Engineering anaerobic fungal-bacterial consortia for medium-chain fatty acid production from lignocellulosic biomass

Anaerobic fermentation of lignocellulosic biomass offers a cost-effective route for sustainable biomanufacturing, yet constructing synthetic consortia for targeted bioproduction remains challenging. Inspired by herbivore rumen microbiome, we engineered bottom-up anaerobic fungal-bacterial consortia for direct conversion of lignocellulose to medium-chain fatty acids (MCFAs). Initial screening experiment identified a newly isolated fungal strain, Neocallimastix sp. FC1, in co-culture with the bacterial strain Megasphaera hexanoica , as an optimal co-culture pair, achieving 21.0 % (carbon-to-carbon basis) lignocellulose-to-MCFAs yield. Lactate, the main fermentation product of Neocallimastix sp. FC1 and primary substrate for M. hexanoica , was undetectable in co-culture suggesting tight cross-feeding. This triggered upregulation of reverse β-oxidation pathway enzymes in M. hexanoica , favoring the production of MCFAs (hexanoate and octanoate) over butyrate. These results suggest that securing high-performing lactate-producing fungi, while maintaining low lactate levels, could be a promising strategy to maximize lignocellulose-to-MCFA conversion. Technoeconomic analysis further supported the process’s economic feasibility and delineated the cost-yield ranges required for profitable implementation.