Exploring substrate cofeeding for enhanced acetogenic C1 bioconversion with AneVO, a low-cost anaerobic parallel...

Acetogenic bacteria are attractive biocatalysts for biochemical production from sustainable single-carbon (C1) feedstocks. The major challenge is their energy-constrained anaerobic lifestyle, which results in slow growth and low productivity. To overcome this limitation, here, we investigate substrate cometabolism in the acetogen Eubacterium limosum, cofeeding either carbon monoxide or glucose as a dopant alongside the primary C1 substrate, methanol or formate. To increase experimental throughput, we developed AneVO, a parallel minibioreactor system that enables benchtop anaerobic batch and fed-batch cultivation, with continuous delivery of custom anaerobic gas blends. In all cofeeding scenarios, E. limosum grew faster, reached 52-254% higher cell densities, and exhibited 2.2- to 3-fold increase in acetate productivity. Most interestingly, cometabolism of CO and methanol synergistically improved growth and production. Together, these results validate AneVO as a low-cost resource for convenient benchtop cultivation of strict anaerobes and present a strategy for enhancing production from C1 feedstocks in an emerging model acetogen.