Technical Assessment of Cellulosic Ethanol Production Using β-Glucosidase Producing Yeast Clavispora NRRL Y-50464 Z. Lewis Liu & Michael A. Cotta Published online: 16 January 2015 # Springer Science+Business Media New York (outside the USA) 2015 Abstract Reducing the cost of cellulosic ethanol production, especially the use of expensive exogenous cellulose hydrolyt- ic enzymes such as cellulase and β-glucosidase, is a critical challenge and vital for a sustainable advanced biofuel indus- try. Here, we report a novel ethanologenic yeast strain Clavispora NRRL Y-50464 that produces sufficient innate β-glucosidase enzyme activity for cellulosic ethanol produc- tion by simultaneous saccharification and fermentation (SSF). In a bottled SSF, strain Y-50464 produced 40.44 g/L ethanol from pure cellulose within 72 h at a conversion rate of 0.04 g/ L/h, applying conventional cellulase without supplementary β-glucosidase. Ethanol conversion from delignified corn sto- ver by Y-50464 showed significantly higher titers and rates at various solids loading levels than that from conventional pretreated corn stover with over 40 to 60 % improved efficien- cy in a bottled SSF. However, the bottled SSF was inefficient for mixing higher levels of cellulose feedstock and should be replaced by a more suitable experimental apparatus. In a 2-L bioreactor SSF using conventional dilute acid pretreated corn stover, strain Y-50464 produced 32 g/L ethanol from 20 % solids loading at 48 h applying cellulase alone without addi- tion of β-glucosidase. This represented a conversion rate of 0.088 g/L/h, the highest rate so far for cellulosic ethanol pro- duction from lignocellulosic materials. Elimination of β- glucosidase in cellulose-to-ethanol fermentation would be ex- pected to reduce cost of cellulose conversion. The robustness, fast growth rate, and the capability of producing both ethanol and β-glucosidase illustrated the potential of strain Y-50464 as a potential candidate biocatalyst for advanced biofuel pro- duction from lignocellulosic biomass. Keywords β-Glucosidase . Cellulosic ethanol . Corn stover . Simultaneous saccharification and fermentation Introduction For cellulosic ethanol production, decomposition of cellulosic polymers by enzymatic hydrolysis and saccharification are necessary for microbes to utilize sugars harbored in lignocel- lulosic materials. Simultaneous saccharification and fermen- tation (SSF) is a well-established process commonly used for cellulosic ethanol conversion [1–4]. In conventional cellulose- to-ethanol SSF procedures, cellulase is added to hydrolyze cellulose polymers into oligosaccharides such as cellobiose and cello-oligosacchaides. Since the commonly used ethanologenic yeast strains are unable to utilize cellobiose, the addition of β-glucosidase is needed to digest cellobiose into the simple sugar glucose, in order to be utilized by the fermentation yeast or other microbes. Currently, the supple- mentary enzyme is considered a major expense in lignocellu- losic biomass conversion [5, 6]. Reducing the cost of cellulos- ic ethanol production is challenging and critical for a sustain- able renewable lignocellulose-to-advanced biofuel industry. The concept of consolidated bioprocessing (CBP) has been widely promoted for lignocellulosic biomass conversion to advanced biofuels [5, 7]. In a CBP process, fermentation or- ganisms would produce all the enzymes needed in order to hydrolyze cellulose into fermentable sugars and produce eth- anol. Thus, removal of external enzymes based on the SSF model is expected to reduce the cost significantly. Over the past two decades, efforts have been made to enable ethanologenic yeast and bacteria to express cellulose hydro- lytic enzymes through genetic engineering. Significant suc- cess has been made, and β-glucosidase enzyme activity was Z. L. Liu (*) : M. A. Cotta Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL 61604, USA e-mail: zlewis.liu@ars.usda.gov Bioenerg. Res. (2015) 8:1203–1211 DOI 10.1007/s12155-014-9575-9