Bio-ethanol production through simultaneous saccharification and fermentation using an encapsulated reconstituted cell-free enzyme system

• An encapsulated reconstituted cell-free enzyme system was developed.
• The system effectively produced bio-ethanol at elevated temperature through SSF.
• The system was successfully evaluated for bioethanol production till 15 repeated batches.
• The kinetics profile of SSF process for this system was studied.
• It offered several advantages and was superior compared to previously reported SSF systems.

An encapsulated reconstituted cell-free enzyme system was developed through liquid-droplet forming method by using endogenous glycolytic and fermentation enzymes from yeast cells and exogenously added saccharification enzymes, cofactors, and ATPase. It was evaluated for bio-ethanol production through simultaneous saccharification and fermentation (SSF) at various temperatures, pH, and cell-free enzyme and substrate concentrations. Using 1% starch as substrate, encapsulated system illustrated maximum efficiency at 45 °C and pH 7.0. SSF with encapsulated and bare reconstituted cell-free enzyme systems produced 3.47 g/L and 2.98 g/L bio-ethanol corresponding to 62% and 53% of maximum theoretical yield, respectively. It was explicable that encapsulated system provided better substrate utilization and product formation at elevated temperatures than bare system. Kinetic profile of SSF process of both systems was affected differently by variations in pH, temperature, and substrate and cell-free enzyme concentrations. Under appropriate conditions, system retained 90%, 64%, and 40% of initial enzyme concentration and produced 3.21, 2.24, and 0.83 g/L bio-ethanol after 5, 10, and 15 consecutive batches, respectively. The current system offered several advantages and was superior compared to previously reported SSF systems. This system can effectively overcome the major barriers associated with successful development of SSF processes for bio-ethanol production on an industrial scale.