Enabling supratheoretical isopropanol yields from carbon-negative glucose fermentations with Clostridium acetobutylicum-Clostridium ljungdahlii cocultures

ABSTRACT Synthetic microbial cocultures, which combine the unique 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 lab-scale 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 genetically-modified Clostridium acetobutylicum [CACas9 Δhbd (p95ace02_atoB), with deleted 4-C metabolism expressing an acetone-formation pathway on the plasmid] with WT Clostridium ljungdahlii using first a pseudo-perfusion approach followed by perfusion culture. CACas9 Δhbd (p95ace02_atoB) produces acetone without any 4-C metabolites and C. ljungdahlii converts that acetone to isopropanol. To explore the mechanism by which these cultures enable supratheoretical isopropanol yields, we first identified NADH-driven hydrogen conversion in CACas9 Δhbd (p95ace02_atoB) as the thermodynamically-limiting step for acetone and thus isopropanol production. We then demonstrated 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 carbon-negative fermentation of glucose to isopropanol as the sole alcohol product in a perfusion bioreactor. Competing Interest Statement The authors have declared no competing interest. Footnotes Author Contact Information: Noah B. Willis (nbwillis{at}udel.edu), BS, Jonathan K. Otten (jkotten{at}udel.edu), BS, Hyeongmin Seo (hyeongmin-seo{at}uiowa.edu), PhD, Pradeep C. Munasinghe (pmunasinghe{at}superbrewedfood.com), PhD, John D. Hill (jdhill{at}udel.edu), BS, Eleftherios T. Papoutsakis (epaps{at}udel.edu), PhD