Dynamics of solvent and hydrogen production profile of a coculture of Clostridium beijerinckii and Clostridium pasteurianum

Exploiting renewable waste streams, such as crude glycerol from biodiesel plants and anaerobic digestate effluent (ADE), as fermentation substrates provides a sustainable route for decreasing their discharge into the environment. This study investigated the coculture of Clostridium beijerinckii NCIMB 8052 and Clostridium pasteurianum ATCC 6013 with the following objectives: (i) to determine whether coculturing enhances glycerol metabolism, (ii) to understand the interactions and population dynamics between the two species, and (iii) to characterize the fermentation products, including acetone, butanol, ethanol (ABE), 1,3-propanediol (1,3-PDO), and gases, during fermentation of mixed glucose and glycerol substrates and in a 100% ADE-borne glucose-glycerol medium. The coculture grew in ADE-borne medium, albeit at a reduced rate, to produce ABE. Real-time quantitative polymerase chain reaction (real-time quantitative PCR) analysis of recA and gyrA copy numbers revealed that the microbial consortium comprised ⁓60% C. beijerinckii and 40% C. pasteurianum during ABE fermentation. Gas chromatography showed that cumulative hydrogen production by the coculture was ⁓1.47- and 1.48-fold higher than in C. pasteurianum and C. beijerinckii monocultures, respectively. Interestingly, the coculture did not produce 1,3-PDO, and gene expression and enzymatic assays indicated significant ( P < 0.05) repression of glycerol-to-1,3-PDO production pathways in C. pasteurianum . These findings demonstrate synergistic interactions within the coculture of C. pasteurianum and C. beijerinckii , as evidenced by enhanced growth, increased butanol and hydrogen production profiles, and the elimination of 1,3-PDO formation. The study further highlights the feasibility of employing ADE as a renewable, waste-derived substitute for conventional potable water and nutrients in ABE fermentation and sustainability.