Optimization of the Protoplast Fusion Conditions of Saccharomyces cerevisiae and Pichia stipitis for Improvement of Bioethanol Production from Biomass

Abstract

Saccharomyces Abstract Bioethanol production using lignocellulosic biomass has gained increased attention because of the abundant supply of this biomass. c e r e v ii s a e is a commonly used microorganism for ethanol production. Nevertheless, S. can not ferment xylose, the second most abundant sugar in plant tissues. In this study, protoplast fusion with the xylose-fermenting yeast performed to improve bioethanol production from biomass. The protoplast formation of S. cerevisiae cerevisiae P. and was achieved using zymolase 20T. The effects of zymolase concentration, enzymatic treatment time and osmotic stabilizers were further stipitis Pichia (ATCC 58785) cells stipitis was investigated. The optimal parameter for the protoplast release of and P. stipitis included 500 µg µLG1 zymolase for 60 min and 750 µg µLG1 zymolase for 120 min, respectively. The maximum protoplast formation ratios were 98.48 and 84.42% for and P. conditions of stipitis P. stipitis , respectively, with 1 mol LG1 sorbitol as the osmotic stabilizer. About 4×106 S. cerevisiae mLG1 protoplasts from S. cerevisiae S. cerevisiae and were isolated. Protoplast fusion frequency was determined using polyethylene glycol (PEG) as fusogen. The optimized fusion S. cerevi s i a e protoplasts with P. stipitis required 35% (w/v) PEG 6000, 10 mM CaCl2 level and 30 min of fusion time. The protoplast fusion rate was 52.21% under the optimized fusion condition.