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B I O T E C H N O L O G Y LETTERS Volume 17 No.ll (Nov.1995) p.1223-1228 receoved 20th September.

INFLUENCE OF MEDIA COMPOSITION ON XYLITOL FERMENTATION BY Candida guiliiermondii USING RESPONSE SURFACE METHODOLOGY

*I.C. ROBERTO 1, S. SATO 2 I.M. de MANCILHA TM,M.E.S. TAQUEDA3

1Dept of Biotechnology-FAENQUIL, 12600-000, Lorena, S.P., 2Faculty of Pharmaceutical Sciences and 3Polytechnic School, Dept of Chemical Engineering, USP-S~o Paulo, S.P. 4 DeptOof Food Engineering, UFV, Vi~osa, M.G., Brazil

SUMMARY

The bioconversion of xylose to xylitol by the yeast Candida guilliermondii FTI 20037 was evaluated under different nutritional conditions using rice straw hemicellulose hydrolysate. Statistical designs were used to determine the fermentation medium composition. Ammonium sulfate and rice bran have been identified as required nutrients in the hydrolysate since there was a significant interaction between them. In the presence of both nutrients, the xylitol yield factor (Yp/s) and volumetric productivities (Qp) were 0.68 g/g and 0.54 g/L.h, respectively.

INTRODUCTION

Rice straw contains a significant portion of hemicellulose fraction that can be easily hydrolyzed to its constituent carbohydrates. The hydrolysis product contains a mixture of sugars with xylose as the major component (Roberto et al., 1994). In recent years, several studies have been performed aimed at the utilization of the xylose-rich hemicellulosic fraction as a substrate for the production of ethanol (Roberto et al., 1991, Lawford and Rousseau, 1993), microbial protein (Meyer et al., 1992), acetic acid (Brownell and N akas, 1991) and xylitol (Strehaino and Dupuy, 1990, Felipe et al., 1993, Roberto et al., 1994, Roberto et al., 1995). The latter represents a product of wide application in the food, pharmaceutical and dental industry owing to its equivalent sweetining power to sucrose and its anticariogenic properties (Bar, 1986, Pepper and Olinger, 1988). Many yeast species are able to convert xylose to xylitol with good yield (Gong eta/., 1983, Barbosa et al., ] 988). The yeast Candida guilliermondii has been frequently used to study this bioconversion in

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synthetic medium due to its high performance (Meyrial et al., 1991, Nolleau and PreziosiBelloy, 1993). We have recently reported the feasibility of converting xylose to xylitol by C.

guilliermondii FTI 20037 using rice straw hydrolysate (Roberto et al. 1994). However, no studies of the effect of nutritional conditions have been investigated yet. Normally, hemicellulosic hydrolysate.are supplemented with the same nutrients employed for synthetic media. The present report aims at defining the medium components for the production of xylitol using the rice straw hemicellulose hydrolysate. Factorial designs have been largely employed for the optimization of the fermentative process due to the possibility of studing several variables with a reduced number of experiments. This methodology also allows one to verify the individual factor effects and their interactions for the response. In this paper, we have studied the effects of nutrient additions on rice straw hydrolysate for xylitol production by the yeast C. guilliermondii using response surface methodology.

MATERIAL AND METHODS Microorganism and Inoculum Preparation

Candida guilliermondii FTI 20037, selected by Barbosa et al., 1988 was used for this study. Cultures were maintained at 4C on malt extract agar slants. The inoculum was prepared by growing cells in 125-mL Erlenmeyer flasks containing 50 mL of medium with the following composition in g/L: xylose, 20; 0qI-I4)2SO4, 3; CaCI2 x 2 H 2 0 , 0.1 and rice bran, 20. Incubation was at 30C on a rotary shaker at 200 rpm for 24 hours. The cultures were inoculated to obtain an initial cell concentration of 0.5 g/L.

HemiceHulose Hydrolysate

Rice straw hemicellulose hydrolysate was obtained by acid hydrolysis according to the method described by Roberto et al., 1995. The concentrated hydrolysate was overtitrated with NaOH (pellets) to a pH 10 and adjusted to 5.0 by sulfuric acid. (72%w/w). After each change in pH, the precipitate was removed by centrifugation at 1000 g for 15 min. The chemical composition of rice straw hydrolysate was in g/L: xylose, 79.3; glucose, o.,_,_,nose,13.4; furfural, 0.13; hydroxymethylfurfural, 0.51 and acetic acid, 1.82. 22.6; v._, t~: ....

Fermentation Conditions

Fermentation was carried out under semiaerobic conditions using 125-mL Erlenmeyer flasks closed with cotton-wool plugs, containing 50 mL of media prepared with hydrolysate supplemented by different nutritional combinations. The fermentations were performed at 30C on a rotary shaker at 200 rpm for 72 hours.

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Experimental Designs and Statistical Analysis


A 23 factorial design (Box et al., 1978, Barros et al., 1995)) was used to study the effect of the combination of medium components on xylitol production. Rice bran, ammonium sulfate and calcium chloride were selected as experimental factors. The levels of these factors were 0 and 20 g/L; 0 and 3 g/L and 0 and 0.1 g/L, respectively. The experimental data were analysed by the response surface regression procedure (Statistica software). This method is based upon the use of the polynomial model: Y = bo + X biXi + X bijXiXj + ei where Y is the response variable (dependent); b, the regression coefficients, X, the independent variables or experimental factor levels and e, experimental error.

Analytical Methods
Xylose and xylitol concentrations were determined by HPLC as described previously (Roberto et al. 1995).

RESULTS AND DISCUSSION


Table 1 shows the distribuition of the factor levels in the experiments according to a 23 design and the data for the response under study. The results indicate that Candida

guilliermondii was able to accumulate xylitol in all the media studied. However, the amount of xylitol formed and xylose consumption were strongly dependent on the medium
composition. When the hydrolysate was supplemented with ammonium sulfate and rice bran (medium 6) the values of xylitol concentration and xylose consumption were 39.3 g/L and 90 %, respectively. These results decreased to 30 g/L and 74 % (medium 3), when these nutrients were omitted from hydrolysate. Such results demonstrate the importance of the presence of both nutrients in the medium for the bioconversion of xylose to xylitol by 6".

guilliermondii. The effect of nitrogen sources on xylitol production by this yeast have been
well documented (Barbosa et al., 1988; Silva et al., 1994). According to Barbosa et al. (1988) small increases in xylitol yields were observed by the addition of casamino acids or yeast extract in the fermentation medium. This observation agrees with our results since rice bran and yeast extract have similar compositions (Miller and Churchill 1986). The addition of the calcium chloride to the rice straw hydrolysate did not promote any positive effect on the parameters evaluated. Since in synthetic medium the presence of this compound is essential for the cell fisiological functions these results suggest that calcium chloride is already present in this hydrolysate.

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Table 1 - Experimental Factorial Design and Results. XYl~lb Xylos~ Produced (g/L) Consumption (%) t 30.21 73 2 + 35.35 80 3 + 30.94 74 4 + + 37.77 84 5 + 36.72 88 6 + + 39.34 90 7 + + 35.91 86 8 + + + 40.05 86 9 0 0 0 37.70 78 aX1 = Rice bran; X2 = Calciumchloride;X3 = Ammoniumsulfate bAveragevalues of triplicates
Xl a X2 X3

Media

The experimental results presented in Table 1 were used to estimate the main effects of variables and their interaction effects. The regression coefficients for a polynomial model in order to predict and quantify the level of the factors on the xylitol production and xylose consumption was also estimated. The effects for ammonium sulfate and rice bran supplementation were positive and significant (Table 2). The model also indicates the existence of a negative interaction between these factors. The antagonistic effect of the interaction between ammonium sulfate and rice bran indicate that simultaneous increases in the levels of these factors do not increase xylitol production and xylose consumption by amounts as large as those expected based on separate increases in these factors. The analysis of variance presented in Table 3 demonstrates that the polynomial model is adequate for describing the relationships between the response under study and the experimental factors. The regression model was significant (p<0.05) with a satisfactory value o f R 2 (0.88 and 0.86) for xylitol production and xylose consumption, respectively.

Table 2 - Estimated Effects and F-values for Xylitol Production and Xylose Consumption. Variables independent Xylose Consumption F-Ratio 17.10" 0.05 67.21" 0.05 10.66" 4.43 1.18 Xylitot Production Effects 35.78 4.69 0.77 4.45 0.79 -1.31 -0.83 -0.03 F-Ratio 57.62* 1.54 51.91" 1.67 4,53* 1.81 0.00

Effects Constant 82.63 X1 4.75 X2 -0.25 X3 9.42 X~X2 0.25 X1X3 -3.75 X2X3 -2.42 X1X2X3 -1.25 *Significantat 5% level, F(1.16~= 4.49.

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Table 3 - Analysis of Variance for the Regression Models of Xylitol Production and Xylose Consumption. Source Model (regression) Residual (error) Total Degree of Freedon 7 16 23 Xylitol 272.56 36.62 309.18 Sum of Squares Xylose 796.96 126.67 923.62

F-ratio 17.01 14.38 p-value 0.0000 0.0000 R2 0.88 0.86 Tests for curvature using center points showed that squared terms are not important for these models.

Since the calcium chloride concentration is not involved in any significant terms of the models, response surfaces can be expressed using only the rice bran and ammonium sulfate variables. Figure 1 shows the response surface of xylitol production and xylose consumption attained with the regression equation: y~ = 82.62 + 2.38X~ + 4.71X3 1.88X~X3 and y2 = 35.78 + 2.34X~ + 2.23X3 - 0.66X~X~ were y~ and y2 are predicted xylose consumption and xylitol production, respectively, and XI and X3 are coded levels for rice bran and ammonium sulfate, respectively. The contour plots show the behaviour of the xylitol concentration and xylose consumption by and rice bran are added in the hydrolysate.

C.guilliermondii when ammonium sulfate

81
::, O

56

f "~.

f,,~

Figure 1 - Influence of ammonium sulfate and rice bran concentration on the xylitol production (A) and xylose consumption (B) by C. guilliermondii.

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CONCLUSION The composition of fermentation medium using rice straw hydrolysate as a substrate requires rice bran and ammonium sulfate supplementation. However, the optimum levels of these nutrients can be determined through additional experiments. Finally, the response surface methodology could be applied to the optimization of other factors, such as pH, aeration level and inoculum concentration which may also play important roles in this bioconversion. ACKNOWLEDGMENT The authors acknowledge financial assistance from CNPq/Brazil and Dr Roy E. Bruns for the helpful revision of this paper.
REFERENCES

1. Bar, A. (1986). Alternative Sweeteners. In: Xylitol, O'Brein Nabors, L.;Gelardi, R., eds, Marcel Dekker Inc, New York, Chapter 10, pp. 185-216. 2. Barbosa, M.F.S., Medeiros, M.B., Mancilha, I.M., Schneider, H. and Lee, H. (1988), d.Ind. Microbiol., 3, 241-251. 3. Barros Nero, B., Scarminio, I.S., Bruns, R,E. (1995). Planejamento e Otimizaqao de Experimentos. Campinas: editora da Unicamp. 4. Box, G.E.P., Hunter W.G. and Hunter, J.S.(1978). Statistics for Experimenters, Wiley and Sons, New York. 5. Brownell, J.E. and Nakas, J.P. (1991), lnd. Microbiol., 7, 1-6. 6. Fdipe, M.G.A., Mancilha, I.M., Vitolo, M., Roberto, I.C. and Silva, S.S. (1993), Arquivos de Biologia e Tecnologia, 36(1 ), 103-114. 7. Gong, C.S., ClaypooI, T.A. Mc Cracken, L.D., Maun, C.M., Ueng, P.P. and Tsao, G.T. (1983), Biotechnol Bioeng., 25, 85-102. 8. Lawford, H.G. and Rousseau, J.D. (1993), Biotechnol. Lett., 15(5), 505-510. 9. Meyer, P.S., du Preez, J.C. and Kilian, S.G. (1992), Syst. AppZ MicrobioZ, 15(1), 161-165. 10. Meyrial, V., Delgenes, J.P., Moletta, R. and Navarro, J.M. (1991), Biotechnot. Lett. 13(4), 281-286. 11. Miller, T.L., Churchill, B.W (1986). Substrates for large-scale fermentation. In: Manual of Industrial Microbiology and Biotechnology, A.L. Demain and N.A. Solomon, eds., Washington: American Society of Microbiology, pp. 130-131. 12. Nolleau, V., Preziosi-BeUoy, L., Delgenes, J.P. and Navarro, J.M. (1993), Curr. MicrobioL, 27, 191-197. 13. Pepper, T. and Olinger, P.M. (1988), Food TechnoL, 42(10), 98-106. 14. Roberto, I.C., Lacis, L., Barbosa, M.F.S. and Mancilha, I.M. (1991), Process Biochem., 26, 15-21. 15. Roberto, I.C., Felipe, M.G.A., Mancilha, I.M., Vitolo, M., Sato, S. and Silva, S.S. (1995), Biores Technol., 51, 255-257. 16. Roberto, I.C., Mancilha, I.M., Souza, C.A., Felipe, M.G.A., Sato, S. and Castro, H.F. (1994), Biotechnol. Lett., 16(11), 1211-1216. 17. Silva, S.S., Mancilha, I.M., Queiroz, M.A., Felipe, M.G.A., Roberto, I.C and Vitolo, M. (1994), d. Basic Microbiol., 34(3), 205-208. 18. Strehaino, P. and Dupuy, M.L. (1990), French Patent 2,641,545.

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