Biocatalytic upgrading of fusel oil from bioethanol production to levulinate esters as renewable fuel oxygenates.

BACKGROUND: The transition toward a sustainable bioeconomy requires efficient strategies for converting renewable biomass and industrial side streams into value-added chemicals and fuels. Fusel oil is an important coproduct of bioethanol fermentation and levulinic acid (LA), a key platform molecule derived from lignocellulosic biomass, can be upgraded into alkyl levulinates, which are promising green solvents, specialty chemicals, and fuel-related applications. RESULTS: In this study, a solvent-free enzymatic approach was developed for the synthesis of alkyl levulinates via lipase-catalyzed esterification of levulinic acid with bioethanol-derived fusel oil and its major alcohol components, including 2-methyl-1-butanol and 3-methyl-1-butanol, using immobilized Candida antarctica lipase B (Novozym® 435). Under optimized conditions (50 °C, LA:alcohol molar ratio 1:10, enzyme loading 10 wt% and molecular sieve addition equal to substrate mass), quantitative levulinic acid conversion and levulinate yield (~ 98-99%) were achieved within 5 h. In comparison, the secondary alcohol 2-butanol, evaluated for mechanistic comparison, reached a lower conversion (~ 70%) under otherwise identical conditions, consistent with its greater steric hindrance within the enzyme active site. Molecular docking simulations supported experimental observations by elucidating substrate-enzyme interactions and differences in binding affinity. The immobilized enzyme exhibited excellent operational stability, maintaining over 97% LA conversion over five consecutive cycles, and successful operation at 100 mL in a bioreactor highlighted the potential for further scale-up. The solvent-free system reduces process complexity, facilitates downstream processing and enhances process sustainability. CONCLUSION: Overall, this work demonstrates an efficient strategy for valorizing fusel oil side stream into high-value levulinate esters, contributing to circular bioeconomy approaches and the development of renewable fuel oxygenates and chemical platforms.