Synergies revealed: RNA-seq study of <i>C. acetobutylicum</i> and <i>C. carboxidivorans</i> co-cultured in the presence of conductive materials

Co-cultures can improve substrate utilization and product yields, yet the dynamics between species remain highly variable and poorly understood. In this study, we investigated the metabolic and transcriptional interplay between <i>Clostridium acetobutylicum</i> and <i>Clostridium carboxidivorans</i> when co-cultured in the presence of activated carbon or magnetite, with the aim of evaluating these materials as metabolism enhancers. Fermentation profiling showed that magnetite accelerated glucose consumption and favored acid over alcohol production, with butyrate and acetate reaching yields of 0.65 and 0.62 mol/mol glucose, respectively. Alcohols (ethanol and butanol) accumulated in late fermentation and occurred concomitantly to a metabolic shift from acidogenesis to solventogenesis, potentially driven by interspecies dynamics as RNA-seq data suggested. RNA-seq analysis detected 7,369 genes and revealed <i>C. carboxidivorans</i> dominated in early fermentation, and <i>C. acetobutylicum</i> was activated later (in view of the number of reads detected for each species). Magnetite-treated samples displayed the most transcriptional variation, and species-specific patterns emerged. Changes in electron-active genes (e.g., <i>hydA</i> and <i>rnf</i> genes) suggest enhanced redox communication, highlighting the capacity of conductive materials to influence metabolic flow and regulatory pathways in microbial consortia, opening possibilities for improved bioproduction and carbon utilization.IMPORTANCEMicrobial co-cultures offer a promising strategy to expand metabolic capabilities beyond those of individual strains, yet their internal coordination remains poorly understood. This study demonstrates that conductive materials not only accelerate substrate utilization but also modulate cooperation in a co-culture of <i>Clostridium carboxidivorans</i> and <i>Clostridium acetobutylicum</i>. According to gene expression levels, we demonstrate a clear temporal division of labor between the two partners, with <i>C. carboxidivorans</i> initiating acidogenesis and <i>C. acetobutylicum</i> later driving solventogenesis. Magnetite and activated carbon addition had little effect, but changes in the expression pattern of electron-active genes (<i>hydA</i> and <i>rnf</i>) could be detected for the two species. Understanding and controlling these dynamics are key to optimizing co-cultures for industrial fermentation and biofuel production.