Bioresource Technology 97 (2006) 19741978

Short Communication

Xylitol production from corn ber and sugarcane bagasse hydrolysates by Candida tropicalis
R. Sreenivas Rao a, Ch. Pavana Jyothi a, R.S. Prakasham b, P.N. Sarma b, L. Venkateswar Rao a,*
b a Department of Microbiology, Osmania University, Hyderabad 500 007, India Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India

Received 3 June 2004; received in revised form 11 August 2005; accepted 17 August 2005 Available online 18 October 2005

Abstract A natural isolate, Candida tropicalis was tested for xylitol production from corn ber and sugarcane bagasse hydrolysates. Fermentation of corn ber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production, though these were very low, even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was found to be 0.43 g/g and 0.45 g/g of xylose utilised with corn ber and sugarcane bagasse hydrolysate respectively. One of the critical factors for low xylitol production was the presence of inhibitors in these hydrolysates. To simulate inuence of hemicellulosic sugar composition on xylitol yield, three dierent combinations of mixed sugar control experiments, without the presence of any inhibitors, have been performed and the strain produced 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. To improve yeast growth and xylitol production with these hydrolysates, which contain inhibitors, the cells were adapted by sub culturing in the hydrolysate containing medium for 25 cycles. After adaptation the organism produced more xylitol 0.58 g/g and 0.65 g/g of xylose with corn ber hydrolysate and sugarcane bagasse hydrolysate respectively. 2005 Elsevier Ltd. All rights reserved.
Keywords: Corn ber; Sugarcane bagasse; Xylitol; Candida tropicalis; Hemicellulose; Adaptation

1. Introduction Xylitol is one of the most expensive polyol sweeteners and has specic health claims in the world market. It is suitable for diabetes, recommended for oral health and paranteral nutrition (Makinen, 2000; Sreenivas Rao et al., 2004). On an industrial scale, xylitol is currently produced through chemical reduction of xylose derived from birchwood chips and sugarcane baggase hemicellulose hydrolysate. It is relatively expensive by about $7 kg1 (Leathers, 2003) comparatively with other natural sweeteners. The chemical process is expensive because of the high

Corresponding author. Tel.: +91 40 27682246; fax: +91 40 27019020. E-mail address: vrlinga@yahoo.com (L.V. Rao).

temperature and pressure required for hydrogenation of xylose. Further more, extensive steps for separation and purication add to the cost. Availability and cost of production are the obstacles impending the increased use of xylitol. Biotechnological production of xylitol could be of economic interest and attractive, as crude hemicellulosic hydrolysates can be used as potential substrates, instead of pure xylose, to reduce the cost of production. Hemicellulose is a plant cell wall polysaccharide and third most abundant polymer in nature. In some plants, it comprises upto 40% of the total dry material. Hemicelluloses are short branched chain heteropolysacharides of mixed hexosans and pentosans that are easily hydrolyzed. The most common form of hemicellulose is xylose polymer (xylan). As hemicellulose is abundant in nature and renewable, extensive research has been undertaken to convert hemicellulose derived carbohydrates, particularly xylose,

0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.08.015

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into useful products. Hydrolysis of hemicellulose, yields glucose, D-xylose, L-arabinose and other minor sugars. During acid hydrolysis, xylose is degraded rapidly to furfural and other condensation byproducts. These degradation products are inhibitory to microorganisms. The inhibitory eect of dierent compounds like furfural, 5hydroxymethyl furfural (HMF), acetate, hydroxybenzaldehyde (HBA), siringaldedyde (SGA) and vanillin on yeast growth is well documented- (Tsao et al., 1999). Various approaches are being considered to remove fermentation inhibitors or minimize their formation, CaO treatment (Silva et al., 1998), use of activated charcoal (Pandey et al., 2000), overliming (Dien et al., 2000), ion exchange resins (Mancilha and Karim, 2003), Ca(OH)2 (Dien et al., 2000), solvent extraction (Cruz et al., 1999), intracellular acidication (Lohmeier-vogel et al., 1998), yeast strain variation (Martin and Jonsson, 2003), laccase enzyme (Cassland and Jonsson, 1999), recombinant strains (Dien et al., 1997) and adaptation of the microbial strains (Rivas et al., 2003) have been reported. Many studies have been conducted utilizing the hemicellulose portion of agricultural residues like Eucalyptus grandis (Silva et al., 1998), rice straw (Roberto et al., 1994), aspen wood hemicellulosic hydrolysate (Preziosi-Belloy et al., 2000), barley bran (Cruz et al., 2000), hybrid polar wood chips (Domnguez et al., 1997) and corn cobs (Rivas et al., 2003) for xylitol production. In the present investigation two substrates, corn ber and sugarcane bagasse have been selected. Corn ber represents a renewable resource that is available in signicant quantities from the corn wet milling industries (Doner et al., 2001). Corn ber contains about 70% fermentable sugars (Dien et al., 1999). Corn ber is rich in pentose sugars (Saha, 2003) in the form of arabinoxylan (hemicellulose). Because of its relatively low commercial value and increasing supply, considerable interest exists in nding new, value-added uses for this material (Dien et al., 1999). The low cost high carbohydrate content of corn ber makes it an attractive feed stock for conversion to xylitol. About 3.4 106 dry tons of corn ber are produced each year in the United States alone (Dien et al., 1999), and about 203.6 Tg of dry corn stover are globally available (Tsao et al., 1999) for D-xylose and xylitol production. Nearly half of all fuel ethanol production in the USA employs a wet-milling process utilizing more than 300 million bushels of corn. Every bushel of maize processed for sweetener, oil or ethanol generate nearly 7 kg of protein and ber rich residues. Many research eorts were made to achieve the production of value added products from sugarcane bagasse (Pandey et al., 2000). The annual global production of dry cut sugarcane is about 328 Tg and Asia (44%) is the primary production region (Kim and Dale, 2004). Each ton of milled sugarcane gives 180280 kg of bagasse residues (Pessoa et al., 1997). The acid hydrolysate from bagasse contains xylose as the main component (Toit et al., 1984). When sugarcane bagasse hemicellulose hydrolysate was

evaluated for cultivation of 18 species of dierent yeasts, Candida sps. emerged as the best performer (Pessoa et al., 1997). Though these two substrates have been studied for the production of dierent products using microorganisms, there are very few reports available with these substrates for xylitol production. Therefore in the present study corn ber and sugarcane bagasse hydrolyastes have been tested for the economical production of xylitol using C. tropicalis. 2. Methods 2.1. Preparation of corn ber hydrolysate Wet corn ber was obtained from Gayatri Starchkem Limited. Corn ber was dried at 55 C for 24 h in a oven and ground in a mixer and passed through a 28 mesh screen. The ground corn ber was mixed with 1% (v/v) H2SO4 at a ratio of 1 g of biomass to 5.0 ml of acid solution and autoclaved for 1 h. After cooling, the liquid was separated from the solids using cheese cloth. Corn ber hydrolysate was prepared as previously described (Saha and Bothast, 1999). Hydrolysate was neutralized using Ca(OH)2, the resulting precipitates were removed by centrifugation at 20,000g. The recovered liquid was treated with activated charcoal. Acid hydrolysate samples (100 ml) were placed in 250 ml Erlenmeyer asks and activated carbon was added to each ask and heated at 80 C for 30 min. The mixture was cooled to room temperature and the activated carbon was vacuum ltered with Bakelite powder bed. Acid hydrolysate was further treated with ion exchange resins (INDION 225 H and INDION 850) to reduce the conductivity of the liquid. The recovered liquid was lter sterilized through a 0.22 lm membrane lter. Forty grams of corn ber mixed with 1% (v/v) H2SO4 (200 ml) solution will give around 110 ml of hydrolysate containing 16.0 dissolved solids (DS) with a conductivity of 8300 lX/cm. The conductivity of the hydrolysate was reduced up to 500 lX/ cm with ion exchange treatment. Monomeric sugar composition in corn ber hydrolysate: xylose 30%, glucose 38%, arabinose 22% and galactose 4%. 2.2. Preparation of sugarcane bagasse hydrolysate The sugarcane bagasse was mixed with 1% (v/v) H2SO4 solution at a ratio of 1 g of biomass to 10.0 ml of acid solution and autoclaved for 1 h. After cooling, the liquid was treated as described above. Twenty grams of sugarcane bagasse mixed with 1% (v/v) H2SO4 (200 ml) solution will give around 150 ml of hydrolyaste containing 3.5 dissolved solids (DS) with a conductivity of 7500 lX/cm. The conductivity of the hydrolysate was reduced up to 500 lX/cm with the ion exchange treatment. Monomeric sugar composition in sugarcane bagasse hydrolysate: xylose 56%, glucose 15% and arabinose 24%.

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2.3. Microorganism and culture conditions C. tropicalis was isolated from a soil sample and identied by using the CANDIFAST test kit (International Microbio, Stago Group, 83870 Signes, France). The identication was conrmed by IMTECH, Chandigarh, India. C. tropicalis was maintained on YM agar and subcultured every 4 weeks. 2.4. Inoculum preparation and fermentation Inoculum (25 ml) was prepared in 100 ml Erlenmeyer asks, with the following media components (g/l): Xylose 30; yeast extract 10; peptone 20; K2HPO4 0.5; KH2PO4 0.5; MgSO4 7H2O 0.5 and ammonium sulfate 2. pH 5.0 and incubated for 24 h on a rotary shaker (250 rpm) at 30 C. After 24 h the cells were recovered by centrifugation. 2.5. Preparation of adapted inoculum for xylitol production The inoculum was grown on corn ber hydrolysate and sugarcane bagasse hydrolysate with supplementation of nutrients (g/l): yeast extract 5; peptone 10; K2HPO4 0.5; KH2PO4 0.5; MgSO4 7H2O 0.5 and ammonium sulfate 2. The cells were recovered by centrifugation and resuspended in fresh hydrolysate. The adaptation was continued for 25 cycles (Fig. 1). 2.6. Fermentation medium Fermentation medium (100 ml) was prepared in 250 ml Erlenmeyer asks with diluted (until about 30 g/l xylose was obtained) corn ber hydrolysate or sugarcane bagasse hydrolysate with supplementation of nutrients (g/l): yeast extract 5; peptone 10; K2HPO4 0.5; KH2PO4 0.5; MgSO4 7H2O 0.5 and ammonium sulfate 2. Adapted cells (5%, 1.0 OD) were used for inoculation and incubated for 48 h at 30 C on a rotary shaker at 250 rpm.

2.7. Model sugar studies Model sugar studies medium (100 ml) was prepared in 250 ml Erlenmeyer asks to simulate inuence of hemicellulosic sugar composition on xylitol yield and to serve as control experiments, due to the absence of inhibitor substances using unadapted strain. The following three dierent combinations of mixed sugar were selected. Un adapted C. tropicalis cells 5% (v/v) were used for inoculation and incubated for 48 h at 30 C on a rotary shaker at 250 rpm. Combinations (g/l) (a) 40 g xylose; 17 g glucose; 1.5 g mannose; 3 g galactose; 20 g L-arabinose; 5 g yeast extract; 10 g peptone; salt solution (as per Section 2.6); (b) 50 g xylose; 30 g glucose; 2.5 g mannose; 5 g galactose; 40 g L-arabinose; 5 g yeast extract; 10 g peptone; salt solution (as per Section 2.6); (c) 60 g xylose; 40 g glucose; 3.5 g mannose; 7 g galactose; 40 g L-arabinose; 5 g yeast extract; 10 g peptone; salt solution (as per Section 2.6). 2.8. Analytical procedures Sugar and sugar alcohols in the culture broth were measured by high performance liquid chromatography (HPLC) using an ion moderated partition chromatography sugar column SHODEX SC 1011 (8 mm 300 mm). The samples were eluted with HPLC water at a ow rate of 0.5 ml/min at 80 C and detected with a dierential refractometer (WATERS 410). The dissolved solids (DS) of the hydrolysate solutions were measured by RFM-332 refractometer (Denver). 2.9. Statistical analysis Minitab 14 package was used for statistical analysis. 3. Results and discussion Corn ber hydrolysate and sugarcane bagasse hydrolysate were neutralized with Ca(OH)2 and treated with activated charcoal and ion exchange resins to reduce inhibitors and conductivity of the hydrolysate. Corn ber has four-fold dissolved solids and more conductivity than sugarcane bagasse hydrolysate. In both cases conductivity was reduced up to 500 lX/cm by ion exchange resins. Sugars derived from corn ber and sugarcane bagasse and other hemicellulose material can be converted to xylitol using a variety of naturally occurring or recombinant yeasts (Jeries and Jinn, 2004). Numerous yeasts convert xylose to xylitol, particularly including sp. of Pichia and Candida. Naturally occurring strains have been reported to produce xylitol under aerobic and semi aerobic conditions (Saha, 2003). Acid hydrolysis produces various toxic substances or inhibitors that interfere with microbial metabolism. Martin and Jonsson (2003) tested 11 industrial and lab strains of Saccharomyces cerevisiae and two Zygosaccharomyces to an inhibitor cocktail. The two most resistant strains were

0.7

Xylitol yield (g/g xylose)

0.65 0.6 0.55 0.5 0.45 0.4 1

Corn fiber Series1 Sugarcane Series2 bagasse

10

15

20

Adaptation cycles

Fig. 1. Xylitol production with sugarcane bagasse and corn ber hydrolysates.

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0.9

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0.65
CF

SB

Xylitol yield (g/g xylose)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Unadapted Adapted SB Adapted CF Xylose

y = yield

0.55

0.45 0 5 10 15 20

Fig. 2. Xylitol yield by parent and adapted C. tropicalis using sugarcane bagasse, corn ber hydrolysates and standard xylose.

x = adaptation cycles
CF (Corn Fiber) y = 0.438095 + 0.0094856x S = 0.0155533, R.Sq. = 97.6% R.Sq(adj) = 97.0% SB (Sugarcane Bagasse) y = 0.424762 + 0.0070857x S = 0.0095867 R.Sq. = 98.4% R.Sq(adj) = 97.9%

S. cerevisiae isolated from spent sulphate liquor plant and one of the laboratory S. cerevisiae strain. Out of 13 strains, some strains were very sensitive, while others were relatively tolerant. When Pessoa et al. (1997) tested 18 strains of yeasts from dierent sp. for evaluation of sugarcane bagasse hydrolysate for cultivation of yeasts, Candida sp. emerged as the best performer. To overcome these problems of inhibitors dierent investigators tried to improve fermentation conditions. Solvent extraction of hemicellulose wood hydrolysate and agarose immobilization (Lohmeier-vogel et al., 1998) partially protected the yeasts from inhibitors. In the present investigation a natural isolate of C. tropicalis was tested for xylitol production from corn ber and suagarcane bagasse hydrolysate. Fermentation of corn ber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production (Fig. 3). These uptake and xylitol production were very low even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was 0.43 g/g and 0.45 g/g of xylose utilised with corn ber hydrolysate and sugarcane bagasse hydrolysate respectively. Three dierent combinations of mixed sugars were used, as shown in Section 2.7 (a, b, c combinations), as control experiments due to the absence of inhibitors. The

Fig. 4. Regression tted line plot for corn ber and sugarcane bagasse.

0.8 0.7

Xylitol yield (g/g)

0.6 0.5 0.4 0.3 0.2 0.1 0 8 16 24

xylitol yield with these three combinations of sugars was 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. However with corn ber and sugarcane bagasse hydrolysate, the xylitol yield was low when compared with mixed sugar control experiments. This is mostly due to the presence of inhibitors in hydrolysates. Xylitol production with pure xylose was reported earlier as 0.78 g/g (Sreenivas Rao et al., 2004) with this strain (Fig. 2). To improve yeast growth and xylitol production from these hydrolysates containing inhibitors, the cells were adapted in the hydrolysate medium by sub culturing for 25 cycles (Fig. 1). After every ve cycles, the organism was tested for xylitol production. With corn ber the xylitol production increased from initial 0.43 g/g to 0.46 g/g, 0.49 g/g, 0.52 g/g of xylose after 5, 10, 15th cycles respectively. After adaptation up to 20th cycle the xylitol production improved to 0.58 g/g of xylose. With sugarcane bagasse, the xylitol production increased from initial 0.45 g/g to 0.48 g/g, 0.52 g/g, 0.57 g/g of xylose after 5, 10, 15th cycles respectively. After adaptation up to 20th cycle the xylitol production was improved to 0.65 g/g of xylose. After 25th cycle, negligible increase in xylitol yield was observed. Xylitol accumulation was more in sugarcane bagasse hydrolysate than in corn ber hydrolysate. The statistical analysis has done by Minitab 14. It is very clear that from statistical analysis the correlation between Y (yield) and X (adaptation cycles) for corn ber and sugarcane bagasse are highly signicant by R2 (Fig. 4). The prediction from the regression equation is most reliable and reproducible with in the small error. 4. Conclusion

Corn Fiber Sugarcane bagasse

32

40

48

Fermentation Time (h)

Fig. 3. Xylitol yield with corn ber hydrolysate and sugarcane bagasse hydrolysate by adapted C. tropicalis.

To produce xylitol by yeasts, in a cost-eective manner the fermentation of xylose from hemicellulosic material is important. The potential of corn ber and sugarcane bagasse hemicellulosic hydrolyzates as substrates for xylitol production by natural isolate and adapted strains of C. tropicalis was demonstrated.

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R.S. Rao et al. / Bioresource Technology 97 (2006) 19741978 Lohmeier-vogel, E.M., Sopher, C.R., Lee, H., 1998. Intracellular acidication as a mechanism for the inhibition by acid hydrolysis-derived inhibitors of xylose fermentation by yeasts. J. Indus. Microbiol. Biotechnol. 20, 7581. Makinen, K.K., 2000. The rocky road of xylitol to its clinical application. J. Den. Res. 79, 13521355. Mancilha, I.M., Karim, M.N., 2003. Evaluation of ion exchange resins for removal of inhibitory compounds from corn stover hydrolysate for xylitol fermentation. Biotechnol. Prog. 19, 18371841. Martin, C., Jonsson, L.J., 2003. Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulosic-derived fermentation inhibitors. Enzyme Microbial Technol. 32, 386395. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., 2000. Biotechnological potential of agro-industrial residues. I. Sugarcane bagasse. Bioresour. Technol. 74, 6980. Pessoa, J.A., de Manchilha, I.M., Sato, S., 1997. Evaluation of sugar cane hemicellulose hydrolyzate for cultivation of yeasts and lamentous fungi. J. Ind. Microbiol. Biotechnol. 18, 360363. Preziosi-Belloy, L., Nolleau, V., Navarro, J.M., 2000. Xylitol production from aspen wood hemicellulose hydrolysate by Candida guilliermondii. Biotech. Lett. 22, 239243. Rivas, B., Torre, P., Domnguez, J.M., Perego, P., Converti, A., Parajo, J.C., 2003. Carbon material and bioenergestic balances of xylitol production from corncob by Debaryomyces hansenii. Biotechnol. Prog. 19, 706713. Roberto, I.C., Mancilha, I.M., de Souza, C.A., 1994. Evaluation of rice straw hemicellulose hydrolysate in the production of xylitol by Candida guilliermondii. Biotechnol. Lett. 16, 12111216. Saha, B.C., Bothast, R.J., 1999. Pretreatment and enzymatic saccharication of corn ber. Appl. Biochem. Biotechnol. 76, 6577. Saha, B.C., 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30, 279291. Silva, S.S., Maria, G.A., Silva, B.A., Arnaldo, M.R., 1998. Acid hydrolysis of Eucalyptus grandis chips for microbial production of xylitol. Process biochem. 33 (1), 6367. Sreenivas Rao, R., Prakasham, R.S., Krishna Prasad, K., Rajesham, S., Sharma, P.N., Venkateswar Rao, L., 2004. Xylitol production by Candida sp.: parameter optimization using Taguchi approach. Process Biochem. 39, 951956. Toit, P.J., Olivier, S.P., Van Biljon, P.L., 1984. Sugarcane bagasse as a possible source of fermentable carbohydrates. I. Characterization of bagasse with regard to monosaccharide, hemicellulose and amino acid composition. Biotechnol. Bioeng. 26, 10711078. Tsao, G.T., Cao, N., Gong, C.S., 1999. Hemicellulose conversion. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseperation, vol. 3. John Wiley & Sons. Inc., ISBN 0-471-138223, pp. 13911400.

Acknowledgements We are grateful to Department of Biotechnology, Government of India and Gayatri Starchkem Ltd., (Corn WetMilling and Sweetener Industry) Hyderabad, AP, India for providing partial nancial assistance and to Y. Bhaskar Reddy, GSL and K. Badhraiah, Swaroop Technologies, Secunderabad for technical support to complete this work. References
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