PRODUCTION OF CANDIDA BIOMASS FROM HYDROLYSED AGRICULTURAL BIOWASTE

Article DOI: 1.2478/v1133-1-8-4 A&EB PRODUCTION OF CANDIDA BIOMASS FROM HYDROLYSED AGRICULTURAL BIOWASTE N.D. Dimova, Z.S. Iovkova, M. Brinkova, Ts.I. Godjevargova University Prof. As. Zlatarov, Department of Biotechnology, Burgas, Bulgaria Corespondence to: Nedyalka D. Dimova E-mail: nelidbg@yahoo.com ABSTRACT Agricultural residues (wheat bran, oats bran and rice husk) rich in carbohydrates was utilized in fermentation processes to produce microbial protein which in turn can be used to upgrade animal feeds and agricultural composts. Two kinds of yeast, identified as Candida tropicalis and Candida utilis were studied for their possible utilization in biomass production. The cells of C. tropicalis produced biomass with higher content of protein (.55 g.g -1 ) than cells of C. utilis (.48 g.g -1 ), cultivated on hydrolyzed wheat bran. The total content of the essential amino acids was highest in the biomass of C. tropicalis, cultivated on hydrolyzed wheat bran (43.5 mg.g -1 DCW). One of the salient features of the amino acid contents of cells of C. tropicalis was the high content of lysine: 82.5, 73.5 and 65.2 mg.g -1 DCW for hydrolyzed wheat bran, oats bran and rice husk, respectively. The results reveal that the C. tropicalis and C. utilis used in this study, grown on agricultural residues (wheat bran, oats bran and rice husk) as substrate, are promising yeast strains for the production of single cell protein. Keywords: agricultural biowaste transfomation, biomass production, Candida sp. Biotechnol. & Biotechnol. Eq. 21, 24(1), 1577-1581 Introduction The study of single cell protein (SCP) production from industrial and agricultural wastes has been studied widely in recent years. Agricultural residues rich in carbohydrates can be utilized in fermentation processes to produce microbial protein which can be used to upgrade both human and animal feeds. As the agricultural residues (rice straw, husk, bran) comprise mainly of lignin, cellulose, and hemicellulose, their direct utilization ratio as animal feedstock is very low. Through appropriate hydrolysis this lignocellulosic biomass could be transferred into fermentable sugar as cultural substrate for growth of microorganisms. Yeasts are common microorganism which can grow on agricultural wastes. Candida sp. is one of the most widely used organisms in the SCP production (1, 2, 15, 16, 2, 21). Over the years efforts have been made to use agricultural residues for production of microbial biomass. There have been several studies about production of microbial biomass from industrial and agricultural wastes such as yeast biomass from rice straw, rice bran (3, 17, 19) hydrolysate salad oil manufacturing wastewater (2), pineapple cannery effluent (15), cabbage (5), ram horn hydrolysate (12). In this study two Candida species (C. tropicalis and C. utilis) were used for SCP production. The main aim of this study was to investigate the suitability of oat bran, wheat bran and rice husk hydrolysates as a substrate in SCP production. Biotechnol. & Biotechnol. Eq. 24/21/1 Materials and Methods Microorganisms, growth medium and cultivation conditions C. tropicalis and C. utilis were obtained from the collection of the Institute of Microbiology, Bulgarian Academy of Sciences. The microorganisms were maintained at 29º C for 24 h on beer agar medium. The cultivation was carried out in 5 ml Erlenmeyer flasks containing 1 ml of sterile glucose solution (2%), on a shaker (2 rpm) for 24 h at 3ºC. The fermentation process was performed as follows: 2 ml of inoculum was transfereed into 5 ml Erlenmeyer flasks containing 5 ml of sterile hydrolysate (at 121ºC for 2 min) as carbon source. This medium was supplemented with.2 % (NH 4 ) 2 SO 4 and.2 % KH 2 PO 4. Solutions of (NH 4 ) 2 SO 4 and KH 2 PO 4 were sterilized separately in a hot air oven at 2ºC for 2 h and added into the medium before start of cultivation. ph was adjusted to 4.5-5. with NaOH solution. Reducing sugar concentration of fermentation medium was adjusted to 2%. Growth was carried out at 3ºC and ph 4.5-5. for 72 h in a shaker (2 rpm). The ph of the medium during cultivation was maintained at 4.5-5. by the addition of 2% NaOH at intervals of an hour. Samples of the yeast culture were withdrawn periodically from the flasks for various analyses. Analytical methods The dry weight of the obtained biomass was determined by the following way: yeast cells were harvested by centrifugation, washed twice with distilled water and after that dryed overnight at 15ºC until constant weight was reached. 1577

The reducing sugar content of the medium was determined using Nelson-Somogyi assay (1). The protein content of the cells was determined by the modified method of Lowry (13). The cell growth was estimated by measuring the optical density spectrophotometrically at 65 nm at different intervals of time. The hydrolysis of oat bran, wheat bran and rice husk was carried out into a tube containing 6N HCl and a small amount of phenol under vacuum, for 22-24 hours at 11ºC. Amino acid content was determined using phenylthiocarbamyl (PTC) derivative of samples by HPLC. Derivatization was performed by reacting the free amino acids, under basic conditions, at room temperature with derivative reagent (phenylisothiocyanate - PITC, water, ethanol, triethylamine, 1:1:7:1) freshly prepared before use. The HPLC analyses were carried out using a Series 4 liquid chromatograph (Perkin Elmer Corp., Norwalk, USA), equipped with an UV detector. Separation was carried out using a LiChroCART -4 Nucleosil 5 C18 column (x4.5 mm i.d., 5μm particle size). The detection wavelength was 4 nm. The chromatographic conditions were: mobile phase: A.5M NH 4 -acetate buffer ph 5. adjusted with o-phosphoric acid; B 65%.1M NH 4 -acetate buffer ph 6.5 adjusted with o-phosphoric acid + 35% CH 3 CN; gradient: 4 min 3% B, from 3% to 47% B for 14 min, from 47% to 1% B for 3 min and 5 min 1% B; flow rate 1 ml.min -1, temperature 4 o C. Results and Discussion Biomass cultivation C. tropicalis and C. utilis were studied for their abilities to grow and utilize some agricultural residues (wheat bran, oats bran and rice husk) as a sole carbon and energy source. It was found that the Candida sp. was capable of growth over a wide ph range of 3. to 6.2 (2). In this study the ph of the medium during cultivation was maintained at 4.5-5. by the addition of 2% NaOH at one-hour intervals. The growth curves of C. tropicalis cells cultivated in the hydrolyzed wheat bran, oats bran and rice husk medium are shown in Fig. 1. A lag phase of 3 hours appears in all growth curves as the microbial cells are adapting during this period. After that, the cells cultivated in the three media grow with high speed and analogically until the 24 th hour. However they reach different cell density, the highest being in hydrolyzed wheat bran (2.54), and the lowest, in hydrolyzed rice husk (1.94). After that the growth slows down and reaches a stationary phase, where the optical density is 2.71, 2.59 and 2.34, for the hydrolyzed wheat bran, hydrolyzed oats bran and hydrolyzed rice husk, respectively. The documented optical densities are higher compared to those from our previous studies, where the optical density for C. lypolytica is 2.1 (7). 1 2 3 4 5 6 7 1578 Biotechnol. & Biotechnol. Eq. 24/21/1 OD 65 nm 3 2,5 2 1,5 1,5 Timе, h Fig. 1. Growth curves of C. tropicalis cells in hydrolyzed: - wheat bran, - oats bran, - rice husk Rather different is the behavior of the C. utilis cells during their cultivation in the same hydrolysates (Fig. 2). In the first twenty hours, long adapting phases are observed in the three of the mediums. During this time the cell density reaches.74,.4 and.21, for the hydrolyzed wheat bran, oats bran and rice husk, respectively. After the adaptation phase, a fast accumulation of biomass is observed in the medium of hydrolyzed wheat and oats bran, by which the optical density of cell suspension reaches 2.3 and 1. for four hours. However, the dynamics of the growth curves of C. utilis cells in the rice husk hydrolysate is different. In this case the growth continues slowly and is almost equal to the growth of the wheat bran hydrolysate by the 63 rd hour. Cell density of 2.73 and 2.76 for wheat bran and rice husk, respectively is reached at the end of the cultivation, while for the oats hydrolysate it is 2.48. Rosma et al. investigated similar processes of continuous adaptation of the cells and slow accumulation of biomass in the initial period of the cultivation of C. utilis in citrus waste (15). OD 65 nm 3 2,5 2 1,5 1,5 1 2 3 4 5 6 7 Fig 2. Growth curves of C. utilis cells in hydrolyzed: - wheat bran, - oats bran, - rice husk It is seen that with the two kinds of yeasts at the end of the process, a cultural liquid with high cell density is obtained. Wijeyaratne and Jayathilake (18) investigated the possibility of receiving a cell protein and reached a cell density at 3ºC, between.8 and 1.. The experiments lead to the conclusion

that highest growth is observed of C. tropicalis in hydrolyzed wheat bran, and the slowest growth - of C. utilis in hydrolyzed rice husk. This may be due to both - the different kinds of sugar, which are present in the substrates, and the presence of different enzyme systems in the cells of the two kinds of yeast. also established that the amount of the accumulated biomass corresponds to the growth. The values of biomass, received by cultivating C. utilis cells, are higher. This responds to the higher cell density of the cultural liquid of this strain. The amount of the absolutely dry biomass at substrate hydrolyzed wheat bran and oats bran is almost equal for both C. tropicalis and C. utilis. Reducing sugar, mg.ml -1 2 15 1 5 1 2 3 4 5 6 7 8 Reducing sugar, mg.ml -1 2 15 1 5 Fig. 3. Content of reducing sugar in C. tropicalis cultural medium using hydrolyzed: - wheat bran, - oats bran, - rice husk Parallel to the growth of the yeast cultures, the content of the reducing sugar in the cultural liquid is also determined at separate stages in the cultivation process. The curves, representing the consumption of the reducing sugar in the cultivation of C. tropicalis, are shown on Fig. 3. The utilization of the three substrates is carried out analogically. The content of the reducing sugar in the cultural media at the beginning and at the end of the cultivation is shown in Table 1. The carbon source is utilized most fully by using hydrolyzed rice husk as a substrate (95.7%). TABLE 1 Content of the reducing sugar in the cultural media at the beginning and end of cultivation (mg.ml -1 ) Cultural media Start of cultivation End of cultivation C. tropicalis C. utisis Hydrolyzed wheat bran 2 2.1 1.7 Hydrolyzed oats bran 2 1.4 1.8 Hydrolyzed rice husk 2.85.6 The curves from Fig. 4, which represent the consumption of the reducing sugar during the cultivation of C. utilis show a good correlation with the growth curves. Obviously, this species needs more time for adaptation to the cells, but with time very fast utilization of the carbon source in hydrolyzed wheat and oats bran is observed. A fluent decrease of the reducing sugar in the cultural liquid occurs at the hydrolyzed rice husk. But in return to this the most fully degrading of reducing sugar is observed in the final stage, compared to all other examined objects (97%). The amount of absolutely dried biomass of the examined cultures is determined at the end of the fermentation. The amount of dry biomass, calculated for 1 g from the hydrolyzed wheat bran, oats bran and rice husk is shown on Table 2. It is 1 2 3 4 5 6 7 8 Fig. 4. Content of reducing sugar in C. utilis cultural medium using hydrolyzed: - wheat bran, - oats bran, - rice husk Amount of dry biomass, g.g -1 TABLE 2 Cultural media C. tropicalis C. utilis Hydrolyzed wheat bran.79.86 Hydrolyzed oats bran.72.79 Hydrolyzed rice husk.47.58 At the same time the content of the protein in the biomass is determined (Table 3). Although the amount of the biomass, received from C. utilis is higher, it contains smaller amounts of protein. The amount of protein in the biomass of this kind of cells according to Kurbanoglu et al. (12) is 49.8% from each gram of dry biomass when C. utilis is cultivated in hydrolyzed ram horn. Chareonsak (4) announce about 46% yield of protein from pineapple waste. According to some other authors the cells of C. tropicalis produce biomass with higher content of protein. Wijeyaratne et al. (18) document yield of 56.9% and 57.7% from biomass, in an investigation on two strains of C. tropicalis. TABLE 3 Content of the protein in the cell biomass (g.g -1 ) Cultural media C. tropicalis C. utilis Hydrolyzed wheat bran.55.48 Hydrolyzed oats bran.48.45 Hydrolyzed rice husk.38.37 Amino acid content The detailed amino acid composition of C. tropicalis and C. utilis in this study, calculated from chromatograms, are given in Table 4. The results presented in Table 4, show that there Biotechnol. & Biotechnol. Eq. 24/21/1 1579

Aminoacid content of biomass from Candida sp., mg.g -1 DCW TABLE 4 Amino acids C. tropicalis C. utilis Wheat bran Oats bran Rice husk Wheat bran Oats bran Rice husk FAO (19) Essential amino acids Lysine 82.5 73.5 65.2 72.5 64.2 57. 58 Leucine 71.2 65. 51.7 63.8 56.5 46.4 65 Isoleucine 78.2 8.4 89. 36.8 32.3 38.3 28 Phenylalanine 35.9 29.3 21.9 36.2 26. 16.8 - Tyrosine 31.7 33.3 27.9 31.8 34. 24. 63 Methionine Cysteine 16.1 5.1 14.3 2.3 15.2 5.2 21.2 4.5 16. 9.2 14.1 4.7 Threonine 34. 43.1 31. 24.8 33.3 21.8 34 Valine 48.8 44. 47.7 4.6 5.7 56.8 35 Non-essential amino acids* Alanine 24.4 23.8 22.2 68.9 1.4 62.3 Aspartic acid 8.7.2 56.4 4.1 78.2 4. Glutamine acid 141.5 11.7 94.9 132.4 114.8 65.2 Serine 52. 43.3 67.3 9.3 84.4 34.6 Glycine 55.8 51.6 82.2 12.9 92.7 36.1 Histidine 75.6 76.9 67.6 33.7 47.8 39.7 Proline 58.2 52.6 2.6 68.8 5.5 59.7 Arginine 54.6 5.6 94.5 3.7 35.4 14.7 *Tryptophan could not be determined in the acid hydrolysates are differences in the amino acid composition of the biomass from different Candida species and when different substrates are used. The biomass that is obtained from the yeast contains all essential amino acids. The biomass is quite rich in lysine. Biomass from C. utilis differs from that of the C. tropicalis. The content of the lysine is higher in the process of cultivation of C. tropicalis upon all cultural media: 82.5, 73.5 and 65.2 mg.g -1 DCW, for hydrolyzed wheat bran, oats bran and rice husk, respectively, compared to that in the biomass of C. utilis: 62.5, 64.2 and 57. mg.g -1 DCW, for hydrolyzed wheat bran, oats bran and rice husk, respectively. Biomass from C. tropicalis had an approximately two times greater concentration of the essential amino acid isoleucine, as compared with the concentration of the same amino acid in biomass from C. utilis. Among the amino acids, glutamic acid was the most abundant: from 11. to 132.4 mg.g -1 of biomass obtained from the two strains cultivated on wheat bran and oats bran. Aspartic acid concentration was quite high in biomass from C. tropicalis on wheat bran and from C. utilis on oats bran. High levels of aspartic acid have also been reported in fungal mycelia (11) and in SCP from some lactobacilli (8). In Table 4, the essential amino acid composition of the biomass from Candida sp. is compared with that of the Food and Agriculture Organization (FAO) (9). The results show that the obtained concentration of essential amino acids from biomass in most experiments is equal to or greater than those in the FAO reference protein, suggesting that the received biomasses are a good protein source. The total content of the essential amino acids is highest in the biomass of C. tropicalis, cultivated on hydrolyzed wheat bran: 43.5 mg.g -1 DCW. Most single cell proteins that are produced to date are deficient in methionine (6). Biomasses from Candida sp. examined in this study are also low in methionine and cysteine, compared to the amount in FAO reference protein. However, yeast biomass is relatively superior to soybean protein and wheat flour concerning their methionine content. Methionine is somewhat liable to acid hydrolysis (14). Therefore, the values presented in Table 4 for methionine, underestimate the actual methionine concentration in the biomass of the samples. Conclusions In conclusion, these results reveal that C. tropicalis and C. utilis used in this study, grown on agricultural residues (wheat bran, oats bran and rice husk) as the substrate, are promising yeast strains for the production of single cell protein. Biomasses from C. tropicalis and C. utilis may be used as a source of protein after sulfur amino acid enrichment. REFERENCES 1. Adoki A. (22) Journal of Applied Sciences & Environmental Management, 6(2), 49-58. 158 Biotechnol. & Biotechnol. Eq. 24/21/1

2. Adoki A. (28) African Journal of Biotechnology, 7(3), 29-295. 3. Anupama P.R. (21) Brazilian Archives of Biology and Technology, 44(1), 79-88. 4. Chareonsak C. (198) J. Natl. Res. Council Thailand, 12(1), 1-24. 5. Choi M.H. and Park Y.H. (23) Biomass Bioenergy,, 221-116. 6. Dalev P. (1992) Peptides, Sofia. 7. Dimova N., Godjevargova Ts., Ivanova D., Georgieva Sv., Brinkova M. (29) Production of microbial proteins hydrolysates. Scientific works, vol. LVI, 1, Plovdiv, 35-311. 8. Erdman M.D., Bergen W.G., Reddy C.A. (1977) Applied and Environmental Microbiology, 33(4), 91-95. 9. FAO/WHO/UNU (1985) Energy and protein requirements. Report of a joint FAO/WHO/UNU expert consultation. Technical report series no. 724. Geneva: World Health Organization. 1. Green F., Clausen C.A., Highley T.L. (1989) Analytical biochemistry, 182, 197-199. 11. Ivarson K.C. and Morita H. (1982) Applied and Environmental Microbiology, 3, 643-647. 12. Kurbanoglu E.B. and Algur O.F. (22) Bioresour. Technol., 85, 1-129. 13. Lowry O., Rosebrough N., Farr A., Randal F.J. (1951) Biol. Chem., 193, 265-275. 14. Menden E. and Cremer H.D. (197) In: Laboratory methods for the evaluation of changes in protein quality (A.A. Albanese, Ed.), Academic Press Inc., New York, 123-161. 15. Rosma A. and Cheong M.W. (27) Malaysian Journal of Microbiology, 3(1), 19-26. 16. Rosma A. and Ooi K.I. (26) Malaysian Journal of Microbiology, 2(2), 15-18. 17. Summers M.D., Jenkins B.M., Hyde P.R., Williams J.F., Mutters R.G., Scardacci S.C., Hair M.W. (23) Biomass Bioenergy, 24, 163-173. 18. Wijeyaratne S.C. and Jayathilake A.N. (2) J. Natn. Sci. Foundatzon Sri Lanka, 28(1), 79-86. 19. Zheng Y.G., Chen X.L., Wang Z. () Journal of Biotechnology, 118, 413-42. 2. Zheng S.K., Yang M., Yang Z.F. () Bioresource Technology, 96, 1183-1187. 21. Zheng S.K., Yang M., Yang, Z.F., Yang Q.X. () Bioresource Technology, 196, 1522-1524. Biotechnol. & Biotechnol. Eq. 24/21/1 1581