SBR operational strategies for directing mixed microbial cultures toward levulinic acid production using hexoses

To date, levulinic acid production by mixed microbial cultures operated under feast and famine regimes has been reported only using mixtures of hexoses and pentoses derived from lignocellulosic hydrolysates. This research presents the first report on the production of LA using a synthetic hexose mixture that mimics the sugar composition of pre-treated beet molasses as the main substrate. Molasses, characterized by its high sugar content and low lignin levels, requires only mild pretreatment, thereby facilitating hexose extraction and minimizing the formation of inhibitors such as hydroxymethylfurfural (HMF). Optimization of molasses pretreatment, combined with adjustments to aeration rate and organic loading rate (OLR), revealed that a moderate aeration rate (3 L/min) and an OLR of 3 g COD/L were optimal for LA production, achieving yields of up to 27.6% ± 1.1% (g LA/g VS). Lower aeration rates (<3 L/min) limited oxygen availability, thereby restricting microbial activity and LA synthesis, whereas higher aeration rates (>3 L/min) may induce oxidative stress, diverting energy from LA production to cellular maintenance. Similarly, high OLR promoted biomass growth, thereby competing with LA production for resources and reducing overall yields. These findings underscore the importance of controlling aeration rate and OLR to optimize LA yields and demonstrate the potential of glucose and fructose derived from hydrolyzed molasses to valorize sugar by-products.