Enzyme and Microbial Technology 41 (2007) 312–317 High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture Yonghong Li a,b , Zongbao (Kent) Zhao b,∗ , Fengwu Bai a,b a Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, PR China b Division of Biotechnology, Dalian Institute of Chemical Physics CAS, Dalian 116023, PR China Received 19 December 2006; received in revised form 22 February 2007; accepted 22 February 2007 Abstract Microbial lipid production by the oleaginous yeast Rhorosporidium toruloides Y4 was studied using glucose as carbon source, in order to realize high-density cell culture. Batch cultures demonstrated that there was little inhibitory effect with a substrate concentration of up to 150 g l−1 . Flask fed-batch cultures were run for 25 days and reached a dry biomass and cellular lipid content of 151.5 g l−1 and 48.0% (w/w), respectively. Using pilot-scale fed-batch cultures in a 15-l stirred-tank fermenter for 134 h resulted in dry biomass, lipid content and lipid productivity of 106.5 g l−1 , 67.5% (w/w) and 0.54 g l−1 h−1 , respectively. The fed-batch culture model used here featured initial nutrient-rich media and a pure carbon source with discontinuous feeding. Gas chromatography analysis revealed that lipids from R. toruloides Y4 contained mainly long-chain fatty acids with 16 and 18 carbon atoms. The four major constituent fatty acids were oleic acid, palmitic acid, stearic acid and linoleic acid. A slight increase in stearic acid production was found during the culture process. Based on these compositional data, microbial lipids from R. toruloides Y4 are a potential alternative oil resource for biodiesel production. © 2007 Elsevier Inc. All rights reserved. Keywords: High-density cell culture; Rhorosporidium toruloides Y4; Fed-batch culture; Microbial lipid 1. Introduction Microorganisms that can accumulate lipids at more than 20% of their biomass are defined as oleaginous species [1]. Some yeast strains, such as Rhodosporidium sp., Rhodotorula sp. and Lipomyces sp. can accumulate intracellular lipids as high as 70% of their biomass dry weight. The majority of those lipids are triacylglycerol (TAG) contained long-chain fatty acids that are comparable to conventional vegetable oils [2]. More importantly, some of those oleaginous species show the ability to metabolize pentoses, demonstrating the potential to produce TAG from lignocellulosic biomass and other cheap materials [3–5]. Recent demand for biodiesel worldwide has turned TAG into an ever-growing and substantial consumption resource [6]. As TAGs from oil crops, woody oil plants or animal fats are currently just balancing the demands of food supply and oleochemical industry, the quest for non-traditional TAG production processes, especially those that can be operated continuously ∗ Corresponding author. Tel.: +86 411 8437 9211; fax: +86 411 8437 9211. E-mail address: zhaozb@dicp.ac.cn (Z. Zhao). 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.02.008 and with no extensive arable land requirement, is pivotal for a sustainable biodiesel industry. In this regard, microbial TAGs may be a prospective alternative feedstock. The basic physiology of lipid accumulation in microorganisms has been well studied. It is known that lipid production requires medium with an excess of sugars or similar components (e.g., glycerol, polysaccharides, etc.) and limited other nutrients, usually nitrogen. Thus, oleaginous potential is critically affected by the carbon-to-nitrogen (C/N) ratio of the culture and other factors like aeration, inorganic salt presence, etc. [7]. Various microorganisms are capable of accumulating huge quantities of lipid when cultured on hydrophobic materials, while lipoid production is restricted when cultivation is carried out in nitrogen-limited sugar-based media [8–10]. The costs of microbial oil production are currently higher than those of vegetable oil but there are many methods to drastically improve the techno-economics of microbial oil production processes. In particular, the exploration of lignocellulose-based carbohydrates as feedstock may greatly lower the costs. Process engineering that leads to a higher lipid production rate and cellular lipid content may also contribute in this regard. Different cultivation modes, including fed-batch Y. Li et al. / Enzyme and Microbial Technology 41 (2007) 312–317 313 and continuous fermentations, have been used to increase cell density of oleaginous microbes. Evans and Ratledge [11] studied Candida curvata D growing on glucose and xylose in a continuous fermentation process and obtained lipid production rates of 0.16 and 0.27 g l−1 h−1 , respectively and a cell density of around 14 g l−1 . Hassan et al. [12] optimized the growth of Apiotrichum curvatum in a continuous culture system using glucose and reached a lipid production rate of 0.42 g l−1 h−1 and lipid content of 31.9% (w/w). Pan et al. [13] obtained a cell density of 185 g l−1 in an 84-h fed-batch culture of Rhodotorula glutinis aerated with oxygen-enriched air. Yamauchi et al. [14] obtained a cell density of 153 g l−1 and a lipid content of 54% (w/w) using fed-batch cultures of Lipomyces starkeyi for 140 h. The red yeast Rhodosporidium toruloides is a known microbial lipid producer and extensive characterization the oleaginous profile of R. toruloides CBS 14 has been reported [15–17], but its high-density fermentation has so far not been published. We have recently identified R. toruloides Y4, a lignocellulose hydrolysate domesticated strain of R. toruloides AS 2.1389, originally from China General Microbiological Culture Collection Center, which can accumulate lipids up to 76.1% of cell dry weight [18]. In this study, we developed an effective, yet simple fed-batch fermentation system for lipid production using R. toruloides Y4. This process greatly increased cell density as well as the rate of lipid production. inoculum. The cultures were incubated in an orbital shaker at a rotary rate of 200 rpm at 30 ◦ C. Twenty-five milliliter of 500 g l−1 glucose was fed in 5-ml aliquots when residual glucose was lower than 20 g l−1 . Samples of 0.2 ml were withdrawn every 12–24 h for residual sugar analysis and estimation of biomass. Pilot scale fed-batch fermentation was carried out in a 15-l stirred-tank bioreactor (FUS-15L (A), Guoqiang Bioengineering Equipment Co. Ltd., Shanghai, China) equipped with an on-line data acquisition and control system. Culture pH and dissolved O2 were monitored with a pH meter (Mettler-Toledo, Switzerland) and an oxygen probe (Mettler-Toledo, Switzerland). The cultivation conditions were as follows: 10% (v/v) seed culture, temperature 30 ◦ C, pH 5.6 (by automatic control using 10.0 M NaOH), aeration at 0.9 vvm and dissolved oxygen at 40–50% saturation. The initial culture contained 7.0 l medium. The substrate concentration was maintained above 20 g l−1 by feeding 1000 g l−1 glucose in 500-ml aliquots in a single pour. Aliquots of 15 ml were taken at 4-h time intervals to estimate glucose concentration, cell dry weight, lipid content and nitrogen concentration. Five cycles of substrate feeding were done and the experiment was run for 134 h. The feeding solution of 1000 g l−1 glucose for pilot scale fermentation was realized, batch-by-batch, with the following procedure. Briefly, the solution was prepared by dissolving 500 g of glucose in water to a final volume of 500 ml at 80 ◦ C or above, autoclaved at 121 ◦ C for 15 min, and cooled. This syruplike solution had a density of 1.31 kg l−1 , and was stable for hours without crystallization at room temperature. 2. Materials and methods Cell optical density was recorded at 600 nm with a V-530 spectrophotometer (Jasco Corp., Tokyo, Japan). Wet cells were collected by centrifugation and washed with the same volume of distilled water. Cell dry weight was obtained from wet cells from a 20 ml culture broth after being dried at 105 ◦ C overnight. The glucose concentration was determined using a SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). Residual nitrogen concentration in the culture medium was determined by estimation of the ammonia formed using direct distillation of cell-free samples by KDN-2C type nitrogen installation (Shanghai, China). To determine fatty acid composition, wet cells were directly transmethylated according to the following procedure. Wet cell pellets from 1 ml of culture were treated in a flask with 0.5 ml of a 5% KOH methanol solution at 65 ◦ C for 50 min, followed by the addition of 0.2 ml BF3 diethyl etherate and 0.5 ml methanol. The mixture was refluxed for 10 min, cooled, diluted with distilled water and extracted with petroleum ether (bp 30–60 ◦ C). The organic layer was washed with distilled water and subjected to fatty acid compositional analysis. Fatty acid methyl esters were analyzed using a 7890F gas chromatography instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary FFAP column (30 m × 0.32 mm × 0.4 m) and flame ionization detector. Operating conditions were as follows: N2 carrier gas 40 ml/min, injection port temperature 230 ◦ C, oven temperature 190 ◦ C and the detector temperature was 230 ◦ C. Fatty acids were identified by comparison of their retention times with those of standard ones, quantified based on their respective peak areas and normalized. 2.1. Organism, media and chemicals Peptone (animal-tissue based containing 3% ammonium-N and 14.5% total nitrogen), yeast extract (containing 3% ammonium-N and 9.0% total nitrogen) and agar were purchased from Aoboxing Bio-tech. Co. Ltd. (Beijing, China). All other chemicals and reagents were bought locally and were of analytical reagent grade. R. toruloides Y4 was maintained at 4 ◦ C on YPD agar slant (1% yeast extract, 1% peptone, 2% glucose and 2% agar) [19], and sub-cultured twice a month. Inoculum was prepared by an overnight culture on YPD medium (1% yeast extract, 1% peptone and 2% glucose, pH 5.5) at 30 ◦ C. Batch experiments were performed using a minimal medium (pH 5.5) containing per liter of distilled water: (NH4 )2 SO4 12.0 g, KH2 PO4 1.0 g, MgSO4 ·7H2 O 1.5 g, yeast extract 0.5 g. Flask fed-batch medium (pH 5.5) contained: glucose 60.0 g, peptone 30.0 g, and yeast extract 30.0 g per of distilled water. Fermenter fed-batch medium (pH 5.5) contained: glucose 60.0 g, peptone 15.7 g and yeast extract 15.7 g/l distilled water. 2.2. Batch culture Flask cultures were conducted in duplicate in 250-ml Erlenmeyer flasks containing 50 ml of minimal medium and inoculated with 5 ml of 20-h-old preculture ((2.0–3.0) × 106 cells). The minimal medium was supplemented with glucose to formulate a medium with an initial substrate concentration of 10, 40, 60, 90, 120, 150, 200, 300 or 400 g l−1 , respectively. The cultures were incubated in an orbital shaker at a rotary rate of 200 rpm at 30 ◦ C. At different time intervals, culture samples (0.2 ml) were withdrawn and the absorbance was measured at 600 nm for optical density comparison. 2.3. Fed-batch culture Flask fed-batch cultures were performed in 250-ml Erlenmeyer flasks. The initial culture contained 22.5 ml medium and 2.5 ml of 28-h-old precultured 2.4. Lipid extraction Total lipid was extracted by the method of Li et al. [20]. Lipid content was expressed as gram lipid per gram dry biomass. 2.5. Analytic methods 3. Results 3.1. Substrate inhibition studies To determine the effect of substrate concentration on growth profile of R. toruloides Y4, batch experiments were performed in conical flasks with glucose concentration ranging from 10 to 400 g l−1 . Fig. 1 shows the plots of cell culture optical density against fermentation time. These results indicated that the yeast 314 Y. Li et al. / Enzyme and Microbial Technology 41 (2007) 312–317 Fig. 1. Effect of initial glucose concentrations on R. toruloides Y4 cell growth. grew well on glucose as a sole source of carbon and energy at a concentration up to 150 g l−1 . When the glucose concentration reached 200 g l−1 , cell growth was greatly repressed and more severe inhibitory effects were observed at even higher glucose concentrations. The data shown in Fig. 1 can also be converted into specific growth rates within the first 20 h. Specifically, when the glucose concentration increased from 40 to 150 g l−1 , the specific growth rate of R. toruloides Y4 showed little decrease, i.e. from 0.135 to 0.119 h−1 . These data suggested that a wide range of substrate concentrations were suitable for lipid production by this strain with no significant substrate inhibition. However, drastic specific growth rate loss was found for the culture when glucose concentrations were higher than 200 g l−1 . Fig. 2 shows the biomass and lipid accumulated by R. toruloides Y4 after 120 h culture against the initial glucose concentration. When the initial glucose concentration increased from 10 to 90 g l−1 , the biomass and lipid yields increased from 4.7 and 1.13 g l−1 to 18.6 and 8.63 g l−1 , respectively. How- Fig. 2. Biomass and lipids accumulated by R. toruloides Y4 vs. initial glucose concentration. Fig. 3. Fed-batch flask fermentation profile for R. toruloides Y4. ever, the biomass and lipid accumulation slightly dropped to 18.0 and 8.60 g l−1 for cultures with an initial glucose concentration of 120 g l−1 . These data were further reduced to 17.7 and 8.30 g l−1 , respectively for cultures with 150 g l−1 glucose. When the substrate concentration reached 200 g l−1 , biomass and lipid production were greatly decreased, suggesting that a considerable inhibitory effect had occurred. 3.2. Fed-batch fermentation The time-courses of biomass and glucose concentration for the flask fed-batch culture over 25 days are shown in Fig. 3. Final cell biomass, lipid and lipid content were 151.5 g l−1 , 72.8 g l−1 and 48.0% (w/w), respectively. Thus, the overall lipid yield and productivity were estimated to be 0.26 g g−1 and 2.91 g l−1 day−1 . These data were higher than those from the batch culture system [18] (yield 0.23 g g−1 , productivity 2.78 g l−1 day−1 ). There was also a noticeable biomass productivity decrease during the whole process. For example, biomass was produced at approximately 9.5 g l−1 day−1 up to 8 days, following which it decreased to about 6.8 g l−1 day−1 . In conclusion, these flask fed-batch experiments demonstrated that R. toruloides Y4 had the robust ability to grow in liquid culture and reach very high cell density and that nitrogen source feeding was not required for this process. Enthused by the good results from flask fed-batch cultures, we also conducted a pilot-scale fed-batch culture using a 15-l stirred-tank fermenter. The typical time-course of glucose concentration, biomass and lipid production for this experiment are shown in Fig. 4. It was obvious that cell growth was fast during the initial stage. Biomass increased rapidly from 0.3 to 32.2 g l−1 within 18 h, and biomass yield was 0.59 g g−1 glucose. In the period of 10–18 h, growth was linear and biomass increased with a high specific growth rate of about 0.13 h−1 . The first batch of feeding medium was introduced at 18 h and repeated four times during the 134-h-long fermentation. The overall biomass yield, lipid yield and cellular lipid content were 0.35 g g−1 , 0.23 g g−1 and 67.5% (w/w), respectively. The overall lipid production rate Y. Li et al. / Enzyme and Microbial Technology 41 (2007) 312–317 Fig. 4. Pilot-scale fed-batch fermentation profile for R. toruloides Y4 in a 15-l fermenter. was 0.54 g l−1 h−1 . Within the 134-h-long fermentation, 2500 g of glucose was fed into the reactor, which corresponds to a final substrate concentration of 307 g l−1 . The ammonium-N (inorganic nitrogen) concentration in the initial medium was 0.068 mol l−1 , however, it dropped to 0.024, 0.015, and 0.005 mol l−1 , at 6, 10, and 18 h, respectively. No media ammonium-N was detectable after 24 h. Such a fast nitrogen source consumption pattern was in well accordance with other R. toruloides strain [15,16]. The course of ammonium-N depletion also correlated with the onset of lipid accumulation. As shown in Table 1, cellular lipid contents for the 6- and 18-h samples are 5.5% (w/w) and 18.1% (w/w), respectively, indicating that the lipid accumulation process was partially established, although excess nitrogen sources in the culture broth favored cell growth. Exhaustion of ammonium-N apparently greatly promoted lipid accumulation, as revealed by a lipid content of 43.8% (w/w) in the 38-h sample. Overall, these data suggested that nitrogen limitation, the key to oleaginity [7], could be realized later through feeding carbon sources, while an initial cell growth stage with excess nitrogen resources was preferred for better efficiency. 3.3. Fatty acid compositional analysis Lipid samples produced by the fermenter fed-batch culture system were analyzed by gas chromatography. Relative fatty 315 acid content is shown in Table 1. It is clear that R. toruloides Y4 is composed mainly of long-chain fatty acids with 16 and 18 carbon atoms. These data show that the distribution of some fatty acids, namely, C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid) and C18:3 (linolenic acid), were almost constant during the whole process. A clear decrease in the relative content of C18:1 (oleic acid) was found, while an increase for C18:0 (stearic acid) was observed over time. It is also interesting to note that C18:2 (linoleic acid) showed an increased presence during the early stage, namely from 8.2% in the 6-h sample to 11.9% in the 18-h sample. However, linoleic acid content drastically decreased when lipid accumulation initiated and turned out to be 4.7 and 8.8%, in the 38 and 78-h samples, respectively. It is known that fatty acid distribution impacts on the saponification number (SN) and iodine value (IV) of the particular lipids [21], which can also determine the cetane number (CN) of the corresponding biodiesel product. According to the empirical equation [22], with the percentage distribution shown in Table 1, all the lipid samples could produce biodiesel with CN values higher than 51.0. Minimal CN values have been set at 47, 49 and 51, by biodiesel standards ASTMD 6751 (USA), DIN 51606 (Germany) and EN 14214 (European Organization), respectively. Therefore, the FAMEs produced from the microbial lipids of R. toruloides Y4 meet these standards. 4. Discussion Nutrient imbalance in the culture medium has long been known to trigger lipid accumulation by oleaginous microorganisms. When cells run out of key nutrients, usually nitrogen, excess substrate continues to be assimilated by the cells and converted into fat for storage. However, under nitrogen-limited conditions, cell propagation is drastically depressed, which in many cases restricts cell density. To achieve a high-density cell culture for microbial lipid fermentation, different substrates and cultivation modes have been used. Fed-batch cultivation modes have been widely applied for microbial lipid production. Pan et al. [13] reported on fedbatch cultures of Rhodotorula glutinis with feeding medium containing 600 g l−1 glucose, 20 g l−1 yeast extract and 9 g l−1 MgSO4 ·7H2 O, and the final cellular lipid content was 40% (w/w). Yamauchi et al. [14] obtained a high cell density of 153 g l−1 and lipid content of 54% (w/w) with Lipomyces starkeyi using a complicated feeding medium and a high seed Table 1 Fatty acid composition of R. toruloides Y4 cells during fed-batch fermentation Cultivation time (h) 6 18 38 78 134 Lipid content (%, w/w) 5.5 18.1 43.8 61.7 67.5 Relative amount of total fatty acids (%, w/w) C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 1.7 1.5 1.6 1.6 1.3 23.6 20.2 23.3 21.6 20.0 0.9 0.3 0.6 1.0 0.6 6.7 7.6 14.3 16.0 14.6 55.3 55.9 53.7 49.3 46.9 8.2 11.9 4.7 8.8 13.1 3.6 2.6 1.7 2.7 3.5 316 Y. Li et al. / Enzyme and Microbial Technology 41 (2007) 312–317 inoculum of 50% (v/v) was also employed in this experiment. Ykema et al. [23] examined lipid production in various culture modes with whey permeate using Candida curvatus. A cell density of 91.4 g l−1 and lipid content of 33% (w/w) were observed using a partial recycling method, while both nitrogen and carbon sources were supplied. Meesters et al. [4] used glycerol as a carbon source in fed-batch fermentation with Candida curvatus and realized a cell density of 118 g l−1 in 50 h with a lipid production rate of 0.59 g l−1 h−1 ; however, the final cellular lipid content was only 25% (w/w). It should be mentioned that these previous fed-batch lipid fermentation processes simultaneously introduced a carbon and nitrogen source, which might produce a disfavored C/N ratio for lipid accumulation. In addition, this feeding operation may also be complicated. To develop an improved culture method for lipid production with R. toruoides Y4, batch flask cultures were firstly carried out to determine the suitable substrate concentration of the initial medium. As these experiments were targeted to define the physiological response of the test strain to a glucose gradient, a minimal medium was employed. It was found that R. toruoides Y4 grew with a similar profile in glucose concentrations up to 150 g l−1 , indicating a good ability to deal with osmotic stress. This made it possible to feed concentrated glucose solution at long time intervals in a discontinuous way during the fed-batch cultivation experiments. Fed-batch flask cultures were then performed to proof-test the cell-density limits for R. toruoides Y4. As R. toruloides has been found to accumulate more lipids when an organic nitrogen source was employed [15,16], we used yeast extract and peptone as nitrogen sources in the initial medium for the shaking flask fed-batch cultures. Such nutrient-rich initial media were also used to promote cell growth rates and to increase the duration of the growth phase. During the feeding stage, only a carbon source was introduced. At the end of the 25-day-long fermentation experiment, dry cell biomass reached 151.5 g l−1 , while cellular lipid content was 48.0% (w/w). Thus, our data formally established a two-stage fed-batch strategy for microbial lipid production. Although the flask fed-batch fermentation obtained a high biomass, it took 25 days for one cycle and the lipid content was relatively low. To further increase the growth rate and lipid content, experiments were done in a 15-l stirred-tank bioreactor. Cell density and lipid content reached 106.5 g l−1 and 67.5% (w/w) over a 134-h fermentation. The overall lipid productivity was 0.54 g l−1 h−1 , which was much higher than that found in the flask fed-batch culture (approximately 0.12 g l−1 h−1 ). Fatty acid compositional analysis revealed that four major constituent fatty acids were oleic acid, palmitic acid, stearic acid and linoleic acid. Individual fatty acid distributions varied with progression of the culture. A slight increase in stearic acid content was found during the culture process; while oleic acid content was decreased. Based on the fatty acid analysis data, FAMEs made from microbial oil by R. toruloides Y4 have a great potential for biodiesel production. In conclusion, we described a simple fed-batch process for lipid production by oleaginous yeast R. toruoides Y4 with high cell density. The process features a nutrient-rich initial medium, sole carbon source feeding and is convenient for large-scale operation. This strategy significantly improves biomass and lipid productivity and will be useful for further engineering of costeffective microbial lipid production processes. Acknowledgements This work was supported by National Basic Research Program of China (973 Program) (no. 2004CB719703) and CAS “100 Talents” program. References [1] Ratledge C, Wynn JP. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 2002;51:1–51. [2] Rattray JBM. In: Ratledge C, Wilkinson SG, editors. Microbial lipids, vol. 1. London/New York: Academic press; 1989. p. 555–697. [3] Zhao ZB. Toward cheaper microbial oil for biodiesel. China Biotechnol 2005;25(2):8–11. [4] Meesters PAEP, Huijberts GNM, Eggink G. High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl Microbiol Biotechnol 1996;45:575–9. [5] Papanikolaou S, Chevalot I, Komaitis M, Marc I. Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl Microbiol Biotechnol 2002;58:308–12. [6] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification—a review. Renew Sust Energy Rev 2006;10(3):248–68. [7] Moreten RS. Physiology of lipid accumulation yeast. In: Moreton RS, editor. Single cell oil. London: Longman; 1988. p. 1–32. [8] Montet D, Ratomahenina R, Galzy P, Pina M, Graille J. A study of the influence of the growth media on the fatty acid composition in Candida lipolytica diddens and lodder. Biotechnol lett 1985;7:733–44. [9] Papanikolaou S, Chevalot I, Komaitis M, Aggelis G, Marc I. Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats. Ant Leeuwen 2001;80:215–24. [10] Papanikolaou S, Galiotou-Panayotou M, Chevalot I, Komaitis M, Marc I, Aggelis G. Influence of glucose and saturated free-fatty acid mixtures on citric acid and lipid production by Yarrowia lipolytica. Curr Microbiol 2006;52:134–42. [11] Evans CT, Ratledge C. A comparison of the oleaginous yeast Candida curvata, grown on different carbon sources in continuous and batch culture. Lipid 1983;18:623–9. [12] Hassan M, Blanc PJ, Granger LM. Lipid production by an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum grown in a single stage continuous culture. Appl Microbiol Biotechnol 1993;40:483–8. [13] Pan JG, Kwak MY, Rhee JS. High density cell culture of Rhodotorula glutinis using oxygen-enriched air. Biotechnol Lett 1986;8:715–8. [14] Yamauchi H, Mori H, Kobayashi T, Shimizu S. Mass production of lipids by Lipomyces starkeyi in microcomputer-aided-fed-batch culture. J Ferment Technol 1983;61:275–80. [15] Evans CT, Ratledge C. Influence of nitrogen metabolism on lipid accumulation in oleaginous yeasts. J Gen Microbiol 1984;130:1693–704. [16] Evans CT, Ratledge C. Influence of nitrogen metabolism on lipid accumulation by Rhodosporidium toruloides CBS 14. J Gen Microbiol 1984;130:1705–10. [17] Evans CT, Ratledge C. Phosphofructokinase and the regulation of the flux of carbon from glucose to lipid in the oleaginous yeast Rhodosporidium toruloides. J Gen Microbiol 1984;130:3251–64. [18] Li YH, Liu B, Zhao ZB, Bai FW. Optimized culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides. Chin J Biotechnol 2006;22(4):650–6. Y. Li et al. / Enzyme and Microbial Technology 41 (2007) 312–317 [19] Sherman F. Getting started with yeast. Meth Enzymol 2002;350:3–41. [20] Li ZF, Zhang L, Shen XJ, Lai BS, Sun SQ. A comparative study on four method of fungi lipid extraction. Microbiology 2001;28(6):72–5. [21] Kalayasiri P, Jayashke N, Krisnangkura K. Survey of seed oils for use as diesel fuels. J Am Oil Chem Soc 1996;73:471–4. 317 [22] Krisnangkura K. A simple method for estimation of Cetane index of vegetable oil methyl esters. J Am Oil Chem Soc 1986;63:552–3. [23] Ykema A, Verbree EC, Kater MM, Smit H. Optimization of lipid production in the oleaginous yeast Apiotrichum curvatum in wheypermeate. Appl Microbiol Biotechnol 1988;29:211–8.