(623f) Syntrophic Consortia Enables Clostridium Ljungdahlii Growth Under Microaerobic Conditions

Due to growing concerns over industrial carbon emissions, several biology-based carbon fixation technologies are in development. Acetogenic gas fermentation is one such technology that has seen recent commercial success converting CO- and CO₂-rich industrial waste gases into bioethanol. However, the impact of these anaerobic microbes is limited by the need for expensive and extensive gas-pretreatment to remove all traces of O₂ prior to fermentation. Furthermore, their productivity is impaired by the low ATP yields inherent in the Wood-Ljungdahl pathway that forms the basis of acetogenic metabolism. In nature, microbial communities enable the mutualistic growth of member species through the exchange of key metabolites and detoxification of the intercellular environment. We hypothesized that an engineered co-culture between an acetogen (Clostridium ljungdahlii) and an aerobe (Escherichia coli) could reduce the need for feedstock purification and increase the range of products available from an acetogenic fermentation-based platform. In this system, C. ljungdahlii produces acetate, which E. coli uses as a substrate to produce higher-value products, while simultaneously reducing the O₂ concentration to levels tolerated by C. ljungdahlii. E coli has a high affinity for O₂, growing aerobically at nanomolar concentrations, and can grow aerobically on acetate—the primary product of the Wood-Ljungdahl pathway. Additionally, this species has been engineered for an array of various biochemicals of interest, which would allow for a “plug-and-play” system for future production platforms. We first examined this system computationally, developing a kinetic model of the co-culture and performing a sensitivity analysis to identify critical parameters that determine the stability of the co-culture. Next, to experimentally test the ability of E. coli to provide low-oxygen conditions while still respiring, we used a sequential culture approach, cultivating E. coli BL21(DE3) ΔxylA in a fully sealed vessel containing aerobic glycerol-xylose media, then subsequently inoculating C. ljungdahlii. The ΔxylA mutant ensures E. coli can only grow on glycerol, which requires oxygen, leaving the xylose for C. ljungdahlii. We found that with E. coli optical densities as low as 0.2, the media was sufficiently reduced such that the C. ljungdahlii was able to grow. Finally, we were able to demonstrate simultaneous growth of both microbes in the co-culture in a bioreactor with continuous sparging of microaerobic gas, additionally observing significant acetate production by C. ljungdahlii. To the best of our knowledge, this is the first report of acetogenic growth under microaerobic conditions. Given that the Wood-Ljungdahl pathway is active during acetogenic metabolism of sugars, we predict that this syntrophic consortium can be adapted to enable autotrophic growth under aerobic conditions. This is being examined in ongoing work. Overall, these results suggest a promising new platform for the conversion of untreated carbon gas mixture into value-added biochemicals.