DefinePK

Abstract

Bioethanol production using lignocellulosic biomass has gained increased attention because of the abundant supply of this biomass. Saccharomyces cerevisiae is a commonly used microorganism for ethanol production. Nevertheless, S. cerevisiae can not ferment xylose, the second most abundant sugar in plant tissues. In this study, protoplast fusion with the xylose-fermenting yeast Pichia stipitis was performed to improve bioethanol production from biomass. The protoplast formation of S. cerevisiae and P. stipitis ATCC 58785 cells was achieved using zymolase 20T. The effects of zymolase concentration, enzymatic treatment time and osmotic stabilizers were further investigated. The optimal parameter for the protoplast release of S. cerevisiae and P. stipitis included 500 g L1501 zymolase for 60 min and 750 g L1501 zymolase for 120 min, respectively. The maximum protoplast formation ratios were 98.48 and 84.42 for S. cerevisiae and P. stipitis, respectively, with 1 mol L1501 sorbitol as the osmotic stabilizer. About 4106 mL1501 protoplasts from S. cerevisiae and P. stipitis were isolated. Protoplast fusion frequency was determined using polyethylene glycol PEG as fusogen. The optimized fusion conditions of S. cerevisiae protoplasts with P. stipitis required 35 wv PEG 6000, 10 mM CaCl2 level and 30 min of fusion time. The protoplast fusion rate was 52.21 under the optimized fusion condition.