Enhancing dark fermentation hydrogen production from lignocellulose through electron bifurcation utilizing lactate in the thermophilic co-culture system

Lactate is one of the most common metabolites produced during dark fermentation, but it can also be used in anaerobic fermentation to produce hydrogen. This study showed that lactate addition significantly promoted the production of hydrogen and butyrate in co-culture, with increases of 22.02% and 51.43%, respectively, compared to the control group. Furthermore, lactate inhibited the growth and spore production of Acetivibrio thermocellus DSM 1313, which could be relieved by co-culture with Thermoanaerobacterium thermosaccharolyticum MJ1. The lactate was completely depleted in the co-culture with HPAC-SCB and avicel cellulose as substrates, while the lactate utilization rates of cellobiose and xylan were only 29.82% and 66.07%, respectively. Transcriptome analysis revealed that gene expression levels associated with lactate uptake, butyrate metabolism, hydrogenase and electron bifurcation were significantly upregulated in co-culture, outlining a proposed pathway for lactate utilization. Finally, the results of enzyme activity, biochemical quantification, electron transfer, and redox analysis confirmed that the co-culture could utilize lactate to produce hydrogen and butyrate through flavin-based electron bifurcation. This study demonstrated that Thermoanaerobacterium thermosaccharolyticum MJ1 could produce hydrogen and butyrate by utilizing lactate through electron bifurcation for the first time.