Self-sufficient fermentation paradigm for cassava stillage valorization into C6 carboxylic acids: regulatory mechanisms and novel microbe identification

Cassava stillage (CS), a carbohydrate-rich byproduct of bioethanol production, holds significant untapped potential as a renewable resource. Upcycling this problematic wastewater offers great promise for addressing both environmental challenges and the demand for sustainable biochemicals. Here, this study proposed a self-sufficient biotechnological paradigm that directly valorizes CS into medium-chain carboxylic acids (MCCAs, e.g., caproic acid) by integrating lactic acid/butyric acid-type fermentation with microbial chain elongation (CE) by two phase fermentation regulatory. Lactic acid and butyric acid were regulated as dominant products with optimal ratio around 2 from CS degradation, and then chain elongated into caproic acid with optimal pH of 6. pH was found to play a crucial role in controlling product distribution in both phases of fermentation and shaping the microbiome. Meanwhile, chain elongation resilience was also found operational pH-dependent. Metagenomic analysis identified the bacterium Clostridium sp. BUCT163 as a putative lactic acid-driven chain elongating microbe. Whole-genome comparison between Clostridium sp. BUCT163 and Clostridium kluyveri species indicated that the genes encoding lactic acid conversion are not widespread among C. kluyveri populations. The combination of metagenomic-binning and comparative genomic analysis Clostridium sp. BUCT163 was distinguished as the novel potential lactic acid/ethanol-driven chain elongating microbe which successfully provided valuable data sets to link bacterial identities with chain elongating microbes. These findings provide foundation for the resource recovery process from CS in a self-sufficient anaerobic fermentation paradigm and the microbial management of chain elongating systems.