 6 N NaOH. Samples were withdrawn from the fermentation broth at 1 day intervals and analyzed to determine the dry cell weight (g/L) and crude sophorolipid (g/L). 2.3. Assays 2.3.1. Fatty acid composition The fatty acid composition of oil and its derived sophorolipid was determined by gas chromatography equipped with flame ionization detector (GC–FID, HP 6890, HP, USA). The fatty acid methyl ester of oils was prepared by esterification; the rapeseed oil (10 g) was dissolved in a 100 mL of methanol with 5% acetyl chloride and the solution was reacted in a 100 °C sand bath under reflux condition for 1 h. After reaction, the fatty acid methyl ester was extracted by n-hexane for GC–FID analysis. To analyze the fatty acid profile of the sophorolipid, 1 g of each sample was dissolved in 15 mL of 0.5 N KOH/methanol and reacted in a 100 °C sand bath under reflux conditions for 3 h to cleave the ester bond. Then, 20 mL of BF3/Methanol was added and reacted at 90 °C for 15 min to form fatty acid methyl esters. The formed methyl ester was extracted by n-hexane for GC analysis. The extracts were analyzed by a GC–FID and operating conditions of the GC were as follows: column, Supelocowax 10 (30 m  0.53 mm  1 lm); initial temp., 170 °C; final temp., 230 °C; program rate, 5 °C/min; injection temp., 250 °C; detection temp., 300 °C (Kim et al., 2005; Coonrod et al., 2008).

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2.3.2. Residual oil The residual oil was extracted from the culture broths using nhexane. The top layer phase, containing vegetable oils was separated by separation funnel, evaporated with a rotary vacuum evaporator (Eyela, Japan), and weighed. 2.3.3. Sophorolipid separation After the residual oil was removed by n-hexane extraction, the culture broths were extracted three times with the same volume of ethyl acetate. To obtain crude sophorolipid, the ethyl acetate layer was evaporated with a rotary vacuum evaporator (Eyela, Japan), and weighed. 2.3.4. Residual glucose and dry cell weight After extraction of sophorolipid from the broths, the residual solvent was removed on the water layer by a rotary vacuum evaporator (Eyela, Japan) and the water layer was then centrifuged to quantify residual glucose. The precipitant was used to measure a dry cell weight (60 °C, 12 h) and residual glucose of the supernatant was quantified by Accu-Check (Loche) for feeding the deficient glucose during cultivation immediately and reconfirmed by HPLC (column, Aminex HPX-87H; mobile phase, 0.01 N of H2SO4, 1 mL/ min). 2.4. Screening of suitable vegetable oil for increasing the productivity of sophorolipid Five vegetable oils (corn oil, soybean oil, rapeseed oil, rice germ oil and soybean dark oil from Shindongbang Co. Ltd., Korea) were screened for selection based on the increase of sophorolipid productivity. The standard medium was employed as a culture medium and culture conditions were as follows: 50 mL/250 mL flask, 250 rpm (shaking incubator), 25 °C, initial pH 5.0, 100 g/L of initial glucose concentration and 100 g/L of initial oil concentration with a sampling point of 7 days. In addition, the fatty acid composition of oil and its derived sophorolipid was determined by GC–FID. 2.5. Establishment of the operation concentration range of glucose The rates of glucose consumption and sophorolipid production in the culture broths were measured using the two-phase batch fermentation for establishing the adequate operating condition of glucose. At the growth phase (0–72 h), the standard medium with 10 g/L of glucose was used for cell growth. After cell growth, different final concentrations (10–200 g/L) of glucose were added to each broth with 50 g/L of rapeseed oil during the production phase (72–96 h) and culture conditions were as follows: 250 rpm, initial pH of 5, 25 °C, 50 mL/250 mL flask of working volume with a 4-day (3 days for growth and 1 day for production) culture period. After cultivation, samples were withdrawn and analyzed using the previously mentioned assay methods to study the effect of glucose concentration on the productivity of sophorolipid. 2.6. Calculation of substrate feeding rate The average molar mass of rapeseed oil was calculated by GC chromatogram under GC conditions which were described above. However, a correlation coefficient was calculated by quantifying of NaOH and rapeseed oil in fed-batch cultivation. The standard medium was employed as a culture medium and culture conditions were as follows: 1 L/2.5 L jar fermenter (Kobiotech., Korea), 550 rpm, 1.0 vvm, 25 °C, initial pH 5.0 and controlled at pH 3.5 by 6 N NaOH, 100 g/L of initial glucose concentration and 50 g/L of initial oil concentration. The feeding conditions were: pulse, 10 g/L/day for 5 and 7 days of the culture period. During the cultivation period, the difference of initial, added and residual oil con-

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centration and added NaOH mass were measured for the construction of the kinetic equation as a first order polynomial equation relating to the production rate of sophorolipid. 2.7. Feeding-rate-controlled fed-batch cultivation Fed-batch cultivation was performed in a 2.5 L jar fermenter with a working volume of 1.0 L. The medium was steam sterilized. Inoculation was performed with 1% (v/v) inoculum. The fermentation was performed at 25 °C with an aeration rate of 1.0 vvm and an agitation rate of 550 rpm for 8 days. The oxygen saturation was controlled at 30% by changing the agitation speed. When necessary, samples were withdrawn from the fermentation broth and analyzed to determine the variables. The initial glucose concentration was 50 g/L and a 30–40 g/L glucose concentration was maintained during the cultivation. In the case of rapeseed oil, 10 g/L of initial concentration was for controlling the foam and the feeding rate of rapeseed oil depended on the decrease of pH in which case the pH was adjusted to 3.5 by adding 6 N NaOH. 3. Results and discussion 3.1. Screening of suitable vegetable oil for increasing the productivity of sophorolipid Sophorolipid are naturally synthesized as mixtures of acidic and lactonic forms with large amounts of variability in their structures (Ashby et al., 2006). In addition, both de novo and bioconversional sophorolipid were produced by C. bombicola synchronously. The de novo sophorolipid means that they consist of de novo fatty acids tails such as palmitic, stearic and oleic acid (Van Bogaert et al., 2008). Oils consist of fatty acids and a glycerol; fatty acids could be used as feedstocks for the bioconversion of sophorolipid and glycerol could be used as a co-substrate because it is known to support microbial growth (Ashby et al., 2006). Therefore, oils could provide a feedstock for sophorolipid production and a nutrient for cell growth. To screen a suitable vegetable oil for increasing production, sophorolipid production by C. bombicola was investigated in the presence of various vegetable oils. Five different oils were employed: corn, soybean, rapeseed, rice germ and soybean dark oil (a byproduct of soybean oil). These oils were chosen according to previously reported results (Kim et al., 2005) and commercial availability in Korea. The oils were added to the culture medium at a concentration of 100 g/L and incubated for 7 days. Variations in product concentrations with different lipophilic substrates are shown in Table 1. Greater production of sophorolipid was obtained with rapeseed and rice germ oil. While soybean oil and soybean dark oil produced the lowest product concentrations, both oils consist of oleic acid as a main fatty acid. In a previous study, corn, soybean, soybean dark oil showed a different fatty acid composition from rapeseed oil, with their main fatty acid being linoleic acid

Table 2 Similarity of fatty acid compositions of substrate oila and its derived sophorolipid. Fatty acidb (%)

Rapeseed oil Sophorolipid

C16:0

C18:0

C18:1

C18:2

C18:3

5.87 8.78

1.67 2.23

51.98 50.70

28.95 26.38

8.74 3.68

a

Molar mass of rapeseed oil: 878. Fatty acid: C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid) and C18:3 (linolenic acid). b

(Kim et al., 2005). Also, sophorolipid production was slightly higher for fermentations of C. bombicola that used C18:1 (oleic acid) instead of C18:0 (stearic acid), C18:2 (linoleic acid) or C18:3 (linolenic acid) (Felse et al., 2007). The fatty acid compositions of rapeseed oil and its derivative sophorolipid are compared in Table 2. Sophorolipid yield (g/L/batch) was observed with an increase in the de novo fatty acid; palmitic acid and the fatty acid compositions of sophorolipid and rapeseed oil showed a similar trend. The de novo fatty acid showed a great influence on sophorolipid production in rapeseed oil which is abundant in oleic acid, and was found to be suitable for increasing the production of sophorolipid. 3.2. Establishment of the operation concentration range of glucose When there are both lipophilic and hydrophilic carbon sources in the production medium, higher yield of sophorolipid was obtained (Felse et al., 2007). Since C. bombicola is able to assimilate the fatty acid directly from the production medium, glucose is an essential feedstock for sophorolipid production (Sarubbo et al., 2007). In literature, there are different works which study the influence of glucose on sophorolipid production (Ashby et al., 2006; Casas and Garcia-Ochoa, 1999; Kim et al., 2005; Sarubbo et al., 2007;). Most of these found an optimal value of initial glucose concentration at 100 g/L for sophorolipid production without optimization of glucose concentration. Consequently, various concentrations of glucose have been tested to increase the productivity at the production phase. The rates of sophorolipid production from the culture broths were measured during the two-phase batch fermentation. At the growth phase (0–72 h), the standard medium with only 10 g/L of glucose as a carbon source was used for cell growth. After cell growth, different final concentrations (10–200 g/L) of glucose were added to the broths with 50 g/L of rapeseed oil at the production phase (72–96 h). As shown in Fig. 1, 30 and 40 g/L of glucose promoted sophorolipid production in the medium, with a production rate of 1.24 and 1.07 g/L/h, respectively. This means that these glucose concentrations are optimal for sophorolipid production during the production phase. Adequate concentration of glucose for sophorolipid production should be maintained during the production phase to increase sophorolipid production as a significant factor. 3.3. Calculation of substrate feeding rate

Table 1 Increased sophorolipid production at higher oleic acid content. Oils b

SDO Soybean oil Corn oil Rice germ oil Rapeseed oil

Contents of oleic acid (%)

Sophorolipid production (g/L)a

16.10 21.90 24.60 40.01 51.98

65.0 ± 7.07 65.0 ± 5.66 98.0 ± 14.14 102.0 ± 4.24 120.0 ± 4.24

Conditions: 100 g/L of initial glucose concentration, 100 g/L of initial rapeseed oil concentration, 200 rpm, initial pH of 5, temp. 25 °C, 50 mL/250 mL flask of working volume, 7 days, batch mode. a Data presented are the average of two experimental replicates. b SDO: Soy bean dark oil, a byproduct of oil processing.

Theoretical neutralization of fatty acid originated rapeseed oil was adjusted by 6 N NaOH. As shown in Table 2, the analyzed average molar mass of rapeseed oil was approximately 877.97 g/mol. Thus 1.76 g of rapeseed oil was equivalent to a 1 mL of 6 N NaOH. However, a correlation coefficient was calculated by quantifying NaOH and rapeseed oil during fed-batch cultivation. During the cultivation, differences of initial, added, and residual oil mass and amount of NaOH added were measured to construct a first order polynomial equation. At the beginning of the cultivation period, the pH was 5.0 and it decreased gradually according to an increase of cell mass. When sophorolipid production began, the pH was maintained at pH 3.5 by adding 6 N NaOH. After 2 days, 12 g/L of

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Total amount of consumed rapeseed oil (g)

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1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

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20 1. y=4.116x+8.271 (R2=0.9400) 2. y=4.7224x (R2=0.8993)

2 0

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Fig. 1. Effect of glucose concentration for establishing the operating concentration range. Conditions: Initial glucose concentration at growth phase 10 g/L (0–72 h), added glucose concentration at production phase 10–200 g/L (72 h), initial rapeseed oil concentration: 100 g/L, 200 rpm, initial pH 5, temp. 25 °C, working volume: 50 mL/250 mL flask, cultural period: 4 days (growth 3 days + production 1 day). * Data presented are the average of triplicate experiments and error bars indicate standard deviation around the mean.

40

40

0

100 125 150 200

Concentration of Glucose (g/L)

50

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0

0.0

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6

Time (days) Fig. 2. Fed-batch fermentation with pulse feeding of glucose. Initial concentration: 100 g/L (glucose); 50 g/L (oil). Oil feeding condition: 10 g/L/day for 5 days.

Fig. 3. Correlation equations of feeding rate of between 6 N NaOH and rapeseed oil. * Equation 1: solid line; equation 2: long-dash.

sults were not obtained, depicted as a long-dash and R2 was 0.89 because of the difference of cell growth phase. The correlation equation that addressed the consumption of NaOH and rapeseed oil fitted well at the stationary growth phase. Therefore, maintenance was assumed to be carried out using glycerol produced in oil catabolism, while the glucose used in cell maintenance can be considered insignificant (Garcia-Ochoa and Casas, 1999). As observed in Fig. 3, the experimental results were a good fit to the equation. The equation was able to adequately reproduce the experimental results during the production phase of C. bombicola. According to these results, it can be established that the feeding rate of NaOH is the main indicator of change in the lipidic composition, which formed a correlation equation that addressed the consumption of NaOH and was expressed by the first order polynomial equation. Theoretically, 1.76 g of rapeseed oil was equivalent to a 1 mL of 6 N NaOH, but the correlation coefficient was higher than 1.76 due to changes in the culture broth such as acetylation of sophorose, synthesis of organic acids and formation of acidic sophorolipid. As a result, calculated and suggested correlation coefficient was a 4.12 g/mL at the production phase. 3.4. Feeding-rate-controlled fed-batch cultivation

dry cell weight was obtained. When cell growth reached the stationary phase, it began to produce sophorolipid from glucose and rapeseed oil concurrently. Sophorolipid production started approximately in the middle of the exponential growth phase and increased significantly after repeated feedings of rapeseed oil. During the production phase, sophorolipid was excreted continuously. A total 100 g of rapeseed oil was added to 1 L of cultivation broth. Fifty grams of rapeseed oil was added initially and the remaining oil was added at a rate of 10 g/L/day for 5 days by pulse feeding. Before feeding, samples were taken and residual oil and sophorolipid were measured. Two hundred and ten grams per liter of extracellular sophorolipid was obtained and the cultivation of C. bombicola was terminated after 7 days (Fig. 2). In addition, the mass of initial, added, residual rapeseed oil, and added NaOH were measured for construction of the first order polynomial equation. As shown in Fig. 3, consumption of NaOH was related to consumption of oil in the stationary growth phase. The experimental data has been fitted by a linear regression technique, correlation equations were distinguished by y0 (y-axis intercept) values and correlation coefficients were represented by R2. When y0 was not zero, data fitted better using a linear regression algorithm, illustrated as a solid line, and R2 was 0.94. When y0 was zero, good re-

The aim of this investigation was to enhance the production of sophorolipid in a fed-batch mode by screening a number of suitable carbon sources and maintaining their optimum concentration. Rapeseed oil was selected and glucose concentration was optimized for sophorolipid production. Fig. 4 shows fed-batch cultivation with an initial glucose concentration of 50 g/L and an initial rapeseed oil concentration of 10 g/L. After inoculation, rapeseed oil was added immediately at a ratio of 1:4.7 (6 N NaOH:rapeseed oil) during the growth phase (0–72 h). In the production phase, the addition rate was changed to a ratio of 1:4.1 (6 N NaOH:rapeseed oil) as a result of Fig. 3. When the feeding-rate-controlled fed-batch cultivation was compared to pulse feeding, the sophorolipid formation was increased by1.8-fold during the stationary phase. As a result, we have produced a crude sophorolipid up to 365 g/L after 8 days through feeding-rate-controlled fed-batch. After 8 days, fermentation was not driven because of high viscosity of the culture broth. The controlled feeding of the hydrophobic carbon source is one of the most important factors for the increased production of sophorolipid using C. bombicola. Also, the consumption of rapeseed oil was correlated with the adjustment of pH by adding NaOH. Consequently, suitable carbon sources (glucose and rapeseed oil) and their optimum operating concentration were determined by

80 6

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14 12 10 8 6 4 2

Dry Cell Weight (g/L)

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Concentration of Glucose (g/L)

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0

150

Time (hrs) Fig. 4. Profile of sophorolipid production using the feeding-rate-controlled fed-batch cultivation of C. bombicola ATCC 22217.

increasing the production of sophorolipid. Based on the coefficient calculation, a novel control method for maintaining optimum concentrations of lipophilic substrates (rapeseed oil) was suggested. 4. Conclusions In this study, rapeseed oil was chosen for increasing sophorolipid production, and the glucose concentration was optimized at 30–40 g/L for the production phase by a two-phase batch fermentation. In addition, the feeding rate of rapeseed oil depended on decreasing the pH during fed-batch cultivation. According to these results, it can be established that NaOH was the main indicator of change in the lipidic compositions, which formed a correlation equation with NaOH, which was expressed by a first order polynomial equation. Consequently, when the feeding-rate-controlled fed-batch cultivation was compared to pulse feeding, the sophorolipid formation increased by 1.8 times, and crude sophorolipid were produced up to 365 g/L over 8 days. In addition, a novel method for substrate feeding was suggested through the calculation of the correlation coefficient. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (MEST) R0A-2007-000-10015-0. References Alcon, A., Santos, V.E., Casas, J.A., Garcia-Ochoa, F., 2004. Use of flow cytometry for growth structured kinetic model development application to Candida bombicola growth. Enzyme Microb. Tech. 34, 399–406. Ashby, R.D., Solaiman, D.K.Y., Foglia, T.A., 2006. The use of fatty acid esters to enhance free acid sophorolipid synthesis. Biotechnol. Lett. 28, 253–260. Ashby, R.D., Nuñez, A., Solaiman, D.K.Y., Foglia, T.A., 2005. Sophorolipid biosynthesis from a biodiesel co-product stream. JAOCS 82, 625–630. Casas, J.A., Garcia-Ochoa, F., 1999. Sophorolipid production by Candida bombicola: medium composition and culture methods. J. Biosci. Bioeng. 88, 488–494. Chen, J., Song, X., Zhang, H., Qu, Y.-B., Miao, J.-Y., 2006. Sophorolipid produced from the new yeast strain Wickerhamiella domercqiae induces apoptosis in H7402 human liver cancer cells. Appl. Microbiol. Biotechnol. 72, 52–59. Coonrod, D., Brick, M.A., Byrne, P.F., DeBonte, L., Chen, Z., 2008. Inheritance of long chain fatty acid content in rapeseed (Brassica napus L.). Euphytica 164, 583–592.

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