Substrate availability drives mixed culture fermentation of glucose to lactate at steady state

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions.