Encapsulated Yeast Cell-free System: A Strategy for Cost-effective and Sustainable Production of Bio-ethanol in Consecutive Batches

This study was intended to develop an encapsulated yeast cell-free system (EyCFS) by confining yeast cell-free lysate within a calcium alginate capsule. The system was evaluated for bio-ethanol production at elevated temperatures and was compared to a bare yeast cell-free system (ByCFS). Fermentation of 10 g/L glucose with shaking (150 rpm), using 2 mg/mL cell-free proteins in the ByCFS produced 3.31 g/L bio-ethanol, corresponding to 65% of the maximal theoretical yield, at 45 degrees C and pH 7.0. On the contrary, the EyCFS produced 4.12 g/L bio-ethanol, corresponding to 81% of the maximal theoretical yield, under the same experimental conditions. The EyCFS also retained 32% of its original activity after 15 consecutive batches. We observed an 11% increase in bio-ethanol production after replenishment of cofactors (ATP and NADH) and ATPase. The weight-based total turnover number (TTNw; 0.82 x 10(3)), cost ratio (R value; 1.22), and yield (80.4%) indicated the economic suitability of the EyCFS for large-scale production. Connecting the EyCFS with an encapsulated saccharification system through separate hydrolysis and fermentation (SHF) resulted in production of 4.87 g/L bio-ethanol, corresponding to 87.6% of the maximal theoretical yield. This system resolved serious limitations of conventional simultaneous saccharification and fermentation in bare cell-free systems. These data demonstrates the superiority of the proposed system in terms of thermal stability, yield, efficacy, and cost-effectiveness.