Expanding the operational window of consolidated bioprocessing hydrogen production from non-detoxified alkali-pretreated tobacco stalks via microbial co-cultivation.

This study investigated the feasibility of producing biohydrogen from non-detoxified alkali-pretreated tobacco stalks 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, demonstrating substantial fermentative capacity under pretreatment-derived inhibitory stress. In CBP, hydrogen production by Acetivibrio thermocellus DSM1313 alone was severely inhibited by 90.71% when non-detoxified stalks were used, whereas co-cultivation with MJ2 markedly alleviated this inhibition and restored hydrogen production to 91.34% of the level obtained from detoxified stalks. To further characterize the tolerance of the co-culture system, a gradient of pretreatment liquor (0-100%, v/v) was introduced as an inhibitory stress factor. Kinetic analysis using the modified Gompertz model showed that the co-culture achieved a maximum hydrogen potential of 125.91 ± 0.54 mM at 40% (v/v) pretreatment liquor, corresponding to a 44.27% increase over the control. A hormetic effect was observed at 20% liquor concentration, whereas a critical threshold was identified at 60%, where hydrogen production sharply declined due to growth arrest of the primary cellulose degrader, DSM1313. These results demonstrate that microbial co-cultivation effectively enhances hydrogen production performance under non-detoxified conditions and expands the operational window of CBP using alkali-pretreated tobacco stalks. The beneficial effect is clearly supported at the functional level and suggests an improved consortium tolerance to pretreatment-derived inhibitory stress.