Sedimentation accelerates mixed-substrate conversion during sequential batch cultivation of a co-culture of sugar-specialist yeast strains

Abstract

Current industrial processes for yeast-based conversion of glucose, xylose, and arabinose mixtures in lignocellulosic hydrolysates mostly rely on monocultures of generalist strains that consume the sugars sequentially. Co-cultivation of "sugar-specialist" strains offers potential advantages regarding volumetric productivity and genetic stability of pentose-fermenting strains during prolonged operation. However, in these cultures, the slow fermentation of pentoses strongly affects the time required for complete sugar conversion. This study explores whether the kinetics of such co-cultures can be improved by increasing the biomass concentration through selective sedimentation of a slow-fermenting strain in sequential batch fermentations. Deletion of ACE2 in a diploid, arabinose-specialist Saccharomyces cerevisiae strain yielded a multicellular, fast-sedimenting phenotype. In monocultures on 20 g∙L-1 arabinose in anaerobic sequential batch bioreactors with a 30-min biomass settling period between cultivation cycles, the sedimenting strain showed a shorter time for full conversion than its non-sedimenting parental strain (11.5 ± 0.4 h and 16.6 ± 0.4 h, respectively). Co-cultures of a glucose-specialist S. cerevisiae strain with the sedimenting arabinose specialist on a glucose-arabinose mixture (10 g·L-1 of each sugar) showed a 29% shorter batch time than corresponding co-cultures with the non-sedimenting arabinose specialist (12.6 ± 0.1 h vs. 17.7 ± 0.1 h). Increasing the biomass settling period to 1 h further reduced the batch time to 8.1 ± 0.1 h and resulted in near-simultaneous depletion of both sugars. These results show that if selective sedimentation of specific slow-fermenting strains can be implemented under relevant process conditions, it has the potential to improve microbial conversion of mixed substrates.IMPORTANCEFermentation of substrate mixtures from lignocellulosic biomass can reduce substrate costs but is often hindered by inefficient, sequential consumption of sugars by "sugar-generalist" Saccharomyces cerevisiae strains. While co-cultures of "sugar-specialist" S. cerevisiae strains can enable parallel consumption in sequential-batch fermentations, the overall volumetric productivity is limited by the slowest fermenter. This study presents a strategy to overcome this limitation by engineering a slow-fermenting arabinose-specialist S. cerevisiae strain for fast sedimentation and implementing a short settling phase between batch cycles in sequential-batch bioreactors, selectively concentrating the specialist's biomass and improving consumption kinetics in subsequent cycles. This approach reduced overall fermentation time by 30% in monoculture and enabled near-simultaneous depletion of arabinose and glucose in co-culture with a glucose-specialist strain. These results show that selective sedimentation of co-culture partners can improve fermentation kinetics in sequential-batch cultures. If compatible with industrial process conditions, this strategy has the potential to improve the economics of co-culture-based fermentations.