In the United States, ethanol from corn grain is an established process with an annual production of approximately 4 billion gallons. However, due to increasing demand for renewable fuel and limited availability of corn starch other feedstock (i.e. lignocellulosic agricultural wastes) are being explored. Newer and better pretreatment technologies are emerging to improve digestibility & fermentability of lignocellulosics. One such novel alkaline pretreatment technology is Ammonia Fiber Expansion (AFEX).

In an industrial process it is critical to perform enzymatic hydrolysis at higher solid loadings in order to achieve economical concentrations of ethanol (>4% (w/w)) after fermentation. In this study, corn stover was pretreated using optimal AFEX conditions, followed by enzymatic hydrolysis at varying glucan loadings (1-12%) in a fed batch process. At higher solid loadings the increased concentration of inhibitors (i.e. lignin phenolics, organic acids) hinders enzyme & microbe performance. In order to reduce this inhibitory effect several strategies were explored. (1) Pretreated biomass was washed to remove pretreatment degradation products and (2) Enzymatic hydrolysis was supplemented with protein rich additives. We will report the sugar yield during the process and ethanol yield by performing Pichia Stipitis fermentation.