Establishment of the optimum initial xylose concentration and nutritional supplementation of brewer’s spent grain hydrolysate for xylitol production by Candida guilliermondii Solange I. Mussatto * , Ine ˆs C. Roberto Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de Sa ˜o Paulo, Lorena, SP, Brazil Received 5 November 2007; received in revised form 20 December 2007; accepted 15 January 2008 Abstract The effects of initial xylose concentration and nutritional supplementation of brewer’s spent grain hydrolysate on xylitol production by Candida guilliermondii were evaluated using experimental design methodology. The hydrolysate containing 55, 75 or 95 g/l xylose, supplemented or not with nutrients (calcium chloride, ammonium sulfate and rice bran extract), was used as fermentation medium. The increase in xylitol yield and productivity was related to the increase of initial xylose concentration, but up to a certain limit, above of which the yeast performance was not improved. The hydrolysate supplementation with nutrients did not interfere with xylose-to-xylitol conversion. By using the statistic tool the best conditions for maximum xylitol production were found, which consisted in using the non-supplemented hydrolysate containing 70 g/l initial xylose concentration. Under these conditions, a xylitol yield of 0.78 g/g and productivity of 0.58 g/(l h) were achieved. # 2008 Elsevier Ltd. All rights reserved. Keywords: Brewer’s spent grain; Hydrolysate; Candida guilliermondii; Xylose; Nutrient; Xylitol 1. Introduction Xylitol can be defined as a rare sugar, as it exists only in low amounts in nature. It has beneficial health properties and represents an alternative to current conventional sweeteners [1]. Industrially, xylitol is produced by chemical hydrogenation of D-xylose with a yield of 50–60% [2]. The biotechnological method of producing xylitol by yeasts has been studied as an alternative to the chemical method. This route is of interest because it requires little energy and is very specific [3], however, it has not yet been able to overcome the advantages of chemical hydrogenation. All published data on xylitol production by yeasts have demonstrated that xylitol accumula- tion is influenced by a number of experimental conditions, such as pH [4], temperature [5], age and inoculum concentration [6], initial substrate concentration [7], agitation/oxygen transfer rate [8] and medium composition [9]. Studying the effect of these conditions is of particular interest as a prerequisite for higher xylitol yields and productivities. Several studies on xylose-rich hemicellulosic fractions have been performed with the goal of their utilization as substrates for biotechnological xylitol production. Brewer’s spent grain (BSG) is a hemicellulose-rich agroindustrial by-product that has been recently evaluated as raw material for this purpose. In a previous work, we found that Candida guilliermondii yeast is able to grow and produce xylitol from BSG hydrolysate [10], but the establishment of the best initial xylose concentration is necessary to produce xylitol with high efficiency. In addition, in that study, the bioconversion was performed in a medium whose composition had not been previously optimized. Hemicellulosic hydrolysates from different raw materials require nutrient supplementation to improve the yeast bioconversion performance [9,11–13]. The present work was directed towards the establishment of the optimum initial xylose concentration and nutrient supplementation of BSG hydrolysate for xylitol production by C. guilliermondii. BSG hydrolysate with xylose concentra- tions varying from 55 up to 95 g/l, supplemented or not with nutrients (ammonium sulfate, calcium chloride and rice bran extract) was used in the experiments, which were planned www.elsevier.com/locate/procbio Process Biochemistry 43 (2008) 540–546 * Corresponding author. Actual address: Universidade do Minho, Departa- mento de Engenharia Biolo ´gica, Campus de Gualtar, 4710-057 Braga, Portugal. Tel.: +351 253 605 413; fax: +351 253 678 986. E-mail addresses: solange@deb.uminho.pt, solangemussatto@hotmail.com (S.I. Mussatto). 1359-5113/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.01.013