In the fermentation processes discussed above, we hypothesized that the production of butanol and hydrogen is strongly influenced by substrates and co-substrates. Subsequently, we used conventional one-at-a-time optimization strategy to identify the best fermentation condition. In this experiment, we chose rice bran as a main substrate and sesame oil cake as a co-substrate to support the production of butanol and hydrogen. To understand interactive effects and also to attain suitable combination of substrate and cosubstrate to maximize the production of butanol and hydrogen, RSM was employed. According to a standard experimental strategy, the concentrations of rice bran and sesame oil cake were varied. The actual values and the corresponding coded levels for each independent variable are given in Table 2. Based on preliminary results, values corresponding to center point on the production of butanol and hydrogen were chosen (Figs. 1B and 2B). All trials according to experimental plan were performed. Saccharolytic enzymes, butanol and hydrogen concentration measured are given in Table 2. It was observed that supplementation of both rice bran and sesame oilcake in RCM significantly enhanced the expression of saccharolytic enzymes from strain BOH3 (Table 2). Highest saccharolytic
activities were measured after 3 days of fermentation for amylase (0.63e2.03 U/ml), cellulase (0.10e0.50 U/ml) and xylanase (1.62e3.95 U/ml), respectively. It was quite surprising that strain BOH3 can express most saccharolytic enzymes including amylase, cellulase and xylanse when RCM was supplemented with both rice bran and sesame oilcake. It suggests that the specific combination of substrate and co-substrate induced secretion of saccharolytic enzymes for strain BOH3. This phenomenon is found to be similar to cellulolytic enzyme expression reported from C. acetobutylicum ATCC824 [36].