Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2014 www.elsevier.com/locate/jbiosc
Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source Hong-Wei Yen* and Jung-Tzu Chang Department of Chemical and Materials Engineering, Tunghai University, Taiwan, ROC Received 19 August 2014; accepted 1 October 2014 Available online xxx
The conversion of abundant lignocellulosic biomass (LCB) to valuable compounds has become a very attractive idea recently. This study successfully used LCB (rice straw) hydrolysate as a carbon source for the cultivation of oleaginous yeast-Rhodotorula glutinis in an airlift bioreactor. The lipid content of 34.3 ± 0.6% was obtained in an airlift batch with 60 g reducing sugars/L of LCB hydrolysate at a 2 vvm aeration rate. While using LCB hydrolysate as the carbon source, oleic acid (C18:1) and linoleic acid (C18:2) were the predominant fatty acids of the microbial lipids. Using LCB hydrolysate in the airlift bioreactor at 2 vvm achieved the highest cell mass growth as compared to the agitation tank. Despite the low lipid content of the batch using LCB hydrolysate, this low cost feedstock has the potential of being adopted for the production of b-carotene instead of lipid accumulation in the airlift bioreactor for the cultivation of R. glutinis. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Hydrolysate; Rhodotorula; Oleaginous; Rice straw; Airlift bioreactor]
Various renewable lipids have been explored for the production of biodiesel to replace fossil-fuel derived diesel, including vegetable oils, animal fats, and kitchen waste oils. However, the major obstacle to commercialization of biodiesel production is its high price compared to the low market price of conventional petroleumbased diesel. It has been estimated that over 70% of the market price of biodiesel lies in the feedstock cost (1). Consequently, much effort has been made toward finding cheap and renewable oil sources for biodiesel production. To this end, the conversion of abundant lignocellulosic biomass (LCB) into biofuels for use as transportation fuels has been presented as a viable option for improving energy security and reducing greenhouse gas emissions (2). LCB is the world’s most abundant and attractive biomass resource that can be used as a raw material for the economic production of microbial oils. The total worldwide production of cellulose and hemicellulose is about 85 l09 tons/annum, with cereal straw estimated to exceed 2.9 l09 tons/annum (3). Although the bioconversion of lignocellulosic residues into ethanol or butanol has been performed successfully, it has been both technically and economically challenging to produce biodiesel from LCB, and the technology has yet to progress from the laboratory stage to the demonstration stage. The major LCB resources come from residues generated by agricultural, forestry, and industrial sources. The difficulty of converting lignocellulosic material to useful biofuel products at economic competitive cost is the major obstacle to widespread utilization of this important resource (4). Hence, there is considerable economic interest in the development of processes * Corresponding author. 181, Taiwan Harbor 3rd Rd, Taichung, Taiwan, ROC. Tel.: þ886 4 23590262x209; fax: þ886 4 23590009. E-mail addresses: [email protected], [email protected] (H.-W. Yen).
that can pretreat and convert inexpensive cellulosic wastes into valuable products to be used as fuel. Some such processes already have been developed, but, so far, the hydrolysis process is still the most challenging step from an economic perspective (5,6). A technology called the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids had been examined by using Cryptococcus curvatus for the lipids accumulation (7). The biological production of single cell oils (SCO) from oleaginous microorganisms as the oil feedstock is considered one of the feasible routes to biodiesel production in lieu of using vegetable oils. Since microbial lipids have many advantages over vegetable oils, such as a short life cycle and no need for agricultural land, they have attracted much interest as a potential non-food feedstock for biodiesel production. Oleaginous microorganisms are classified as strains that have a microbial lipid content in excess of 20% (g/g) (8). Numerous oleaginous yeasts and microalgae have been reported to grow and accumulate significant amounts of lipids. More specifically, the characteristics of rapid growth and the ability to utilize a range of carbon sources (e.g., glucose, glycerol) makes Rhodotorula glutinis an attractive candidate for microbial oil production (9e12). Further, an oil content as high as 72% obtained in R. glutinis has been reported (9). Pan and his colleagues obtained a cell density of 185 g/ l in an 84-h fed-batch culture of R. glutinis aerated with oxygenenriched air (13). In general, the components of fatty acids extracted from R. glutinis are mainly palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linolenic acid (C18:2), where palmitic acid and oleic acid often account for over 80% of total fatty acids (14). To further convert the microbial oils to biodiesel, a direct methanolysis process by the assisting of acids had been explored, which reached up to 97% of
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
Please cite this article in press as: Yen, H.-W., and Chang, J.-T., Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
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yield under several specific conditions (15). Besides the accumulation of high lipids, R. glutinis is also well-known in the industry as a b-carotene producer (16), which b-carotene is regarded as a valuable compound in the health industry. In this study, the hydrolysate of LCB (rice straw) was explored for the cultivation of oleaginous yeast- R. glutinis to produce SCO and b-carotene. Due to the potential inhibitors existing in the hydrolysate solution, the suitability of hydrolysate as a carbon source for the growth of R. glutinis is firstly examined. The C/N ratio effects by varying the adding amount of yeast extract on total lipid production and b-carotene accumulation are also evaluated. To achieve the scaled-up production, the performance of R. glutinis in the internal-loop airlift bioreactor and in the conventional agitation tank using LCB hydrolysate is also compared in this study. MATERIALS AND METHODS Microorganism and medium Freeze-dried R. glutinis BCRC 22360 was obtained from the Bioresource Collection and Research Center, Taiwan (BCRC). The seed medium composition and the cultivation methods followed suggestions provided by the BCRC. The fermentation medium (per liter) comprised defined amounts of different carbon sources (including glucose, glycerol, crude glycerol and LCB hydrolysate), 2 g of yeast extract, 2 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4$7H2O, 0.1 g of CaCl2 and 0.1 g of NaCl (17). Sodium hydroxide at 1.0 N or hydrogen chloride at 1.0 N was used to adjust the pH. LCB hydrolysate was kindly provided by the Institute of Energy Research, Taiwan, which was prepared through 2 stages of LCB hydrolysis of agriculture wastes and vegetable residues (pretreatment by dilute acid to remove hemicellulose and using cellulase for the following cellulose hydrolysis). The detail hydrolysis procedure was still the confidential process to the Institute of Energy Research, Taiwan, which could not be revealed here. The components of LCB hydrolysate is shown in Table 1. Before the use of LCB hydrolysate, it would be diluted properly to the designed reducing sugar concentration. The crude glycerol was purchased from a local biodiesel manufacture company (Yu-Hwa biodiesel company, Taiwan), with crude glycerol being the by-product of the conventional base catalyst transesterification process. No any extra purification process was performed before crude glycerol was used as one of the medium ingredients. Fermentation in 5-L conventional agitation fermentor The batch fermentation was operated in a conventional 5-L stirred desk-top fermentor (model BTF-A, Biotop Ltd., Taiwan) with a 2-L working volume, the fermentation medium of which was described in the above section. The pH level was maintained at 5.5 by automatically feeding NaOH solution (1.0 N). The fermentor was operated at 24 C with dissolved oxygen controlled at over 30% by adjusting the agitation rate in the range of 200e500 rpm with a 1 vvm aeration rate. Fermentation in 5-L internal-loop airlift bioreactor Batch fermentation was respectively carried out in a in a 5-L internal-loop glass airlift bioreactor (30 cm in height, with a 10 cm outer diameter and 7.7 cm inner tube diameter) with a working volume of 3 L. All experiments were controlled at 24 C and the pH was controlled at 5.5 by using 1 N NaOH solution. The aeration rate was performed at 1.5 vvm to explore the effects of using LCB hydrolysate on cell growth and on the accumulation of total lipids. Analytical methods Infrared balance was adopted to rapidly measure the cell mass concentration. Five ml of broth was centrifuged at 7000 rpm for 10 min. After removing the supernatant, about an equal volume of distilled water was added to eliminate the impurities. The washing procedure was performed several times, and the final liquor was dried by using infrared balance at 150 C to evaporate the water content. The total lipid analysis was based on a modification of the procedure used by Bligh and Dyer (18). The dry cell mass was ground into a fine powder; then, 0.05 g of the powder was blended with 5 ml chloroform/methanol (2:1), and the mixture was agitated for 20 min at room temperature in an orbital shaker. The solvent phase was recovered by centrifugation. The same process was repeated twice, and the whole solvent was evaporated and dried under vacuum conditions. The dinitrosalicylic acid (DNS) analysis was adopted for the measurement of reducing sugars of LCB hydrolysate. The glycerol concentration was measured by HPLC (Agilent series 1100, Agilent Technologies, Santa Clara, CA, USA) with a refractive index detector, while the analysis was performed in a C-18 column (Vercopak N5 ODS, 250 mm 4.6 mm, Taiwan). The mobile phase was composed of 0.01 N H2SO4 with a flow rate of 0.4 ml/
min (19). The measurement of b-carotene was performed as following: 50 mg of cells, after freeze-drying, were mixed with a 2 ml mixture consisting of acetonitrile, isopropyl alcohol and ethyl acetate (40:40:20 v/v), followed by ultrasonication (power 5, reaction time 2 min) to crush the cells for pigment extraction. The extract was centrifuged and the supernatant filtered through a 0.45-mm membrane filter and subjected to HPLC analyses, which were performed on a reversed-phase C18 analytical column [N5 ODS (C-18) 4.6 mm i.d. 250 mm]. The mobile phase was composed of acetonitrile, isopropanol and ethyl acetate (40:40:20 v/v) and had a flow rate of 0.7 ml/min. The column thermostat was set at 25 C, while the detector was operated at a wavelength of 457 nm. Analysis of lipid composition 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 4 ml of a 0.5 N KOH/ methanol solution at 100 C for 15 min, followed by the addition of 5 ml BF3 diethyl etherate and 5 ml methanol. The mixture was refluxed for 15 min, cooled, diluted with distilled water and then extracted with n-Hexane. The organic layer was washed with distilled water and subjected to fatty acid compositional analysis. Fatty acid methyl esters were analyzed using a gas chromatography instrument (Focus GC, Thermo, USA) equipped with a cross-linked capillary Column SEG BP20 (25 m 0.22 mm 0.25 mm) and flame ionization detector. Operating conditions were as follows: N2 carrier gas at 40 ml/min; injection port temperature of 230 C; oven temperature of 200 C; and, a detector temperature of 230 C. Fatty acids were identified by a comparison of their retention times with those of standard ones, quantified based on their respective peak areas, and then normalized.
RESULTS AND DISCUSSION Using hydrolysate from LCB as the carbon source Several studies have revealed results of conventional carbon sources being used as the carbon source for the growth of R. glutinis in the literature. In this study, glucose, glycerol, crude glycerol and LCB hydrolysate each at 30 g/L (hydrolysate solution was prepared at the equivalent of 30 g/L of reducing sugars) were compared for the growth of R. glutinis. As shown in Fig. 1, LCB hydrolysate solution can be a good carbon source for the growth of R. glutinis, which can produce a similar cell mass as compared to the batch using glucose. As seen in Table 1 of LCB hydrolysate components analysis, the glucose concentration was about 50% of reducing sugars (measured by the DNS method), which implied that there was still some other reducing sugars unclassified in the LCB hydrolysate. The concentration of glucose in the hydrolysate was even higher than that of xylose, which were 71.5 and 18.1 g/L, respectively. The high cell mass obtained in the batch using LCB hydrolysate suggests that the potential inhibitors (e.g., HMF, furfural) derived from the hydrolysis process would not inhibit the growth of R. glutinis under these cultivation conditions. Due to the recalcitrant nature of LCB products, different pretreatment process was often adopted for the destruction of complex cells was and exposed the cellulose for the further degradation of cellulolytic enzymes. On the other hand, lignocellulose pretreatment has the detrimental effect of also releasing a wide range of hydrolysis compounds, which are inhibitory to fermenting microorganisms and cellulolytic enzymes (20,21). Nevertheless, such kind of potential inhibitors seems not affecting the growth of R. glutinis in this study under the investigated conditions. It had been also reported that lipid production of Rhodosporidium toruloides afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile also indicated that those inhibitors had little effects on lipid biosynthesis (22). The lipid composition analyses for each batch with different carbon source are shown in Table 2, which indicates that oleic acid (C18:1) dominated the fatty acid components among all batches.
TABLE 1. Components of LCB hydrolysate solution (g/L). Reducing sugar
Glucose
Xylose
Arabinose
Formic acid
Acetic acid
HMF
Furfural
148.5 4.6
71 3.2
18.1 1.5
0.5 0.1
2.3 0.2
4.9 0.1
0.11 0.1
0.2 0
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FIG. 1. Effects of hydrolysate on the growth of R. glutinis as compared to other carbon sources.
Besides oleic acid, linoleic acid (C18:2) comprised a relative high ratio in the batch using LCB, of which the percentage of linoleic acid was far higher than that of the other batches. The ratios of saturated to unsaturated fatty acids among all batches are similar. More unsaturated fatty acid would lead to a lower cold filter plugging point (CFPP) and a higher instability of oil products due to the easier oxidation of unsaturated fatty acid (23). A similar percentage of saturated and unsaturated fatty acids indicates that the microbial lipids derived from the batches with different carbon sources might have a similar quality. Although, the growth of R. glutinis was not severely affected by using LCB hydrolysate, it was noted that the batch using hydrolysate had the lowest lipid content among all batches, which was as low as only w10%. It was reported that using undetoxified cornstover hydrolysate as substrate, the yeast of Rhodotorula graminis achieved a lipid content of 34% w/w (24). Even in the detoxified liquid hydrolysate from the pretreatment of wheat straw, about 20%
of lipid content was obtained in the cultivation of R. glutinis (5). In general, R. glutinis is well-known as an oleaginous microorganism for its ability of being the producer for total lipid production. A remarkably high lipid content of 72% for R. glutinis has been reported in the literature. Therefore, such a low lipid content indicated that the batch using LCB hydrolysate had a low C/N ratio, which led to the low total lipid content observed. The idea of increasing the C/N ratio to enhance total lipids production has been validated by many researchers. Therefore, the effects of nitrogen source-yeast extract (YE) on lipid production are investigated in next section. Effects of YE addition amount with hydrolysate as the carbon source To raise the lipid content obtained in the batch with LCB hydrolysate, the adjustment of YE addition in the medium containing hydrolysate at 30 g/L of reducing sugars was performed. Results are shown in Fig. 2, which reveals no significant difference in
TABLE 2. Components of fatty acid distribution with different carbon sources. Relative percentage (%)
Glucose Glycerol LCB hydrolysate
C 14:0
C 16:0
C 16:1
C 18:0
C 18:1
C 18:2
C 18:3
0.8 0.8 1.04
15 13.2 13.3
0.4 1.7 0.95
6.1 4.6 5.1
72.2 68.2 55.5
3.5 8.8 20.2
1.9 2.7 6.3
Saturated
Unsaturated
21.9 18.6 19.4
78.1 81.4 80.6
FIG. 2. Effects of nitrogen source concentration on the growth of R. glutinis while using hydrolysate as the carbon source.
Please cite this article in press as: Yen, H.-W., and Chang, J.-T., Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
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J. BIOSCI. BIOENG., TABLE 3. The comparison of b-carotene and lipid content among all batches with LCB.
b-Carotene (mg/g) Lipid content (%)
Stirred tank
Airlift 1 vvm
Airlift 2 vvm
1.243 0.168 27.4 3.9
1.016 0.081 26.0 1.6
0.486 0.198 34.3 0.6
with no YE adding can yield the highest b-carotene content of 1.3 0.01 mg/g among all batches. The overall implications derived from Figs. 1 and 2 are that the LCB hydrolysate might not be a suitable carbon source for total lipid production in the cultivation of oleaginous R. glutinis, but it might be a good substrate for b-carotene production.
the lipid content measured among all batches with varied YE addition, ranging from 0 to 2 g/L. Increased amounts of YE in the medium produced a higher cell mass, the highest of which attained 9.2 g/L in the shaker trial with 2 g/L of YE addition. Nevertheless, the lipid content remained at the approximate level of 10% in all batches, regardless of the amount of added YE. This implies that a simple C/N ratio adjustment with LCB hydrolysate as the carbon source could not raise the lipid content in the cultivation of R. glutinis. However, in the cultivation of oleaginous yeast-Trichosporon cutaneum using corncob residues hydrolysate, the lipid content was verified to be enhanced by increasing the C/ N ratio, of which the highest lipid content was 32.1% (25). Even the value of 32.1% obtained in the batch with corncob residues hydrolysate was still lower than the regular lipid content obtained in other studies using conventional carbon sources. Conclusively, LCB hydrolysate seems not to be a good carbon source for the growth of R. glutinis as regarding to the accumulation of high lipid content for biodiesel production. Nevertheless, even LCB hydrolysate cannot have the high lipid content obtained, the b-carotene production is another attractive product of R. glutinis cultivation. Fig. 2 also reveals that the batch
Comparison of R. glutinis cultivation in the airlift and agitation tank by using cellulose hydrolysate Despite the accumulation of total lipids using LCB hydrolysate as the carbon source seeming unsuccessful in the shaker trials, the growth of R. glutinis using LCB hydrolysate at 60 g/L of reducing sugars in the airlift bioreactor (at the aeration rates of 1 and 2 vvm, respectively) and in a conventional agitation tank were examined. Results are shown in Figs. 3 and 4. As seen in Fig. 3 featuring the cells growth curve, a higher cell mass was obtained in the airlift tank with 2 vvm than that of the other two operations, for which the airlift batch at 2 vvm had the highest 20 g/L of cell mass after 72 h cultivation. The reducing sugars measurement also revealed the fastest consumption curve in the airlift (2 vvm) batch, which suggests that the 2 vvm airlift batch can achieve the highest cell mass (as shown in Fig. 4). In addition to the highest cell mass observed in airlift batch with 2 vvm, it also had the highest cell growth rate of 0.283 g/L h as compared to the 0.111 and 0.139 g/L h for the airlift batch at 1 vvm and of the agitation tank, respectively. For comparison, the lipid content and b-carotene content among all batches are shown in Table 3, which indicates that the highest lipid content of 34.3 0.6% was obtained in the airlift batch with 2 vvm; far higher than that of the shaker trials. In contrast to the highest lipid content, the airlift batch with 2 vvm had the lowest b-carotene content at 0.486 0.198 mg/g. Among all batches, the highest b-carotene content achieved, as observed in the agitation tank, was 1.243 0.168 mg/g, which is quite competitive compared to other studies (26,27). To further confirm that the airlift batch with 2 vvm could have the highest lipid production, the consumption profile of major carbon sources in the airlift batch with 2 vvm was examined. Results are shown in Fig. 5, and
FIG. 4. Consumption of reducing sugars for the growth of R. glutinis in the stirred tank and in the airlift bioreactor by using hydrolysate as the carbon source.
FIG. 5. Components profile of airlift bioreactor at 2 vvm with 60 g/L LCB reducing sugars.
FIG. 3. Growth of R. glutinis in the stirred tank and in the airlift bioreactor by using hydrolysate as the carbon source.
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indicate that the major carbon sources of glucose and xylose would be simultaneously utilized for the growth of R. glutinis at almost the same rate. It was estimated that the lipids yield in the airlift tank with 2 vvm was about 12% (gram of total lipid produced per gram of reducing sugar consumed), which was similar to the value of 15% obtained in the batch with corn-stover hydrolysate by using T. cutaneum (22). The operation of the airlift bioreactor at 2 vvm achieved the highest cell mass growth as compared to the agitation tank. In conclusion, the LCB hydrolysate has the potential of being adopted for the production of b-carotene instead of lipid accumulation in the airlift bioreactor for the cultivation of R. glutinis. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from Taiwan’s MOST under grant number 103-2623-E-029-001-ET and 103-3113-E-006-006. References 1. Miao, X. and Wu, Q.: Biodiesel production from heterotrophic microalgal oil, Bioresour. Technol., 97, 841e846 (2006). 2. Yousuf, A.: Biodiesel from lignocellulosic biomasseprospects and challenges, Waste Manag., 32, 2061e2067 (2012). 3. Sun, X. F., Sun, R. C., and Tomkinson, J.: Degradation of wheat straw lignin and hemicellulosic polymers by a totally chlorine-free method, J. Polym. Degrad. Stab., 83, 47e57 (2004). 4. Sun, Y. and Cheng, J.: Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol., 83, 1e11 (2002). 5. Yu, X., Zheng, Y., Dorgan, K. M., and Chen, S.: Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid, Bioresour. Technol., 102, 6134e6140 (2011). 6. Xie, H., Shen, H., Gong, Z., Wang, Q., Zhao, Z. K., and Bai, F.: Enzymatic hydrolysates of corn stover pretreated by a N-methylpyrrolidoneeionic liquid solution for microbial lipid production, Green Chem., 14, 1202e1210 (2012). 7. Gong, Z., Shen, H., Wang, Q., Yang, X., Xie, H., and Zhao, Z. K.: Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process, Biotechnol. Biofuels, 6, 36e46 (2013). 8. Ratledge, C. and Cohen, Z.: Microbial and algal oils: do they have a future for biodiesel or as commodity oils? Lipid Technol., 20, 155e160 (2008). 9. Meng, X., Yang, J., Xu, X., Zhang, L., Nie, Q., and Xian, M.: Biodiesel production from oleaginous microorganisms, Renew. Energy, 34, 1e5 (2009).
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10. Yen, H. W., Yang, Y. C., and Yu, Y. H.: Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis, J. Biosci. Bioeng., 114, 453e456 (2012). 11. Yen, H. W. and Zhang, Z.: Enhancement of cell growth rate by light irradiation in the cultivation of Rhodotorula glutinis, Bioresour. Technol., 102, 9279e9281 (2011). 12. Yen, H. W. and Zhang, Z.: Effects of dissolved oxygen level on cell growth and total lipid accumulation in the cultivation of Rhodotorula glutinis, J. Biosci. Bioeng., 112, 71e74 (2011). 13. Pan, J. G., Kwak, M. Y., and Rhee, J. S.: High density cell culture of Rhodotorula glutinis using oxygen-enriched air, Biotechnol. Lett., 8, 715e718 (1986). 14. Li, M., Liu, G. L., Chi, Z., and Chi, Z. M.: Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a, Biomass Bioenergy, 34, 101e107 (2010). 15. Liu, B. and Zhao, Z. K.: Biodiesel production by direct methanolysis of oleaginous microbial biomass, J. Chem. Technol. Biotechnol., 82, 775e780 (2007). 16. Aksu, Z. and Eren, A. T.: Production of carotenoids by the isolated yeast of Rhodotorula glutinis, Biochem. Eng. J., 35, 107e113 (2007). 17. Kim, B. K., Park, P. K., Chae, H. J., and Kim, E. Y.: Effect of phenol on bcarotene content in total carotenoids production in cultivation of Rhodotorula glutinis, Korean J. Chem. Eng., 21, 689e692 (2004). 18. Bligh, E. G. and Dyer, W. J.: A rapid method for total lipid extraction and purification, Can. J. Biochem. Physiol., 37, 911e917 (1959). 19. Athalye, S. K., Garcia, R. A., and Wen, Z.: Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare, J. Agric. Food Chem., 57, 2739e2744 (2009). 20. Cao, G., Ximenes, E., Nichols, N. N., Zhang, L., and Ladisch, M.: Biological abatement of cellulase inhibitors, Bioresour. Technol., 146, 604e610 (2013). 21. Castro, E., Diaz, M. J., Cara, C., Ruiz, E., Romero, I., and Moya, M.: Dilute acid pretreatment of rapeseed straw for fermentable sugar generation, Bioresour. Technol., 102, 1270e1276 (2011). 22. Hu, C., Wu, S., Wang, Q., Jin, G., Shen, H., and Zhao, Z. K.: Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum, Biotechnol. Biofuels, 4, 25e32 (2011). 23. Schleicher, T., Werkmeister, R., Russ, W., and Meyer-Pittroff, R.: Microbiological stability of biodiesel-diesel-mixtures, Bioresour. Technol., 100, 724e730 (2009). 24. Galafassi, S., Cucchetti, D., Pizza, F., Franzosi, G., Bianchi, D., and Compagno, C.: Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis, Bioresour. Technol., 111, 398e403 (2012). 25. Gao, Q., Cui, Z., Zhang, J., and Bao, J.: Lipid fermentation of corncob residues hydrolysate by oleaginous yeast Trichosporon cutaneum, Bioresour. Technol., 152, 552e556 (2014). 26. Bhosale, P. and Gadre, R.: b-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant, J. Ind. Microbiol. Biotechnol., 26, 327e332 (2001). 27. Bhosale, P. B. and Gadre, R. V.: Production of b-carotene by a mutant of Rhodotorula glutinis, Appl. Microbiol. Biotechnol., 55, 423e427 (2001).
Please cite this article in press as: Yen, H.-W., and Chang, J.-T., Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source Hong-Wei Yen* and Jung-Tzu Chang Department of Chemical and Materials Engineering, Tunghai University, Taiwan, ROC Received 19 August 2014; accepted 1 October 2014 Available online xxx
The conversion of abundant lignocellulosic biomass (LCB) to valuable compounds has become a very attractive idea recently. This study successfully used LCB (rice straw) hydrolysate as a carbon source for the cultivation of oleaginous yeast-Rhodotorula glutinis in an airlift bioreactor. The lipid content of 34.3 ± 0.6% was obtained in an airlift batch with 60 g reducing sugars/L of LCB hydrolysate at a 2 vvm aeration rate. While using LCB hydrolysate as the carbon source, oleic acid (C18:1) and linoleic acid (C18:2) were the predominant fatty acids of the microbial lipids. Using LCB hydrolysate in the airlift bioreactor at 2 vvm achieved the highest cell mass growth as compared to the agitation tank. Despite the low lipid content of the batch using LCB hydrolysate, this low cost feedstock has the potential of being adopted for the production of b-carotene instead of lipid accumulation in the airlift bioreactor for the cultivation of R. glutinis. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Hydrolysate; Rhodotorula; Oleaginous; Rice straw; Airlift bioreactor]
Various renewable lipids have been explored for the production of biodiesel to replace fossil-fuel derived diesel, including vegetable oils, animal fats, and kitchen waste oils. However, the major obstacle to commercialization of biodiesel production is its high price compared to the low market price of conventional petroleumbased diesel. It has been estimated that over 70% of the market price of biodiesel lies in the feedstock cost (1). Consequently, much effort has been made toward finding cheap and renewable oil sources for biodiesel production. To this end, the conversion of abundant lignocellulosic biomass (LCB) into biofuels for use as transportation fuels has been presented as a viable option for improving energy security and reducing greenhouse gas emissions (2). LCB is the world’s most abundant and attractive biomass resource that can be used as a raw material for the economic production of microbial oils. The total worldwide production of cellulose and hemicellulose is about 85 l09 tons/annum, with cereal straw estimated to exceed 2.9 l09 tons/annum (3). Although the bioconversion of lignocellulosic residues into ethanol or butanol has been performed successfully, it has been both technically and economically challenging to produce biodiesel from LCB, and the technology has yet to progress from the laboratory stage to the demonstration stage. The major LCB resources come from residues generated by agricultural, forestry, and industrial sources. The difficulty of converting lignocellulosic material to useful biofuel products at economic competitive cost is the major obstacle to widespread utilization of this important resource (4). Hence, there is considerable economic interest in the development of processes * Corresponding author. 181, Taiwan Harbor 3rd Rd, Taichung, Taiwan, ROC. Tel.: þ886 4 23590262x209; fax: þ886 4 23590009. E-mail addresses: [email protected], [email protected] (H.-W. Yen).
that can pretreat and convert inexpensive cellulosic wastes into valuable products to be used as fuel. Some such processes already have been developed, but, so far, the hydrolysis process is still the most challenging step from an economic perspective (5,6). A technology called the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids had been examined by using Cryptococcus curvatus for the lipids accumulation (7). The biological production of single cell oils (SCO) from oleaginous microorganisms as the oil feedstock is considered one of the feasible routes to biodiesel production in lieu of using vegetable oils. Since microbial lipids have many advantages over vegetable oils, such as a short life cycle and no need for agricultural land, they have attracted much interest as a potential non-food feedstock for biodiesel production. Oleaginous microorganisms are classified as strains that have a microbial lipid content in excess of 20% (g/g) (8). Numerous oleaginous yeasts and microalgae have been reported to grow and accumulate significant amounts of lipids. More specifically, the characteristics of rapid growth and the ability to utilize a range of carbon sources (e.g., glucose, glycerol) makes Rhodotorula glutinis an attractive candidate for microbial oil production (9e12). Further, an oil content as high as 72% obtained in R. glutinis has been reported (9). Pan and his colleagues obtained a cell density of 185 g/ l in an 84-h fed-batch culture of R. glutinis aerated with oxygenenriched air (13). In general, the components of fatty acids extracted from R. glutinis are mainly palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linolenic acid (C18:2), where palmitic acid and oleic acid often account for over 80% of total fatty acids (14). To further convert the microbial oils to biodiesel, a direct methanolysis process by the assisting of acids had been explored, which reached up to 97% of
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
Please cite this article in press as: Yen, H.-W., and Chang, J.-T., Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.10.001
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yield under several specific conditions (15). Besides the accumulation of high lipids, R. glutinis is also well-known in the industry as a b-carotene producer (16), which b-carotene is regarded as a valuable compound in the health industry. In this study, the hydrolysate of LCB (rice straw) was explored for the cultivation of oleaginous yeast- R. glutinis to produce SCO and b-carotene. Due to the potential inhibitors existing in the hydrolysate solution, the suitability of hydrolysate as a carbon source for the growth of R. glutinis is firstly examined. The C/N ratio effects by varying the adding amount of yeast extract on total lipid production and b-carotene accumulation are also evaluated. To achieve the scaled-up production, the performance of R. glutinis in the internal-loop airlift bioreactor and in the conventional agitation tank using LCB hydrolysate is also compared in this study. MATERIALS AND METHODS Microorganism and medium Freeze-dried R. glutinis BCRC 22360 was obtained from the Bioresource Collection and Research Center, Taiwan (BCRC). The seed medium composition and the cultivation methods followed suggestions provided by the BCRC. The fermentation medium (per liter) comprised defined amounts of different carbon sources (including glucose, glycerol, crude glycerol and LCB hydrolysate), 2 g of yeast extract, 2 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4$7H2O, 0.1 g of CaCl2 and 0.1 g of NaCl (17). Sodium hydroxide at 1.0 N or hydrogen chloride at 1.0 N was used to adjust the pH. LCB hydrolysate was kindly provided by the Institute of Energy Research, Taiwan, which was prepared through 2 stages of LCB hydrolysis of agriculture wastes and vegetable residues (pretreatment by dilute acid to remove hemicellulose and using cellulase for the following cellulose hydrolysis). The detail hydrolysis procedure was still the confidential process to the Institute of Energy Research, Taiwan, which could not be revealed here. The components of LCB hydrolysate is shown in Table 1. Before the use of LCB hydrolysate, it would be diluted properly to the designed reducing sugar concentration. The crude glycerol was purchased from a local biodiesel manufacture company (Yu-Hwa biodiesel company, Taiwan), with crude glycerol being the by-product of the conventional base catalyst transesterification process. No any extra purification process was performed before crude glycerol was used as one of the medium ingredients. Fermentation in 5-L conventional agitation fermentor The batch fermentation was operated in a conventional 5-L stirred desk-top fermentor (model BTF-A, Biotop Ltd., Taiwan) with a 2-L working volume, the fermentation medium of which was described in the above section. The pH level was maintained at 5.5 by automatically feeding NaOH solution (1.0 N). The fermentor was operated at 24 C with dissolved oxygen controlled at over 30% by adjusting the agitation rate in the range of 200e500 rpm with a 1 vvm aeration rate. Fermentation in 5-L internal-loop airlift bioreactor Batch fermentation was respectively carried out in a in a 5-L internal-loop glass airlift bioreactor (30 cm in height, with a 10 cm outer diameter and 7.7 cm inner tube diameter) with a working volume of 3 L. All experiments were controlled at 24 C and the pH was controlled at 5.5 by using 1 N NaOH solution. The aeration rate was performed at 1.5 vvm to explore the effects of using LCB hydrolysate on cell growth and on the accumulation of total lipids. Analytical methods Infrared balance was adopted to rapidly measure the cell mass concentration. Five ml of broth was centrifuged at 7000 rpm for 10 min. After removing the supernatant, about an equal volume of distilled water was added to eliminate the impurities. The washing procedure was performed several times, and the final liquor was dried by using infrared balance at 150 C to evaporate the water content. The total lipid analysis was based on a modification of the procedure used by Bligh and Dyer (18). The dry cell mass was ground into a fine powder; then, 0.05 g of the powder was blended with 5 ml chloroform/methanol (2:1), and the mixture was agitated for 20 min at room temperature in an orbital shaker. The solvent phase was recovered by centrifugation. The same process was repeated twice, and the whole solvent was evaporated and dried under vacuum conditions. The dinitrosalicylic acid (DNS) analysis was adopted for the measurement of reducing sugars of LCB hydrolysate. The glycerol concentration was measured by HPLC (Agilent series 1100, Agilent Technologies, Santa Clara, CA, USA) with a refractive index detector, while the analysis was performed in a C-18 column (Vercopak N5 ODS, 250 mm 4.6 mm, Taiwan). The mobile phase was composed of 0.01 N H2SO4 with a flow rate of 0.4 ml/
min (19). The measurement of b-carotene was performed as following: 50 mg of cells, after freeze-drying, were mixed with a 2 ml mixture consisting of acetonitrile, isopropyl alcohol and ethyl acetate (40:40:20 v/v), followed by ultrasonication (power 5, reaction time 2 min) to crush the cells for pigment extraction. The extract was centrifuged and the supernatant filtered through a 0.45-mm membrane filter and subjected to HPLC analyses, which were performed on a reversed-phase C18 analytical column [N5 ODS (C-18) 4.6 mm i.d. 250 mm]. The mobile phase was composed of acetonitrile, isopropanol and ethyl acetate (40:40:20 v/v) and had a flow rate of 0.7 ml/min. The column thermostat was set at 25 C, while the detector was operated at a wavelength of 457 nm. Analysis of lipid composition 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 4 ml of a 0.5 N KOH/ methanol solution at 100 C for 15 min, followed by the addition of 5 ml BF3 diethyl etherate and 5 ml methanol. The mixture was refluxed for 15 min, cooled, diluted with distilled water and then extracted with n-Hexane. The organic layer was washed with distilled water and subjected to fatty acid compositional analysis. Fatty acid methyl esters were analyzed using a gas chromatography instrument (Focus GC, Thermo, USA) equipped with a cross-linked capillary Column SEG BP20 (25 m 0.22 mm 0.25 mm) and flame ionization detector. Operating conditions were as follows: N2 carrier gas at 40 ml/min; injection port temperature of 230 C; oven temperature of 200 C; and, a detector temperature of 230 C. Fatty acids were identified by a comparison of their retention times with those of standard ones, quantified based on their respective peak areas, and then normalized.
RESULTS AND DISCUSSION Using hydrolysate from LCB as the carbon source Several studies have revealed results of conventional carbon sources being used as the carbon source for the growth of R. glutinis in the literature. In this study, glucose, glycerol, crude glycerol and LCB hydrolysate each at 30 g/L (hydrolysate solution was prepared at the equivalent of 30 g/L of reducing sugars) were compared for the growth of R. glutinis. As shown in Fig. 1, LCB hydrolysate solution can be a good carbon source for the growth of R. glutinis, which can produce a similar cell mass as compared to the batch using glucose. As seen in Table 1 of LCB hydrolysate components analysis, the glucose concentration was about 50% of reducing sugars (measured by the DNS method), which implied that there was still some other reducing sugars unclassified in the LCB hydrolysate. The concentration of glucose in the hydrolysate was even higher than that of xylose, which were 71.5 and 18.1 g/L, respectively. The high cell mass obtained in the batch using LCB hydrolysate suggests that the potential inhibitors (e.g., HMF, furfural) derived from the hydrolysis process would not inhibit the growth of R. glutinis under these cultivation conditions. Due to the recalcitrant nature of LCB products, different pretreatment process was often adopted for the destruction of complex cells was and exposed the cellulose for the further degradation of cellulolytic enzymes. On the other hand, lignocellulose pretreatment has the detrimental effect of also releasing a wide range of hydrolysis compounds, which are inhibitory to fermenting microorganisms and cellulolytic enzymes (20,21). Nevertheless, such kind of potential inhibitors seems not affecting the growth of R. glutinis in this study under the investigated conditions. It had been also reported that lipid production of Rhodosporidium toruloides afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile also indicated that those inhibitors had little effects on lipid biosynthesis (22). The lipid composition analyses for each batch with different carbon source are shown in Table 2, which indicates that oleic acid (C18:1) dominated the fatty acid components among all batches.
TABLE 1. Components of LCB hydrolysate solution (g/L). Reducing sugar
Glucose
Xylose
Arabinose
Formic acid
Acetic acid
HMF
Furfural
148.5 4.6
71 3.2
18.1 1.5
0.5 0.1
2.3 0.2
4.9 0.1
0.11 0.1
0.2 0
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FIG. 1. Effects of hydrolysate on the growth of R. glutinis as compared to other carbon sources.
Besides oleic acid, linoleic acid (C18:2) comprised a relative high ratio in the batch using LCB, of which the percentage of linoleic acid was far higher than that of the other batches. The ratios of saturated to unsaturated fatty acids among all batches are similar. More unsaturated fatty acid would lead to a lower cold filter plugging point (CFPP) and a higher instability of oil products due to the easier oxidation of unsaturated fatty acid (23). A similar percentage of saturated and unsaturated fatty acids indicates that the microbial lipids derived from the batches with different carbon sources might have a similar quality. Although, the growth of R. glutinis was not severely affected by using LCB hydrolysate, it was noted that the batch using hydrolysate had the lowest lipid content among all batches, which was as low as only w10%. It was reported that using undetoxified cornstover hydrolysate as substrate, the yeast of Rhodotorula graminis achieved a lipid content of 34% w/w (24). Even in the detoxified liquid hydrolysate from the pretreatment of wheat straw, about 20%
of lipid content was obtained in the cultivation of R. glutinis (5). In general, R. glutinis is well-known as an oleaginous microorganism for its ability of being the producer for total lipid production. A remarkably high lipid content of 72% for R. glutinis has been reported in the literature. Therefore, such a low lipid content indicated that the batch using LCB hydrolysate had a low C/N ratio, which led to the low total lipid content observed. The idea of increasing the C/N ratio to enhance total lipids production has been validated by many researchers. Therefore, the effects of nitrogen source-yeast extract (YE) on lipid production are investigated in next section. Effects of YE addition amount with hydrolysate as the carbon source To raise the lipid content obtained in the batch with LCB hydrolysate, the adjustment of YE addition in the medium containing hydrolysate at 30 g/L of reducing sugars was performed. Results are shown in Fig. 2, which reveals no significant difference in
TABLE 2. Components of fatty acid distribution with different carbon sources. Relative percentage (%)
Glucose Glycerol LCB hydrolysate
C 14:0
C 16:0
C 16:1
C 18:0
C 18:1
C 18:2
C 18:3
0.8 0.8 1.04
15 13.2 13.3
0.4 1.7 0.95
6.1 4.6 5.1
72.2 68.2 55.5
3.5 8.8 20.2
1.9 2.7 6.3
Saturated
Unsaturated
21.9 18.6 19.4
78.1 81.4 80.6
FIG. 2. Effects of nitrogen source concentration on the growth of R. glutinis while using hydrolysate as the carbon source.
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J. BIOSCI. BIOENG., TABLE 3. The comparison of b-carotene and lipid content among all batches with LCB.
b-Carotene (mg/g) Lipid content (%)
Stirred tank
Airlift 1 vvm
Airlift 2 vvm
1.243 0.168 27.4 3.9
1.016 0.081 26.0 1.6
0.486 0.198 34.3 0.6
with no YE adding can yield the highest b-carotene content of 1.3 0.01 mg/g among all batches. The overall implications derived from Figs. 1 and 2 are that the LCB hydrolysate might not be a suitable carbon source for total lipid production in the cultivation of oleaginous R. glutinis, but it might be a good substrate for b-carotene production.
the lipid content measured among all batches with varied YE addition, ranging from 0 to 2 g/L. Increased amounts of YE in the medium produced a higher cell mass, the highest of which attained 9.2 g/L in the shaker trial with 2 g/L of YE addition. Nevertheless, the lipid content remained at the approximate level of 10% in all batches, regardless of the amount of added YE. This implies that a simple C/N ratio adjustment with LCB hydrolysate as the carbon source could not raise the lipid content in the cultivation of R. glutinis. However, in the cultivation of oleaginous yeast-Trichosporon cutaneum using corncob residues hydrolysate, the lipid content was verified to be enhanced by increasing the C/ N ratio, of which the highest lipid content was 32.1% (25). Even the value of 32.1% obtained in the batch with corncob residues hydrolysate was still lower than the regular lipid content obtained in other studies using conventional carbon sources. Conclusively, LCB hydrolysate seems not to be a good carbon source for the growth of R. glutinis as regarding to the accumulation of high lipid content for biodiesel production. Nevertheless, even LCB hydrolysate cannot have the high lipid content obtained, the b-carotene production is another attractive product of R. glutinis cultivation. Fig. 2 also reveals that the batch
Comparison of R. glutinis cultivation in the airlift and agitation tank by using cellulose hydrolysate Despite the accumulation of total lipids using LCB hydrolysate as the carbon source seeming unsuccessful in the shaker trials, the growth of R. glutinis using LCB hydrolysate at 60 g/L of reducing sugars in the airlift bioreactor (at the aeration rates of 1 and 2 vvm, respectively) and in a conventional agitation tank were examined. Results are shown in Figs. 3 and 4. As seen in Fig. 3 featuring the cells growth curve, a higher cell mass was obtained in the airlift tank with 2 vvm than that of the other two operations, for which the airlift batch at 2 vvm had the highest 20 g/L of cell mass after 72 h cultivation. The reducing sugars measurement also revealed the fastest consumption curve in the airlift (2 vvm) batch, which suggests that the 2 vvm airlift batch can achieve the highest cell mass (as shown in Fig. 4). In addition to the highest cell mass observed in airlift batch with 2 vvm, it also had the highest cell growth rate of 0.283 g/L h as compared to the 0.111 and 0.139 g/L h for the airlift batch at 1 vvm and of the agitation tank, respectively. For comparison, the lipid content and b-carotene content among all batches are shown in Table 3, which indicates that the highest lipid content of 34.3 0.6% was obtained in the airlift batch with 2 vvm; far higher than that of the shaker trials. In contrast to the highest lipid content, the airlift batch with 2 vvm had the lowest b-carotene content at 0.486 0.198 mg/g. Among all batches, the highest b-carotene content achieved, as observed in the agitation tank, was 1.243 0.168 mg/g, which is quite competitive compared to other studies (26,27). To further confirm that the airlift batch with 2 vvm could have the highest lipid production, the consumption profile of major carbon sources in the airlift batch with 2 vvm was examined. Results are shown in Fig. 5, and
FIG. 4. Consumption of reducing sugars for the growth of R. glutinis in the stirred tank and in the airlift bioreactor by using hydrolysate as the carbon source.
FIG. 5. Components profile of airlift bioreactor at 2 vvm with 60 g/L LCB reducing sugars.
FIG. 3. Growth of R. glutinis in the stirred tank and in the airlift bioreactor by using hydrolysate as the carbon source.
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indicate that the major carbon sources of glucose and xylose would be simultaneously utilized for the growth of R. glutinis at almost the same rate. It was estimated that the lipids yield in the airlift tank with 2 vvm was about 12% (gram of total lipid produced per gram of reducing sugar consumed), which was similar to the value of 15% obtained in the batch with corn-stover hydrolysate by using T. cutaneum (22). The operation of the airlift bioreactor at 2 vvm achieved the highest cell mass growth as compared to the agitation tank. In conclusion, the LCB hydrolysate has the potential of being adopted for the production of b-carotene instead of lipid accumulation in the airlift bioreactor for the cultivation of R. glutinis. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from Taiwan’s MOST under grant number 103-2623-E-029-001-ET and 103-3113-E-006-006. References 1. Miao, X. and Wu, Q.: Biodiesel production from heterotrophic microalgal oil, Bioresour. Technol., 97, 841e846 (2006). 2. Yousuf, A.: Biodiesel from lignocellulosic biomasseprospects and challenges, Waste Manag., 32, 2061e2067 (2012). 3. Sun, X. F., Sun, R. C., and Tomkinson, J.: Degradation of wheat straw lignin and hemicellulosic polymers by a totally chlorine-free method, J. Polym. Degrad. Stab., 83, 47e57 (2004). 4. Sun, Y. and Cheng, J.: Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol., 83, 1e11 (2002). 5. Yu, X., Zheng, Y., Dorgan, K. M., and Chen, S.: Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid, Bioresour. Technol., 102, 6134e6140 (2011). 6. Xie, H., Shen, H., Gong, Z., Wang, Q., Zhao, Z. K., and Bai, F.: Enzymatic hydrolysates of corn stover pretreated by a N-methylpyrrolidoneeionic liquid solution for microbial lipid production, Green Chem., 14, 1202e1210 (2012). 7. Gong, Z., Shen, H., Wang, Q., Yang, X., Xie, H., and Zhao, Z. K.: Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process, Biotechnol. Biofuels, 6, 36e46 (2013). 8. Ratledge, C. and Cohen, Z.: Microbial and algal oils: do they have a future for biodiesel or as commodity oils? Lipid Technol., 20, 155e160 (2008). 9. Meng, X., Yang, J., Xu, X., Zhang, L., Nie, Q., and Xian, M.: Biodiesel production from oleaginous microorganisms, Renew. Energy, 34, 1e5 (2009).
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