Co-Immobilization of Clostridium carboxidivorans and Clostridium kluyveri in a Synthetic Dual-Layer Biofilm for Syngas Conversion

Syngas fermentation in combination with chain elongation offers great promise for sustainable medium-chain fatty acid production. While immobilization has proven effective for stabilizing monocultures of C. kluyveri for chain elongation, its applicability to co-cultures involving C. carboxidivorans for simultaneous syngas fermentation remains unexplored. This study investigates the physiological compatibility of C. carboxidivorans with agar-based hydrogel immobilization and its co-cultivation potential with C. kluyveri in a synthetic dual-layer biofilm reactor. First, we conducted autotrophic batch fermentations using suspended and immobilized cells, proving metabolic activity similar for both. Applying different sulfur feeding rates, experiments showed best ethanol formation with C. carboxidivorans at increased sulfur feeding, enabling better conditions for chain elongation with C. kluyveri. In the synthetic dual-layer biofilm reactor, with the C. carboxidivorans biofilm in contact with the CO-containing gas phase above the C. kluyveri biofilm, the formation of 1-butyrate and 1-hexanoate was observed with product formation rates of 0.46 g L−1 d−1 1-butyrate, and 0.91 g L−1 d−1 1-hexanoate, respectively. The formation rate of 1-hexanoate in the dual-layer biofilm reactor was approximately 7.6 times higher than that reported with suspended cells in a stirred tank bioreactor. Spatial analysis revealed species-specific migration behavior and confirmed that C. carboxidivorans reduced local CO concentrations, improving the environment for C. kluyveri.