(688d) A Synthetic Symbiotic Co-Culture of Aerobes and Anaerobes for Bioproduction from CO2 and H2

Biology-based carbon fixation technologies, especially non-photosynthetic microbial carbon capture using renewable H_2, are an attractive solution to controlling industrial CO₂ emissions. Among CO₂-fixing microbes, acetogenic bacteria are particularly promising candidates due to their use of the highly energy efficient Wood-Ljungdahl pathway. However, as obligate anaerobes, acetogens are limited in the range of products they can make with the fixed carbon at industrially relevant metrics because of the oxygen-sensitive enzymes of the pathway and energy-limited metabolic process.

To overcome this challenge, we explore the concept of a synthetic aerobic-anaerobic co-culture system, where the acetogen is paired with an aerobic microbe in a microaerobic reactor. In this system, the aerobe upgrades the main product from the acetogen (acetate or ethanol), to higher-value chemicals by using oxygen as a better electron acceptor, while rapidly consuming the available oxygen and providing a low steady state dissolved oxygen (DO) level which permits acetogenic growth. We first demonstrate the feasibility and of this approach in a model system composed of Escherichia coli and Clostridium ljungdahlii, showing robust co-culture growth under heterotrophic conditions and acetogen autotrophic growth under microaerobic environment via qPCR and HPLC analysis. Under fully autotrophic conditions, slow acetate uptake and oxygen use by E. coli under microaerobic conditions prevented the growth of C. ljungdahlii due to relatively high oxygen tensions. We pursued a multi-pronged approach to this problem, including rational engineering of E. coli acetate catabolism under microaerobic conditions, exploration of different growth media and inlet oxygen concentration, alternative pairs of microbes, and evaluation of ethanol as an alternative intermediate metabolite. These efforts identified a more promising microaerobic acetate utilizer under low oxygen levels, Pseudomonas putida, and another model acetogen, Eubacterium limosum, with better oxygen tolerance. This pairing allowed the successful demonstration of a fully autotrophic system, with CO₂ as the major carbon source for the system. This work paves the way for a simple, low-cost system for upgrading CO₂ to value-added chemicals with best-in-class performance metrics, as well as a model system for investigating the adaptation of strictly anaerobic microbes to microaerobic conditions.