Enabling supratheoretical isopropanol yields from carbon-negative glucose fermentations with a Clostridium acetobutylicum- Clostridium ljungdahlii coculture.

Synthetic microbial cocultures, which combine the designed capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of Clostridium ljungdahlii with a genetically modified Clostridium acetobutylicum (CACas9 Δhbd-p95ace02_atoB), which lacks 4-C metabolism while overexpressing an acetone-formation pathway on a plasmid. CACas9 Δhbd-p95ace02_atoB produces acetone without 4-C metabolites and C. ljungdahlii converts the acetone to isopropanol. To explore the potential of this coculture system to achieve supratheoretical isopropanol yields, we identify NADH-driven hydrogen conversion in CACas9 Δhbd-p95ace02_atoB as the thermodynamically limiting step for acetone production in monocultures and thus isopropanol production in cocultures. We then demonstrate the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 Δhbd-p95ace02_atoB monocultures. Finally, we demonstrate a continuous carbon-negative fermentation of glucose to isopropanol as the sole alcohol produced from glucose in a 3.6-L scale perfusion bioreactor.