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Advances in Environmental Biology, 5(11): 3613-3619, 2011 ISSN 1995-0756 3613 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Microbial Production of Xylitol from Corn Cob Hydrolysate Using Pichia sp 1 P. Jeevan, 2 R. Nelson and 3 A. Edith Rena 1 P.G. and Research Department of Microbiology, J.J. College of Arts and Science, Pudukottai 622 404, Tamil Nadu, India. 2 Department of Botany, Government Arts College, Ariyalur, Tamil Nadu, India. 3 PG and Research Department of Biotechnology, JJ College of Arts and Science, Pudukottai 622 404, Tamil Nadu, India. P. Jeevan, R. Nelson and A. Edith Rena: Microbial Production of Xylitol from Corn Cob Hydrolysate Using Pichia Sp. ABSTRACT Xylitol is being used as a highly valued ingredient with some interesting and useful properties in food and pharmaceutical products. It can be produced from xylose rich (hemicelluloses fraction of lignocelluloses) sources by chemical or microbial method. Microbial production of xylitol from corn cobs hemicellulosic hydrolysate using the yeast Pichia sp was experimentally investigated. Acid hydrolysis of hemicellulosic fraction of corn cobs using sulfuric acid was performed under different conditions of temperature, reaction time and acid concentration. 40gl -1 xylose was obtained from 100gl -1 corn cobs by acid hydrolysis using 2 % sulfuric acid at 121 C for 60 mins. Batch fermenation was carried out in shake flasks for xylitol production using Pichia sp in corn cobs hydrolysate medium and optimized under different cultivation conditions. The results showed that the xylose consumption by the yeast and xylitol production was maximum at the optimum ph 6, temperature 28 C and agitation 150 rpm. The bioconversion yield was 35 gl -1 xylitol from 40 gl -1 xylose within 72 h and the bioconversion efficiency was 90%. Key words: Xylitol, corn cob hydrolysate, fermentation, xylose, Pichia. Introduction Xylitol (C 5 H 12 O 5 ) a five carbon sugar polyalcohol, is found in various fruits and some vegetables [28]. Because of its high sweetening power and no insulin requirement for its digestion, xylitol has recently become an attractive as an alternative sweetener for the treatment of diabetics [11]. It is anticariogenic prevents the formation of acids that attack the tooth enamel [15.24] and can be used as a sugar substitute in dietetic food for diabetics [18]. Xylitol can also be used in the treatment of osteoporosis since it considerably improves the biomechanical properties of the bones and prevents reduction both in their density and in their contents of minerals, calcium and phosphorus [16]. Moreover xylitol inhibits the growth of the bacterial species like Streptococcus pneumoniae and Haemophilus influenzae, which cause acute medium otitis, so it could be employed, instead of antibiotics, to combat this disease [9,26]. Owing to all these characteristics, xylitol is a feedstock of particular interest to the food odontological and pharmaceutical industries. Xylitol is currently produced on an industrial scale by a catalytic reduction (hydrogenation) of xylose obtained from wood sources such as white birches. However, alternative processes have been extensively explored because of the high production cost and environmental impact associated with the excessive utilization of natural wood sources [13]. Lignocellulosic materials are mainly composed of cellulose, hemicellulose and lignin. Hemicellulose is composed of linear and branched heteropolymers of L-arabinose, D-galactose, D-glucose, D-mannose and D-xylose. The composition of hemicellulose varies according to the plant species, for instance in wheat straw (32%), barley straw (32%), rice straw (25%), corn cobs (37%), sugarcane (22%) and eucalyptus wood (15-22%) [25]. The hemicellulose fraction can be hydrolyzed and used for fermentation of xylose, with a view of producing xylitol [4]. Microbial xylitol production from agricultural wastes containing hemicelluloses could be a possible candidate because it has the potential to realize cheaper production of xylitol with low environmental impact by the effective utilization of renewable resources such as agricultural wastes [20,6]. Among agricultural resources (wastes) corn cobs are Corresponding Author P. Jeevan, P.G. and Research Department of Microbiology, J.J. College of Arts and Science, Pudukottai 622 404,TamilNadu, India. E-mail: jeevancftri@yahoo.com; Tel.: +91-4322-260 103; Mobile: +91-9791886609

regarded as a promising agricultural resource for microbial xylitol production because corn is widely cultivated and corn cobs are rich in hemicelluoses but are not effectively utilized. In microbial xylitol production from corncobs the cobs are first bydrolysed to produce xylose from hemicelluloses by acid hydrolysis and the corn cob hydrolysate is then used as the medium for xylitol production by xylose utilizing organisms [17]. Therefore, to evaluate the feasibility of microbial xylitol production form corn cobs it is essential to examine the hydrolysis conditions of corn cobs and the xylitol production yield using the corn cob hydrolysate. The objective of the present study was to evaluate the bioconversion of xylose to xylitol by Pichia sp., in the corn cob hydrolysate. Materials and Methods Raw material: Corn cobs were locally collected from pudukkottai, Tamil Nadu, India, dried in the sunlight, milled to give a particle size of approximately 1 mm thickness and used as a raw material in this study. Their average composition determined according to standard methodology [12] was 32% cellulose, 35% hemicelluloses, 20% lignin, 4% ash and 3.4 % of acetyl groups (oven-dry basis) Preparation of Corn Cob Hydrolysate: 100gl -1 corn cobs were hydrolyzed under various sulfuric acid concentrations (1%-4%) at different temperatures (121 C) and for different reaction times in an autoclave. After hydrolysis, the liquid fraction (corn cob hydrolysate) was filtered through Whatman No: 1 filter paper and the ph was raised to 9 with calcium oxide and decreased to ph 5.5 with sulfuric acid. The hydrolysate was concentrated under vacuum at 70 C to increase xylose concentration. After these treatments, the hydrolysate was mixed with 10% activated charcoal, agitated (200 rpm, 30 C, 1 h) and then filtered. The filtrate was autoclaved at 120 C for 15 min. The filterate (corn cob hydrolysate) was used as fermentation medium for xylitol production. Microorganism and Culture Conditions: The yeast strain used in this study was isolated from a soil sample of agricultural fields, Pudukkottai, Tamil Nadu, India. The selected strain was identified as Pichia sp according to taxonomic identification. Taxonomic identification was carried out by Department of Micrbiology, JJ college of Arts and Science, Pudukkottai, Tamil Nadu, India. The strain was maintained on YPD agar containing (10gl -1 yeast extract; 20gl -1 bacteriological peptone, 20gl -1 glucose 3614 and 15gl -1 agar) at 30 C for 24 h, maintained at 4 C and subcultured at regualr intervals. Inoculum Development: From the subculture, one loopful of yeast cells were inoculated into 30 ml test tubes containing 5 ml of preculture medium (30gl -1 xylose, 10gl -1 yeast extract, 20gl -1 peptone and ph 6.0) and cultivated at 30 C for 24 h on a rotary shaker (Orbitek, India) at 200 rpm. Inoculum (25 ml) was prepared in 100 ml Erlenmeyer flasks containing 30 g l -1 xylose, 10 g l -1 yeast extract, 20 g l -1 peptone, 0.5gl -1 K 2 HPO 4, 0.5gl -1 KH 2 PO 4, 2gl -1 (NH 4) 2 SO 4, 0.5gl -1 MgSO 4.7H 2 O and ph 6.0 and it was inoculated with 5 ml of preculture and incubated for 24 h on a rotary shaker (200 rpm) at 30 C. After 24 h, the flask culture was centrifuged and washed twice with distilled water and then used as inoculum for fermentation. Fermentation: In shaking flask batch fermentations were performed in 250ml Erlenmeyer flask containing 100ml of corn cob hydrolysate medium by adding 10 g l -1 yeast extract, 20 g l -1 peptone, 0.5 g l -1 K 2 HPO 4, 0.5 g l -1 KH 2 PO 4, 2 g l -1 (NH 4) 2 SO 4, 0.5 g l -1 MgSO 4.7H 2 O and ph 6.0 and cultivated under aerobic condition on rotary-shaker at 200 rpm, 30 C for 96 hours. It was inoculated to a final concentration of 10 7 cells/ml. The samples were withdrawn at regular intervals such as 24, 48, 72 and 96 h of incubation. The cell growth was determined spectrophotometrically (OD) at 600 nm. The periodically drawn samples were centrifuged at 12000rpm for 10 mins and the supernatant was used for HPLC analysis for determining the xylose consumption and xylitol concentration in fermented broth. Analytical Procedures: Xylose and Xylitol concentrations were determined using High Pressure Liquid Chromatography (HPLC) with an Aminex HPX- 87H, (Biorad, USA) carbohydrate column (300X7.8mm) at 45 C, using 5mM H 2 SO 4, as an eluent. A flow rate of 0.6 ml/min and a sample volume of 20µl were maintained. The eluate was monitored with Refractive Index (RI) detector. The peaks were identified and quantified by comparing with retention times of authentic standards (Xylose and Xylitol) [1]. The cell growth (Biomass) was determined by measuring optical density (OD) at 600 nm and correlated with dry weight (One OD unit = 1.55 g drycell/l).

Optimization of Fermentation conditions: Effect of Acid Concentration: Different concentrations of sulphuric acid such as 1%, 2%, 3% and 4% were used for corncob hydrolysate preparation to determine the highest yield of xylose during acid hydrolysis. After pretreatment powdered corn cobs were hydrolysed under different temperature based on the acid concentration such as 1% H 2 SO 4 concentration at 100 C for 30 mins, 2% at 110 C for 60 mins, 3% at 120 C for 90 mins, and 4% at 126 C for 120 mins in stainless steel pressure cooker. Effect of Temperature: The general method was repeated accordingly for the optimization of incubation temperature. The culture flasks were incubated at different temperature like 28 C, 33 C and 37 C, at 120rpm for 96 h. The culture supernatant was taken after 24, 48, 72 and 96 h of incubation and then biomass xylose consumption and xylitol concentrations were determined. Effect of ph: 3615 To determine the effect of ph, the fermentation media was prepared with various ph such as 5, 6, and 7. All flasks were inoculated and incubated at 30 C, 200 rpm for 96 h. The culture supernatant was taken after 24, 48, 72 and 96 h of incubation and and then biomass xylose consumption and xylitol concentrations were determined Effect of Agitation: To determine the effect of agitation on the growth of yeast and xylitol production, the fermentation media were incubated at various shaking speeds such as 100, 150 and 200 rpm at 28 C for 96 h. The culture supernatant was taken after 24, 48, 72 and 96 h of incubation and and then biomass xylose consumption and xylitol concentrations were determined. Results and Discussion Batch fermentation of corncob hemicellulose hydrolysate by the yeast Pichia sp for xylitol production was performed using shaking flask experiments. Cell growth (OD), sugar utilization (xylose consumption) and xylitol concentration were monitored during fermentation. Table 1: Bioconversion of Xylose into Xylitol by different Yeasts Using Different Raw Materials. Microorganism Raw Material Xylitol yield ( gl -1 ) References Pichia sp., Corn cobs 35 Our work Candida tropicalis Corn cobs 15.0 Farooq Latif & Mohamed Ibrahim, 2001 Candida sp Corn cobs 70 Dominguuez et al.,1997 C. guilliermondii Sugarcane bagasse 13.5 Alves et al., 1998 C. guilliermondii Eucalyptus wood 8.5 Canettieri et al., 2001 D. hansenii Corn cobs 64 Rivas et al., Effect of acid concentration on xylitol production: The xylose concentration in the corncob hydrolysates resulting from the hydrolysis of 100 gl -1 corncobs under various sulphuric acid concentrations is shown in Figure.1. In the ranges of 1.0-3.0% sulphuric acid, the xylose concentration were approximately 40gl -1 xylose and constant. The xylose yield was 0.25g xylose/g corncobs. Since, hemicellulose account for approximately 35% of the dry corncobs, the xylose yield for the hemicelluloses in the corncob becomes 0.71g xylose/g hemicelluloses (Kiyosi Tada et al., 2004). Naturally this is because the increase in sulphuric acid concentration enhanced the decomposition of lignin and the sugars released from the corncobs during acid hydrolysis. The maximum xylose concentration of 25gl -1 xylose was obtained at 2% sulphuric acid concentration. However the sugar concentration (xylose) dropped with a further increase in the acid concentration of 3-4%. Hence, microbial xylitol production was then performed using the hydrolysates obtained by 2% sulphuric acid treatment. Effect of temperature on xylitol production: Batch fermentation was performed at 4 different temperatures (28 C, 32 C, 36 C and 40 C) to determine the effect of temperature on xylitol production. The highest biomass 9.72g was obtained at the temperature 28 C followed by 8.3g at 32 C at 72 h. The growth was absent at 40 C. The maximum xylitol production (24.8 gl -1, 21.2 gl -1 and 18 gl -1 ) was obtained at 28 C, 32 C and 36 C respectively (Fig.2 A,B,C). According to our results 28 C was an optimal temperature for xylitol production by Pichia sp from corncob hydrolysates. Cao et al., found that xylitol production by Candida sp.b-22 kept relatively constant in the temperature range 35 40 C and appreciably decreased over 45 C. A similar trend was observed for D. hansenii NRRL Y-7426 by Dominguez, et al., in the range 28 37 C, where 100 gl -1 xylitol were produced from 130 gl -1 xylose, while at 44 C xylitol production decreased down to 41.9 gl - 1. Barbosa, et al., observed for C. guilliermondii maximum xylitol formation (23gl -1 ) and specific growth rate (0.78 h -1 ) within 30 35 C and that both parameters decreased over 40 C.

Effect of ph on xylitol Production: Batch fermentation was performed using 3 different ph values (4, 5 & 6 ) to determine the effect of ph on xylitol production (Fig.3A,B,C). The xylitol yield 30 gl -1 was obtained in ph 6 (Fig.3C). The highest biomass concentration 17.8 gl -1 was obtained after 72 hours in ph of 6. The xylose consumption rate was the same for all the ph value evaluated, it increased slightly when the ph was raised. The 3616 balance between xylitol production and cell growth indicates that the xylose is consumed at ph 5, 6 and 7. According to our results, 6 was an optimal ph value for xylitol production by Pichia sp from corncob hydrolysates. In previous reports [23] also attained very high xylitol concentration at a ph varying from 4 to 6. Vongsuvanlert and Tani (1989) reported that Candida boidinii grew best at initial ph of 6.5 but gave a maximum xylitol yield (0.13g/g) at ph 7. Fig. 1: Effect of Acid Hydrolysis on Corncob Hydrolysate. Fig. 2: Biomass, xylose consumption and xylitol production during batch fermenation of corncob hydrolysate using Pichia sp at different temperatures (A)28 C (B) 32 C (C) 36 C.

3617 Fig. 3: Biomass, xylose consumption and xylitol production during batch fermenation of corn cob hydrolysate using Pichia sp at different ph A)pH4 B) ph5 C) ph6. Effect of Agitation on xylitol Production: The effect of agitation on xylitol production at different shaking speed (100, 150 and 200rpm) was performed (Fig.4A,B,C). The maximum xylitol production (35.2 gl -1 ) was observed in flasks maitnained at agitation 150rpm for 72 hours. The highest biomass (16.3gl -1 ) was obtained at 150rpm (Fig.4B). The cell density and xylitol concentration increased with xylose consumption and remained constant. The xylose consumption was completely reduced after 72 hours. Similar observation has been reported for xylitol production from xylose by Candida guilliermondii [3]. They reported that maximum xylitol yield was observed at agitation speed of 150rpm. The xylitol yield sharply decreased with increasing agitation speed. This is probably due to oxygen mediated NADH consumption that lowers the level of NAD+, thus accelerating the further metabolism of xylose to xylitol [21]. The bioconversion of xylose into xylitol concerning our work and the work of other authors are in Table 1. A xylitol production of 35 gl -1 was achieved in the present study, were lower than those obtained in corncobs hydrolysates using Candida sp (70 gl -1 ) and D.hansenii (64 gl -1 ). At the same time, the values obtained in our work were higher than those observed in corn cobs (15 gl -1 ), sugarcane bagasse (13.5 gl -1 ) and eucalyptus hydrolysates (8.5 gl -1 ). It is difficult to compare the results obtained by different researchers since many variables influence the xylose to xylitol bioconversion in hemicellulosic hydrolysates. While a considerable high bioconversion yield could be achieved in the corn cob hydrolysate using a relatively low initial cell concentration (0.5 gl -1 ), the poor bioconversions observed in the eucalyptus hydrolysate were attributed to a very high concentration of inhibiting hydrolysis by-products. As a whole, the present study points to the fact that the corn cobs hydrolysate is a potential raw material to be used as a source of xylose for xylitol bioproduction using Pichia sp. The toxic compounds present in the hydrolysate can be efficiently removed by a simple detoxification strategy based on ph alteration and active charcoal adsorption. instead of a complex chemically defined medium. Xylitol production from corncob has some advantages such as the reduction in the cost of the medium, cheaper and easily available raw material and the ease of purification in the culture broth.

CCCC 3618 Fig. 4: Biomass, xylose consumption and xylitol production during batch fermenation of corncob hydrolysate using Pichia sp at different agitations A)100 rpm B) 150 rpm C) 200 rpm. Acknowledgments The authors extend their thanks to the management of Government Arts College, Ariyalur, Tamil Nadu, India, for providing the facilities to do the research work in the Department of Botany. References 1. Agblevor, F.A., B.R. Hames, D. Schell and H.L. Chum, 2007. Analysis of Biomass sugars using a novel HPLC method. Applied Biochemistry and Biotechnology, 136: 309-325. 2. Alves, L.A., M.G.A. Felipe, J.B. Almeida e Silva, S.S. Silva and A.M.R. Prata, 1998. Pretreatment of sugarcane bagasse hemicelluloses hydrolysate for xylitol production by Candida guilliermondii. Applied Biochemistry and Biotechnology, 70(72): 89-98. 3. Barbosa, M.F.S., M.B. Medeiros, I.M. Mancilha, H. Schneider and H. Lee, 1988. Screening of yeasts for production of xylitol from D-xylose and some factors which affect xylitol yield in Candida guilliermondii. Journal of Industrial Microbiology, 3: 241-251. 4. Canettieri, E.V., J.B. Almeida e Silva and M.G.A. Felipe, 2001. Application of Factorial Design to the Study of Xylitol Production from Eucalyptus Hemicellulosic Hydrolysate. Applied Biochemistry and Biotechnology, 24: 159-168. 5. Cao, N.J., R. Tang, C.S. Gong and L.F. Chen, 1994. The effect of cell density on the production of xylitol from D-xylose by yeasts. Appl Biochem Biotechnol, 45(46): 515-59. 6. Cho, C., S. Aruga, M. Hatsu, T. Suzuki, K.K. awai and K. Takamizawa, 2000. Development of medium by the combined treatment of steam explosion and chemical decompostition of peanut shells for production of xylitol by Candida tropicalis. J.Jpn.Soc.Waste Manage.Experts, 11: 11-20. 7. Dominguez, J.M., N. Cao, C.S., Gong and G.T. Tsao, 1997. Dilute acid hemicellulose hydrolysates from corn cobs for xylitol production by yeasts. Biores. Technol., 61: 85-90.

8. Dominguez, J.M, C.S. Gong and G.T. Tsao, 1997. Production of xylitol from D-xylose by Debaryomyces hansenii. Appl Biochem Biotechnol., 63(65):117-27. 9. Erramoupse, J. and C.A. Heyneman, 2000. Treatment and prevention of otitis media. Annals of Pharmacotherapy, 34(12): 1452-1468. 10. Farooq, L. and I.R. Mohammad, 2001. Production of ethanol and xylitol from corn cobs by yeasts, Bioresour. Technol., 77: 57-63. 11. Gales, M.A. and T.M. Nguyen, 2000. Sorbitol compared with xylitol in prevention of dental caries. Annals of Pharmacotherapy, 34(1): 98-100. 12. Garrote, G., H. Dominguuez and J.C. Parajo, 2002. Autohydrolysis of Corn cob study of nonisothermal operation of xylooligosaccharide production, J.Food. Eng., 52: 211-8. 13. Jyri-Pekka, M., S. Tapio and S. Rainer, 2001. Effect of solvent polarity on the hydrogenation of xylose. J.chemical.Technol. Biotehcnol., 76: 90-100. 14. Kiyoshi Tada, Jun-Ichi Horiuchi, Tohru Kanno and Masayoshi Kobayashi, 2004. Microbial xylitol production from conr cobs using Candida magnoliae, Journal of Bioscience and Bioengineering, 98(3): 228-230. 15. Makinen, K.K., 1992. Latest dental studies on xylitol and mechanism of action of xylitol in caries limitation. In: Greenby, T.H. (ed.), Progress in sweeteners, 2 nd. Edn. Elsevier Applied Science. London, N.Y., pp: 331-362. 16. Mattila, P.T., M.J. Svanberg, T. Jamsa and M.L.E. Knuuttila, 2002. Improved bone biomechanical properties in xylitol-fed aged rats. Metabolism-Clinical and Experimental., 51(1): 92-96. 17. Nakano, K., R. Katsu, K. Tada and M. Matsumura, 2000. Production of highly concentrated xylitol by Candida magnoliae under a microaerobic condition mainained by simple fuzzy control. Journal of Bioscience and Bioengineering, 89: 372-376. 18. Parajó, J.C., H. Dominguez and J.M. Dominguez, 1998. Biotechnological Production of Xylitol. Part 1: Interest of Xylitol and Fundamentals of its Biosynthesis. Bioresource Technology, (65): 191-201. 3619 19. Rivas, B., P. Torre, J.M. Dominguez, P.P. erego, A. Converti and J.C. Parajo, 2003. Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii. Biotechnol Prog., 19: 706-13. 20. Roberto, I.C., M.G.A. Felipe, I.M. Machilha, M. Vitolo, S. Sato and S.S. Silva, 1995. Xylitol production by Candida guilliermondii as an approach for the utilization of agroindustrial residues. Bioresour.Technol., 51: 255-257. 21. Saha, B.C. and R.J. Bothast, 1997. Fuels and chemicals from biomass. ACS symposium series. American chemical society, 66: 307-319. 22. Sene, L., M.G. Felipe, M. Vitolo, S.S. Silva and I.M. Manciha, 2000. Adaptation and reutilization of Candida guilliermondii cells for xylitol production in bagasse hydrolysate. J. Bas. Microbiol., 38: 61-69. 23. Sene, L., M.G.A. Felipe, S.S. Silvio and M. Vitolo, 2001. Preliminary Kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production. Applied Biochemistry and Biotechnology, 91(93): 671-680. 24. Shen, P., F. Cai, A. Nowicki, J. Vincent and E.C. Reynolds, 2001. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopepticleamorphous calcium phosphate. Journal of Dental Research, 80(7): 2066-2070. 25. Sun, R.C., 1999. Extraction, fraction, isolation and structural characterization of polysaccharides and lignin from wheat straw. PhD thesis, University of Wales, Bangor. 26. Tapiainen, T., L. Luotonen, T. Kontiokari, M. Renko and M.Uhari, 2002. Xylitol administered only during respiratory infections failed to prevent acute otitis media. Pediatrics, 109: 1-6. 27. Vongsuvalert, V. and Y. Tani, 1989. Xylitol production by a methanol yeast Candida boidinii (Kloekera sp.) No. 2201. J. Ferment. Bioengg., 67: 35-39. 28. Washuttle, J., P. Riederer and E. Banchen, 1997. A qualtative and quantitative study of sugar alcohols in several foods.j. Food. Sci.., 38: 1262.