(624a) Roller Bottle Method to Generate High-Solids, Fermentable Enzymatic Hydrolysates and Rapidly Screen Plant Materials

Most studies that screen large numbers of lignocellulosic feedstocks for biofuel production either use biomass composition or enzymatic hydrolysis sugar yields as a predictor for fuel production during fermentation. We have previously found that these values do not always predict negative microbial responses to hydrolysates because they do not effectively capture the level or type of inhibition microbes experience during growth. There is a need to rapidly screen plant materials through the entire biofuel production process, including fermentation, under industrially relevant conditions. However, there are a number of major difficulties associated with this approach. First, biomass at high solids conditions are difficult to mix effectively down. Second, contamination by lactic acid bacteria or other microbes can be a major issue during enzymatic hydrolysis. Addition of antibiotics to control contamination can negatively affect fermentation microbes and the environment. Every time the fermentation vessel is opened, there is the potential for contamination, which makes fed batch addition of biomass risky. Finally, pH control, which is important for adequate enzyme activity, is difficult in small-scale screening. And the most effective buffers are inhibitory to fermentation microbes. In order to screen large numbers of samples quickly, we developed a roller bottle enzymatic hydrolysis system that is able to operate at 6% and 9% glucan loading (~18% and ~27% solids, respectively). To validate this method we loaded AFEX-treated corn stover or switchgrass and water into 85 mL Oak Ridge centrifuge tubes. The loaded tubes were sterilized in an autoclave to eliminate potential contamination by lactic acid bacteria, an approach that was previously found to be effective and not result in sugar degradation. Following autoclaving, phosphate buffer and enzymes were added and the mixture placed on a roller bottle apparatus inside a static incubator at 50°C. After 72 hr, the tubes were removed, centrifuged at 12,000 rpm and 4°C for 50 min. The liquid was prefiltered through a 0.5 μm glass filter followed by sterile filtration using a 50 mL Autofil filter system (0.2 μm). Samples were evaluated for their sugar concentration and fermentation performance using S. cerevisiae.