Synthetic Biofilm Reactor with Independent Supply of Gas and Liquid Phase for Studying Chain Elongation with Immobilized Clostridium kluyveri at Defined Reaction Conditions

In this study, we explore the use of C. kluyveri in synthetic biofilms for the production of 1-butyrate and 1-hexanoate, investigating the impact of inoculation temperature during biofilm formation and the presence of yeast extract. Therefore, a novel synthetic biofilm reactor has been designed and constructed. Prior to investigating synthetic biofilms in this reactor, we carried out preliminary batch experiments in anaerobic flasks containing an inoculated agar hydrogel fixed at the bottom and overlaid medium. For the operation of the novel synthetic biofilm reactor, specific volumes of inoculated agar hydrogel were dispensed into a cylindrical mold with a diameter of 102 mm, forming the synthetic biofilm with a height of 4 mm, which was then transferred into the biofilm reaction chamber onto the support grid. The biofilm support grid separates the gas phase (CO2, N2) above the synthetic biofilm from the aqueous phase (medium) below. Our results show that C. kluyveri remains metabolically active at biofilm preparation temperatures of up to 45 °C, with extended lag phases observed at 70 °C. The synthetic biofilm demonstrated efficient chain elongation in batch processes, converting ethanol and acetate into 1-butyrate and 1-hexanoate, with final concentrations of 2.7 g L−1 and 10.1 g L−1, respectively, with yeast extract in the circulating liquid medium of the synthetic biofilm reactor setup. The maximum estimated space-time yields for 1-butyrate and 1-hexanoate, referenced to the biofilm volume, were 1.331 g L−1 h−1 and 4.947 g L−1 h−1, respectively. Experiments without yeast extract lead to final concentrations of 2.0 g L−1 1-butyrate, and 7.3 g L−1 1-hexanoate and maximum estimated space-time yields, referenced to the biofilm volume, were 0.332 g L−1 h−1 and 1.123 g L−1 h−1, respectively. The use of synthetic biofilms, even without yeast extract, eliminates the need for significant cell growth during chain elongation. However, product concentrations were lower without yeast extract.