Lactate vs butyrate production during mixed culture glucose fermentation driven by substrate availability as determined by metaproteomics

Mixed-culture fermentation provides a means to recycle carbon from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, individual systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive product expression changes under differing conditions are currently poorly understood. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by CO2, 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 work, a continuous stirred bioreactor was operated at low pH (5.5) with substrate concentration varied between limiting and non-limiting conditions. Using glucose as a model substrate, two sustained operational states were defined: butyrate production during substrate limitation, and lactate production in the non-limited state. Through SWATH-MS metaproteomics and 16S rDNA community profiling, the mechanism of change between butyrate and lactate was described primarily by redirected carbon flow through the methylglyoxal bypass by Megasphaera under substrate non-limiting concentrations. Crucially, butyrate production resumed upon return to substrate-limited conditions, demonstrating the reversibility of this transition.