POTENTIAL LIPID PRODUCTION OF OLEAGINOUS YEAST LIPOMYCES STARKEYI FROM GLUCOSE AND XYLOSE

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1 POTENTIAL LIPID PRODUCTION OF OLEAGINOUS YEAST LIPOMYCES STARKEYI FROM GLUCOSE AND XYLOSE Noppan Peawsuphon, Anusith Thanapimmetha, Maythee Saisriyoot, Penjit Srinophakun * *Department of chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok 19, Thailand ABSTRACT This study evaluates the capability of oleaginous yeast Lipomyces starkeyi to synthesize the microbial lipids using glucose and xylose as its carbon source individually. Firstly, the effect of inoculum concentration (%, 6%, % and 1% v/v) was determined by using glucose as carbon source at 3 g/l. Secondly, the glucose concentration (, 6, and 1 g/l) was varied to get the highest cell dry weight. Finally, the cultivation of L. starkeyi was performed to enhance the growth at higher glucose concentration of 6 g/l. After that, the cultivation was carried out at 6 g/l of xylose and the cell dry weight was compared between glucose and xylose media. From the result, 1% of inoculum concentration was an optimum content for effectiveness of yeast. g/l of glucose was the best concentration for cultivation and gave 1.1 g/l biomass concentration at hrs. The initial glucose and xylose of 6 g/l gave the highest cell dry weight at 1.3 g/l and 15.2, respectively and the highest lipid contents were 17.7% in glucose and 1.3 g/l in xylose. From the results, xylose was found to be suitable carbon sources for lipid accumulation while glucose was proper for the high cell growth. Keywords: Lipomyces starkeyi, Microbial lipids, Oleaginous yeast *fengpjs@ku.ac.th INTRODUCTION Nowadays, the abundant use of fossil fuels such as petroleum, coal and natural gas, which is caused from the high energy demand in industrialized world, raises the pollution problems [1]. Therefore, it is continuously necessary to develop renewable energy sources for example biodiesel and ethanol. As biodiesel can be an alternative fuel in the transport sector practically. In this context, alternative fuel must be economically competitive, environmental acceptable and available readily [2]. Biodiesel fatty acid methyl esters (FAME) is the product from transesterification of triacylglycerol (TAGs) with an alcohol at the presence of some alkaline catalysts [3]. In comparison with fossil fuels, biodiesel caused the low toxicity, the low formation of pollutants during combustion as well as its renewable characteristics. Consequently, biodiesel is an excellent substitute to fossil fuels []. However, the cost of biodiesel production is somewhat expensive (mainly virgin vegetable oil) and requirement of the land for cultivation plants because of TAGs came from oil crops. Although the waste oils are used to reduce the cost, the process requires the cheap raw materials [5]. Recently, many researchers are interested the microbial oils that derived from some Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 1

2 microorganisms, which have potential application in biodiesel production in terms of the microbial oils conversion to fatty acid methyl ester instead of using vegetable oils [6]. Some microorganisms that can accumulate lipids more than 2% in their dry cell mass are defined as oleaginous species [7]. Oleaginous yeasts are one type of all oleaginous microbes, which can produce the intracellular lipids as high as 7% of cell dry weight for example Lipomyces Starkeyi, Rhodosporidium toruloides and Cryptoccoccus albidus []. TAG is the major produced lipid of fatty acid composition in those yeast cells and contained long-chain fatty acid that are identical with vegetable oil for biodiesel production [9]. The aim of this study was to evaluate the ability of oleaginous yeast Lipomyces starkeyi to synthesize the microbial lipids using glucose and xylose as its sole carbon source. EXPERIMENTAL A. Microorganism and inoculum preparation The yeast strain utilized in this work was Lipomyces Starkeyi DSM 7295 and purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Colonies of L. starkeyi was grown on YM agar plate at 3 o C, stored in the refrigerator and subculture monthly. The inoculum preparation of each batch of all experiments was followed by two successive cell propagations in liquid media (3 g/l of yeast extract, 3 g/l of malt extract, 5 g/l of peptone from soybean and 1 g/l of glucose) at 3 o C, once at h and second time at 3 h. All other chemicals and reagent were analytical grade. B. Fermentation medium preparation Fermentation medium was prepared according to Wild et al., (21) [1]. The composition of fermentation medium was: 3 g/l of glucose (or xylose), 1 g/l of (NH) 2 SO, 1.5 g/l of yeast extract, 2.5 g/l of Na 2 HPO, 7 g/l of KH 2 PO,.15 g/l of MgSO 7H 2 O,.1 g/l of CaCl 2 2H 2 O,.2 mg/l of FeSO 7H 2 O, 1 mg/l of ZnSO 7H 2 O, 7 mg/l of MnSO H 2 O, 9.1 mg/l of CoCl 2 6H 2 O and 1 mg/l of CuSO 5H 2 O. Initial ph 5.5 was adjusted by adding 1 M NaOH in fermentation medium. C. Cultivation conditions Inoculum of yeast cells after two successive cell propagations were inoculated to 25 ml Erlenmeyer flasks containing 5 ml fermentation medium (1% v/v). Batch experiments were performed under aerobic conditions in fermentation medium using an orbital shaker at an agitation rate of 1 rpm and the incubated temperature was 3 o C. D. Analytical methods Wet yeast cells were collected by centrifugation at 5 rpm for 15 min and washed with the same volume of distilled water. Cell dry weight was obtained from wet cells Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 2

3 Cell dry weight (g/l) from a 2 ml culture broth after using the freeze-drying (Lyophilization). Extraction of lipids from cell dry weight was performed according to the procedure of Bligh and Dyer [11]. Lipid was extracted with chloroform-methanol solution. The total lipid was estimated by gravimetric method. The sugar concentration (Glucose and Xylose) was determined by using DNS method []. RESULTS AND DISCUSSION A. Effect of inoculum concentration on biomass Microbial oil production is profoundly related to the inoculum concentration, which is the main factor for lipid accumulation by L. starkeyi using glucose as a carbon source. These following conditions were used in this study: 2-1% of inoculum concentration, 3 g of initial glucose, 3 o C of temperature, 1 rpm of agitation, 5.5 of initial ph and h of cultivation. The effect of inoculum concentration on biomass was shown in Fig. 1. It could be seen that biomass or cell dry weight raised up with the increased inoculum concentration in the same way. At last h of cultivation, the maximum cell dry weight of 1., 1.5, 1.1 and 1. g/l were obtained at 2%, %, 6%, % and 1% of inoculum concentration respectively. However, among these inoculum concentrations, at 6 h of cultivation, 1% reached the stationary phase before the rest of inoculum and glucose concentration approached to zero as can been seen in Fig. 2. Hence, the optimal inoculum concentration of 1% was chosen in the next experiment Fig. 1 Effect of inoculum concentrations on biomass ( ) %; ( ) 6%; ( ) %; ( ) 1% of inoculum concentration Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 3

4 Cell dry weight (g/l) Glucose content (g/l) Fig. 2 Effect of inoculum concentrations on sugar concentration ( ) %; ( ) 6%; ( ) %; ( ) 1% of inoculum concentration B. Effect of initial glucose concentration on biomass Carbon source is a major nutrient for the growth of yeast in terms of reproduction and metabolism [13]. Moreover, the species and concentration of carbon source played an important role in cell growth and lipid accumulation. As previous studies, glucose was the most suitable carbon source for cell growth and lipid synthesis due to its structure [1]. Therefore, glucose was used as a carbon source and its concentration was varied from -1 g/l with the same cultivation conditions (1% inoculum, 3 o C, 1 rpm, initial ph 5.5 and h of cultivation). The effect of glucose concentration on biomass was shown in Fig. 3. It was shown that at last h of cultivation. The highest cell dry weight was obtained at, 6 and g/l glucose, which was equal to about 1 g/l. Biomass around 13 g/l was derived by 1 g/l glucose. As figure, 6 g/l glucose was still excess at hours of cultivation time. Consequently, the next part of this study was performed on more investigation of yeast growth Fig. 3 Effect of initial glucose concentrations on biomass ( ) g/l; ( ) 6 g/l; ( ) g/l; ( ) 1 g/l initial glucose Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page

5 Glucose content (g/l) Cell dry weight abd Lipid (g/l) Glucose content (g/l) Fig. The change of glucose concentration ( ) g/l; ( ) 6 g/l; ( ) g/l; ( ) 1 g/l initial glucose C. Effect of glucose and xylose concentration as the sole carbon source Both of glucose and xylose are the reduced sugar that can be easily used by microorganism. Glucose and xylose were used as the sole carbon sources in yeast cultivation and the results are shown in Fig. 5 and 6. The effect of different carbon sources to cell growths and the lipid accumulation processes. Comparing the cell growths and lipid accumulations of L. starykeyi cultured on medium with xylose (CDW, 15.2 g/l and lipid content, 25.7%) and glucose (CDW, 1.3 g/l and lipid content, 17.7%), the CDW using xylose as the carbon source was lower but lipid content was higher than glucose. Possibly, xylose was converted into xylulose through a two-step reduction and oxidation, and xylose was further converted into glucose through the pentose phosphate pathway in the catabolism of xylose in yeasts [15]. The cell growth on xylose resulting in lower CDW than that on glucose, suggesting that the metabolic utilization of xylose might probably passed the pentose-phosphate pathway and not through the phosphoketolase reaction in L. starkeyi [16]. However, the mechanism of xylose catabolism in L. starkeyi is not clear. As a result, xylose was able to be used as carbon sources for microbial oil production and glucose was suitable for the cell growth Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 5

6 Xylose concentration (g/l) Cell dry weight abd Lipid (g/l) Fig. 5 Effect of glucose concentration as the sole carbon source ( ) Glucose content; ( ) Cell dry weight; ( ) Lipid content Fig. 6 Effect of xylose concentration as the sole carbon source ( ) Xylose content; ( ) Cell dry weight; ( ) Lipid content CONCLUSIONS This work was studied the effectiveness of oleaginous yeast, Lipomyces starkeyi, to synthesize the microbial lipids using glucose and xylose as its sole carbon source individually. L. starkeyi can use both of glucose and xylose to produce microbial oils by fermentation. Xylose was found to be suitable carbon sources for lipid accumulation and in the same time, glucose was proper in the high cell growth. ACKNOWLEDGEMENTS The authors would like to acknowledge Department of Chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand. REFERENCES [1] Karatay, S. E. and Dӧnmez G. (21) J. Bioresour. Technol. 11, [2] Meher, L. C., Vidya Sagar, D., Naik, S.N. (26) J. Renew. Sust. Energy Rev. 1, [3] Economou, Ch.N., Makri, A., Aggelis, G., Pavlou, S., Vayenas, D.V. (21) J. Bioresour. Technol. 11, [] Zhu, L.Y., Zong, M.H., Wu, H. (2) J. Bioresour. Technol. 99, [5] Liu, B., Zhao, Z.K. (27) J. Chem. Technol. Biotechnol. 2, [6] Peng, X., Chen, Y. (2) J. Bioresour. Technol. 99, [7] Ratledge C, Wynn JP. (22) J. Adv Appl Microbiol. 51 (1) 51. [] Li, Y., Zhao Z. and Bai, F. (27) J. Enzy. and Micro. Technol. 1, [9] Rattray JBM. In: Ratledge C, Wilkinson SG, editors. Micro bial lipids, vol.1. London/New York: Academic press; 199. p Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 6

7 [1] Wild, R., Patil, S., Popoviv, M., Zappi, M. and Dufreche, S. (21) J. Food Technol. Biotechnol. (3), [11] Bligh, E.G. and Dyer, W.J. (1959) J. Biochem. Phys. 37, [] Miller, G.L. (1959) J. Anal. Chem. 31, [13] Liu, J., Yue, Q., Gao, B., Ma, Z. and Zhang P. (2) J. Bioresour. Technol. 16, [1] Li, Y.H., Liu, B., Sun, Y., et al. (25) J. China Biotechnol. 25 (), [15] Papanikolaou, S., and Aggelis, G. (211) J. Lipid Sci. Tech. 113 (), [16] Wang, R., Wang, J., Xu, R., Fang, Z. and Liu, A. (21) J. Bioresour. Technol. 9 (), Petrochemical and Materials Technology Tuesday May 23, 217, Pathumwan Princess Hotel, Bangkok, Thailand Page 7