Statistical Optimization of Fermentation Process Parameters by Taguchi Orthogonal Array Design for Improved Bioethanol Production

The statistical optimization of different fermentation process parameters in SSF of mixed MAA and organosolv pretreated 1% (w v？1) wild grass, namely, recombinant Clostridium thermocellum hydrolytic enzymes’ volume (GH5 cellulase, GH43 hemicellulase), fermentative microbes’ inoculum volume (Saccharomyces cerevisiae, Candida shehatae), pH, and temperature, was accomplished by Taguchi orthogonal array design. The optimized parameters in 100？mL of fermentation medium were (%, v v？1) as follows: 1.0, recombinant GH5 cellulase (5.7？mg？1, 0.45？mg mL？1); 2.0, recombinant GH43 hemicellulase (3.7？U mg？1, 0.32？mg mL？1); 1.5, S. cerevisiae (3.9 × 108 cells mL？1); 0.25, C. shehatae (2.7 × 107 cells mL？1); pH, 4.3; and temperature, 35°C. pH with p-value 0.001 was found to be the most significant factor affecting SSF. The ethanol titre obtained in Taguchi optimized shake flask SSF was 2.0？g L？1 implying a 1.3-fold increase as compared to ethanol titre of 1.5？g L？1 in unoptimized shake flask SSF. A 1.5-fold gain in ethanol titre (3.1？g L？1) was obtained with the same substrate concentration in lab scale bioreactor on scaling up the shake flask SSF with Taguchi optimized process parameters. 1. Introduction Cost-effective fermentation of lignocellulosic hydrolysate to a value-added product, bioethanol, necessitates the conspicuous enhancement in the activities of various hydrolytic enzymes along with efficient mixed sugar utilization by various fermentative microbes [1]. Simultaneous saccharification and fermentation (SSF) is a single step combination of enzymatic hydrolysis of complex polysaccharides with concurrent fermentation of derived monosaccharides to ethanol [2]. The northeast part of India has a wide abundance of lignocellulosic substrate, namely, wild grass (Achnatherum hymenoides), rich in cellulose and hemicellulose [3]. As compared to the commercially employed hydrolytic enzyme of the corresponding Trichoderma system, the cellulosome of the anaerobic thermophilic bacterium, Clostridium thermocellum, exhibits a 50-fold higher specific activity against crystalline cellulose [4]. The advancement in molecular biology has familiarized new area of enzyme production in transformed cells with overexpression and their subsequent use for the breakdown of structural carbohydrates, namely, cellulose and hemicellulose, into simple sugars [3, 5]. According to CAZy database, glycoside hydrolase family 5 (GH5) exhibits activities of chitosanase (EC 3.2.1.132), cellulase (EC 3.2.1.4), glucan 1, 3- -glucosidase (EC 3.2.1.58), and licheninase (EC 3.2.1.73) whereas glycoside