Lactic acid bacteria in dark fermentation: From competitor to syntrophic partner for biohydrogen production from complex sugars

Dark fermentation of pretreated lignocellulosic hydrolysates represents a promising pathway for renewable biohydrogen production. However, the complex reducing sugar (RS) compositions and metabolic interactions between microbiota remain unresolved. This study systematically evaluated the biohydrogen production performance from mesophilic dark fermentation and microbial network dynamics of different RS systems at relevant concentrations, aiming to reveal the underlying microbial drivers behind variations in hydrogen production efficiency. Batch fermentation experiments showed that hexoses-based substrates outperformed pentose-based substrates in hydrogen production, yielding 102.09% and 60.25% more hydrogen at concentrations of 3.5 g/L and 20 g/L, respectively. Under low-concentration conditions (3.5 g/L), the galactose-fed group exhibited greater hydrogen production potential than the glucose-fed group, with a 12.76% increase in hydrogen yield. Additionally, the mixed sugar systems effectively enhanced pentose utilization potential, achieving hydrogen yields only 6.46%-13.89% lower than pure hexose-based systems. Notably, despite lactate accumulation reaching 972.47 mg/L under high RS concentration (20 g/L), hydrogen production increased substantially from 702.68 mL H 2 /L fermentation broth to 2287.79 mL H 2 /L fermentation broth. Three-dimensional excitation-emission matrix (3D-EEM) analysis further indicated a 41.98% enhancement in humic-like substance-associated fluorescence intensity under the 20 g/L condition. Furthermore, microbial network analysis revealed that Lactobacillus could contribute positively to hydrogen production efficiency through synergistic interactions with certain hydrogen-producing bacteria. These findings reframe Lactobacillus sp. from a conventional carbon competitor to a collaborative participant within hydrogen-producing consortia, highlighting the potential of strategies based on substrate composition and microbial network regulation for biohydrogen production from lignocellulosic waste. • Hexoses outperformed pentoses in BHP yield with an increasement of 60.25% - 102.09%. • BHP efficiency increased by 2.26 times with average LA concentration of 972.47 mg/L. • LAB genera exhibited substrate-specific abundance and partnership with Clostridium. • Microbial networks were shaped by both sugar composition and concentration. • Tuning hexose-pentose ratios and using LAB- Clostridium syntropy proved to be crucial.