Insights into Chain Elongation Mechanisms of Weak Electric-Field-Stimulated Continuous Caproate Biosynthesis: Key Enzymes, Specific Species Functions, and Microbial Collaboration

Chain elongation (CE) technology can recover high-value biochemicals such as caproate, contributing to the realization of carbon neutrality. Electro-assisted fermentation shows extraordinary potential to enhance microbial activity in anaerobic fermentation systems. However, little is known of the effects of key functional genes of enzymes, specific species functions, and microbial collaboration on caproate biosynthesis in the continuously fed expanded granular sludge bed (EGSB) reactor with mixed culture under a weak electric field. In this study, caproate production was enhanced by 21% with 0.05 V electric field introduction in the CE bioreactor continuously operated for 150 days. Mechanism investigation revealed the abundance of functional genes involved in converting substrates to key intermediates (acetyl CoA and malonyl CoA) and in the fatty acid biosynthesis pathway (FAB), and the activities of transmembrane transport and energy metabolism were upregulated. Ruminococcaceae_bacterium played the most significant role in enhancing caproate biosynthesis with electric field introduction. Some essential functional genes were undetected within Ruminococcaceae_bacterium, which implied that harmonious microbial collaboration existed to supplement the lacking functional genes to complete CE processes. Firmicutes_bacterium, Pseudomonas_aeruginosa, Caproiciproducens_sp._NJN-50 and Candidatus_Neoanaerotignum_tabaqchaliae played complementary and collaborative roles in constructing the  microbial collaboration mechanisms in the CE process with the electric field. This study offered a deep understanding of the CE mechanisms in the mixed-culture continuously fed reactor and unveiled the respective roles of different species and the microbial interaction and collaboration mechanisms with weak electric field stimulation and provided a promising strategy for enhancing caproate biosynthesis.