Fermentation of biomass sugars to ethanol using native industrial yeast strains Dawei Yuan a,1 , Kripa Rao b,1 , Patricia Relue a , Sasidhar Varanasi b,⇑ a Department of Bioengineering, University of Toledo, 1610 N. Westwood Ave. MS 303, Toledo, OH 43606, United States b Department of Chemical and Environmental Engineering, University of Toledo, 1610 N. Westwood Ave. MS 305, Toledo, OH 43606, United States article info Article history: Received 1 July 2010 Received in revised form 7 November 2010 Accepted 9 November 2010 Available online 13 November 2010 Keywords: Cellulosic ethanol Xylose isomerase Simultaneous isomerization and fermentation (SIF) with native yeast Co-immobilized enzymes Mixed sugar fermentation (MSF) abstract In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn–ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction One of the crucial issues in utilizing lignocellulosic biomass as a feedstock is the ability to ferment to ethanol both the C6 and C5 sugars resulting from biomass saccharification. Research efforts have focused on the utilization of xylose and native strains capable of fermenting xylose have been identified among bacteria, yeast and filamentous fungi (Skoog and Hahn-Hägerdal, 1988). However, these strains do not display many industrially desired characteris- tics, such as resistance to inhibitors commonly found in hydroly- zates (Hahn-Hägerdal et al., 2007a; Olofsson et al., 2008). As a result none of these strains are likely to find industrial use. Signif- icant progress has been made recently in the development and testing of genetically modified yeast and bacterial strains capable of metabolizing both sugars (Bera et al., 2010; Dutta et al., 2010; Govindaswamy and Vane, 2007; Hahn-Hägerdal et al., 2007b; Kuyper et al., 2005; Lau and Dale, 2010; Zhang et al., 1995). However, the long-term viability of these engineered-strains in the demanding operating environment associated with ‘‘cellulosic ethanol’’ production is yet to be established (Lau et al., 2008). Therefore, approaches that are able to employ native strains as op- posed to genetically modified organisms (GMOs) are still desirable. Saccharomyces cerevisiae is well-adapted to industrial use due to its near theoretical ethanol yields and tolerance to a wide spectrum of inhibitors and elevated osmotic pressure (Hahn-Hägerdal et al., 2007a). S. cerevisiae contains all necessary enzymes for the conver- sion of xylose to ethanol, except xylose isomerase (XI) (Gong et al., 1981). Hence, addition of exogenous xylose isomerase for isomer- ization of xylose to xylulose and fermentation of xylulose to etha- nol using native yeast strains was proposed (Gong et al., 1981). Until now, due to the unfavorable xylose:xylulose (6:1) equilib- rium, xylulose formation has been the bottleneck in commercially implementing this strategy. Two different approaches have been used to partially overcome this unfavorable isomerization equilibrium. In one approach, re- agents that selectively complex to xylulose, such as borax, are em- ployed to shift the isomerization equilibrium in favor of more xylulose formation (Gong et al., 1981). Indeed, borax, when added to an isomerization medium at pH 7.5 and 34 °C at a molar ratio to xylose of 1:8, is able to improve the xylulose yield at equilibrium from 14% to about 50% (Rao et al., 2008). In a second approach, simultaneous isomerization and fermen- tation (SIF) has been attempted to improve xylose utilization (Hahn-Hägerdal et al., 1986). Unfortunately, the pH optima for the isomerization (7.5) and the fermentation (4.5) steps are vastly different. In SIF operating under suboptimal pH conditions, the utilization of xylose is unsatisfactory due to the limited con- centrations of xylulose available to the yeast (Chiang et al., 1981; Gong et al., 1981). In a previous paper, a novel technique was introduced to sustain two pH microenvironments in a single vessel. The technique in- volves coating immobilized XI particles with urease and dispersing the particles in a low pH fermentation broth which contains urea (Rao et al., 2008; Varanasi et al., 2009). As hydrogen ions diffuse 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.034 ⇑ Corresponding author. Tel.: +1 419 530 8093; fax: +1 419 530 8086. E-mail address: sasidhar.varanasi@utoledo.edu (S. Varanasi). 1 These authors contributed equally to the research. Bioresource Technology 102 (2011) 3246–3253 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech