Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation

This study evaluates the feasibility of using non-detoxified alkali-pretreated tobacco stalks for biohydrogen production through separate hydrolysis and fermentation (SHF) and consolidated bioprocessing (CBP). In SHF, Thermoanaerobacterium thermosaccharolyticum MJ2 produced 215.26 ± 49.61 mM hydrogen from non-detoxified enzymatic hydrolysates, showing high tolerance to pretreatment inhibitors. In CBP, while the hydrogen production of Acetivibrio thermocellus DSM1313 alone was inhibited by 90.71% when using non-detoxified stalks, the co-culture of DSM1313 and MJ2 significantly mitigated this inhibitory effect, reaching 91.34% of the yield obtained from detoxified stalks. To further elucidate the detoxification mechanism, the system's tolerance was quantitatively assessed by introducing a gradient of pretreatment liquor (0–100%, v/v). Kinetic analysis using the modified Gompertz model revealed that the co-culture achieved a maximum hydrogen potential of 125.91 ± 0.54 mM under 40% (v/v) pretreatment liquor stress—a 44.27% increase over the control. While a hormetic effect was observed at 20% liquor concentration, a critical threshold was identified at 60%, where the system failed due to the growth arrest of the primary degrader, DSM1313. Collectively, these results demonstrate that microbial co-cultivation significantly expands the operational window of non-detoxified CBP. By providing a higher system-level tolerance threshold to pretreatment-derived inhibitors, this strategy reduces the dependence on intensive detoxification processes, offering a robust approach for cost-effective biohydrogen production.