Impact of acetogenic partner and pH on syngas fermentation in Clostridium kluyveri co-cultures

The co-cultivation of acetogens with Clostridium kluyveri has gained attention as a strategy to broaden the product spectrum of syngas fermentation, enabling the production of medium-chain carboxylic acids (MCCAs) and their alcohols. Extracellular pH is a critical operational parameter during co-cultivation, as strain-specific responses to pH can influence the performance of the microbial system. Here, a selection of acetogens with distinct optimal pH ranges was screened in co-culture with C. kluyveri. The acetogens assessed comprised Clostridium autoethanogenum, Clostridium carboxidivorans, Acetobacterium wieringae JM, Eubacterium limosum and Alkalibaculum bacchi. Co-cultures were grown in batch bottles under CO:H₂ (66:34 (v/v); 170 kPa) across an initial pH range of 6-8. Results revealed remarkable differences in substrate utilisation and product formation by the different acetogen-C. kluyveri co-cultures. Only co-cultures with A. wieringae JM and C. autoethanogenum showed complete CO/H₂ utilisation, with the highest butyrate/caproate titres obtained at pH 7-7.5 and 6, respectively. Upon two CO/H₂ refills of the headspace, the A. wieringae JM-C. kluyveri co-culture at pH 7.5 produced 16.9 mM butanol (1.25 g L^-1) and 14.8 mM hexanol (1.51 g L^-1) with 90.7 % product specificity (mol C/mol C total soluble products). Further batch bioreactor experiments with continuous gas feeding of the A. wieringae JM-C. kluyveri co-culture achieved some of the highest MCCA titres from syngas to date (3.4 g L^-1 butyrate; 3.4 g L^-1 caproate). The findings of this study highlight the distinct capabilities of acetogens in syngas-fermenting, chain-elongating co-cultures, and the effect of pH on MCCA productivity, providing insights for rational co-culture design.