Surfactant-induced extremozyme production and aqueous separation for bioprocess intensification

Extremophiles constitute a valuable source of robust biocatalysts, although their industrial implementation is still constrained by limited production yields and costly downstream processing. In this work, a sustainable strategy combining surfactant-induced fermentation and deep eutectic solvent-based extraction was developed for the intensified production and recovery of an extracellular thermolipase from Bacillus thermoamylovorans CH6B. Triton X-102 was first evaluated as a lipase production inducer, leading to extracellular activities above 550 U L⁻¹ after 48 h at 55 °C. Biomass growth and enzyme production were successfully described by logistic and Luedeking-Piret models, confirming that lipase synthesis is predominantly growth-associated. Subsequently, Aqueous Biphasic Systems composed of Triton X-102 and choline chloride-based deep eutectic solvents were investigated at 45-65 °C. Among the solvents evaluated, choline chloride:ethylene glycol exhibited the best phase-forming ability together with the greatest thermal stabilization effect on the enzyme, increasing the Gibbs free energy of thermal deactivation to 172.1 kJ mol⁻¹. Under optimized tie-line conditions at 55 °C, the selected system recovered approximately 95 % of the extremolipase into the deep eutectic solvent-rich phase while preserving enzyme activity. Finally, a preliminary process simulation, intended as a proof-of-concept assessment, indicated the potential technical viability of integrating fermentation and extraction at a larger scale, estimating a production of 7.75 t of enzyme-rich extract from 131 batches/year; a comprehensive evaluation of industrial and economic feasibility would require further study. These results highlight surfactant-assisted fermentation coupled with deep eutectic solvent-based extraction as a promising strategy for sustainable extremozyme downstream processing, warranting further evaluation at larger scale.