Development of a high temperature biological process for efficient butanol production

A biological process for butanol production was investigated using a set of thermotolerant enzymes, capable of hydrolyzing cellulose and hemicellulose.  Enzymes were expressed in E. coli for purification and optimal enzyme mixtures for hydrolyzing pretreated biomass were evaluated using a high-throughput robotic system with a 96-well microtiter plate format.  An augmented simplex lattice design of experiments was used to sample a reduced number of points on the n-dimensional simplex design, allowing coverage of a wide range of enzyme mixtures while requiring fewer experiments.  Ionic liquid pretreated switchgrass was incubated with enzyme mixtures at pH 6, 70°C for 24 to72 hours and the hydrolysates were analyzed for glucose, xylose, and cellobiose yields. The data were used to generate a model for predicting optimal enzyme ratios for maximum sugar yield. Once an optimal enzyme mixture was identified, enzyme production was scaled up to produce sufficient quantities for enzymatic hydrolysis of 10 g of ionic-liquid pretreated switchgrass.  The scale up experiment resulted in one-third lower sugar content than was measured from the microtiter plate experiments.  Subsequently, the enzymatic hydrolysates generated from ionic liquid pretreatment were assessed for their ability to support fermentation of sugars to butanol using a microbial strain.  After 48 hours of fermentation with 17 g/L total sugar, butanol levels were 4 g/L and acetone was 1 g/L with minor ethanol production.  The solvent yield from the pretreated enzymatic hydrolysate was identical to the glucose only supplemented control indicating that no inhibitory compounds were present.