Synthetic and Syntrophic Cocultures of Clostridia Enable Enhanced Production of Three-to-Six Carbon Chemicals Through Carbon Capture, Cell Fusion, and Metabolite Sharing Across Fermentation Scales

Abstract

Historically and currently, industrially-relevant energy and feedstock chemicals such as butanol, isopropanol, ethanol, acetone, hexanol, and caproate have been produced though the refining of petrochemicals. While this approach has produced low-cost energy abundance, we are rapidly exhausting our stores of accessible petroleum and warming the planet through our poor uses of fossil fuels. Past efforts by chemical engineers offer a partial solution to this conundrum. Around the time of World War I and World War II, the Weizmann process of acetone-butanol-ethanol fermentation was used to produce large amounts of acetone required to produce cordite from renewable feedstocks such as molasses. While successful at the time, this fermentation by Clostridium acetobutylicum had several issues. It was outcompeted by petroleum-based processes partially because a third of all sugar carbon was lost as carbon dioxide with the corresponding electrons lost as hydrogen. Without modern metabolic engineering techniques, the balance of products could not be adjusted to meet market demands. These problems can now be potentially solved. C. acetobutylicum remains an extremely productive solventogen that can utilize a broad array of 5- and 6-C sugars in addition to complex polysaccharides like hemicellulose. Genetic engineering through the transformation of plasmids or through chromosomal editing through CRISPR allows us to tune the ratio of its products. Through coculture with other bacteria, the environment found in nature can be emulated. The acetogen Clostridium ljungdahlii utilizes only fructose, carbon monoxide, carbon dioxide, and hydrogen for growth. When paired with C. acetobutylicum, it can capture its waste carbon dioxide and hydrogen, allowing for full carbon utilization. It can also utilize exogenously-sparged carbon dioxide and hydrogen. Moreover, C. ljungdahlii produces non-native metabolites in coculture while also sharing metabolites, reducing equivalents, genetic material, and proteins with C. acetobutylicum. Through this process, the balance of products can be shifted with minimal genetic engineering. With more genetic engineering, undesired products can be suppressed entirely. In addition to a coculture of C. acetobutylicum and C. ljungdahlii, C. kluyveri can also be cocultured with C. acetobutylicum and Clostridium saccharolyticum. In this fermentation, the reverse β-oxidation pathway of C. kluyveri is used to generate high-value 6-C chemicals from lower-value precursors such as ethanol and acetate. Throughout this work, bioreactor engineering and the parametric design of cocultures is employed to maximize the concentrations and productivity of target chemicals. First, C. kluyveri is cocultured with novel partners for the rapid production of caproate and hexanol. After that, cocultures of C. acetobutylicum and C. ljungdahlii are employed to transform the ABE fermentation into the IBEA (isopropanol, butanol, ethanol, and acetone) fermentation with accompanying flux analysis to analyze the changes in gene expression and culture productivity with and without cocultures and with the manipulation of different parameters. Finally, after genetic engineering to remove butanol and ethanol production from the coculture, cell density as increased through the use of resuspensions and a retentostat as long-term carbon-negative production of isopropanol is demonstrated at a large scale through the use of a packed bed reactor and an engineered C. acetobutylicum and C. ljungdahlii coculture. These coculture interactions and bioreactor designs portend new possibilities for sustainable chemical production as the natural and engineered interactions of coculture species are studied.