Novel Two-Stage Fermentation Process for Bioethanol Production Using Saccharomyces pastorianus Yogender Kumar Gowtham Dept. of Bioengineering, Clemson University, 301 Rhodes Research Center, Clemson, SC 29634 Kristen P. Miller Dept. of Biological Sciences, Clemson University, Clemson, SC 29634 David B. Hodge Dept. of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824 Dept. of Biosystems & Agricultural Engineering, Michigan State University, East Lansing, MI 48824 DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI 48824 Dept. of Civil, Environmental and Natural Resource Engineering, Lulea ˚ University of Technology, Lulea ˚ 97752, Sweden J. Michael Henson Dept. of Biological Sciences, Clemson University, Clemson, SC 29634 Sarah W. Harcum Dept. of Bioengineering, Clemson University, 301 Rhodes Research Center, Clemson, SC 29634 DOI 10.1002/btpr.1850 Published online December 20, 2013 in Wiley Online Library (wileyonlinelibrary.com) Bioethanol produced from lignocellulosic materials has the potential to be economically fea- sible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose-metabolizing pathways. In this study, a novel two-stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non-Saccharomyces microor- ganism and coupled to a glucose-utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomer- ase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol pro- ductivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two-stage fermentation process has the capability to be a competitive alternative to recombinant Saccha- romyces cerevisiae-based fermentations. V C 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014 Keywords: sustainable energy, xylose, hydrolysates, Escherichia coli Introduction Nonrenewable, fossil energy sources such as coal, gas, and petroleum are being consumed at alarming rates. Some experts anticipate these nonrenewable sources will be depleted in the next 30 years. 1 Furthermore, the United States consumes nearly 19% of the world’s total fossil fuel consumption, making it one of the largest consumers. 2 Also, in the United States, approximately one-fourth of the fossil fuels are used in the transportation sector. 3 Consequently, there is a need to develop alternative energy sources to meet these energy demands. Biofuels are a class of renewable fuels that are produced from naturally occurring resources such as woody plants, agricultural crops, and waste and oil seeds. 4 Bioethanol is the most commonly produced biofuel and could potentially replace fossil fuels in many energy applications, particularly in the transportation sector. 5 Correspondence concerning this article should be addressed to S. W. Harcum at harcum@clemson.edu. 300 V C 2013 American Institute of Chemical Engineers