Exploring C1 substrate cofeeding in Eubacterium limosum with AneVO, a low-cost anaerobic parallel bioreactor platform.

Acetogenic bacteria have emerged as attractive biocatalysts for renewable biochemical production, using the highly efficient Wood-Ljungdahl pathway to convert a range of sustainable single-carbon (C1) feedstocks. The major challenge is their energy-constrained anaerobic lifestyle, which results in slow growth and limits the product spectrum. To overcome this limitation, here we investigate substrate co-metabolism in the acetogen Eubacterium limosum, cofeeding either carbon monoxide (CO) or glucose alongside the primary C1 substrate (methanol or formate). To increase experimental throughput, we developed AneVO, a parallel mini bioreactor system based on eVOLVER that enables benchtop anaerobic batch and fed-batch cultivation, along with continuous delivery of anaerobic gas blends. With all substrate pairs tested, E. limosum grew faster and reached 52-254% higher cell densities with cofeeding, while maintaining or improving the volumetric uptake rate of the main C1 substrate. Product formation also improved, with an increase in volumetric acetate productivity from glucose cofeeding of 2.2-fold with formate and 2.4-fold with methanol, and 3-fold from CO cofeeding with methanol. Together these results validate AneVO as a low-cost platform for convenient benchtop cultivation of strict anaerobic microbes in multiple growth modes, and present a strategy for enhancing C1 bioconversion rates in E. limosum, an emerging model acetogen.