Fe3+-modified biochar for enhanced hydrogen production from alkaline-pretreated sugar beet pulp via Klebsiella sp. HDJT1: process optimization and mechanisms

Pretreatment is a critical step in dark fermentation-based hydrogen production, as it directly affects the accessibility of biomass substrates to microbial degradation, with the presence of Fe 3+ -modified biochar. This study aimed to enhance hydrogen production from sugar beet pulp (SBP) via dark fermentation by optimizing alkali pretreatment, screening cellulolytic bacteria, and applying modified biochar. NaOH pretreatment of SBP was optimized, followed by isolation of Klebsiella sp. HDJT1 from sugar beet rhizosphere. Optimal NaOH pretreatment (6% NaOH, 50 °C, 1.5 h) reduced lignin content by 65.8% and increased cellulose exposure, as confirmed by scanning electron microscopy (SEM), revealing a disrupted lignocellulosic structure. Dark fermentation with 10 g/L pretreated SBP yielded 1177 mM H 2 , a 76.4% increase over non-inoculated controls. Fe 3+ -modified biochar (100 mg/L Fe 3+ ) further boosted H 2 production to 1728 mM, enhancing yields by 159% versus controls by improving enzyme stability and microbial community structure, specifically enriching Paraclostridium bifermentans and Pseudomonas veronii —the alkali pretreatment combined with Klebsiella sp. HDJT1 and Fe 3+ -biochar significantly improved SBP valorization for hydrogen production. This integrated approach could optimize substrate accessibility, microbial activity, and catalytic efficiency, providing a sustainable strategy for agricultural waste-to-energy conversion.