Bacillus subtilis T7 encodes [FeFe]-hydrogenase and lignin-targeting LPMOs for consolidated bioprocessing

This study presents Bacillus subtilis T7 as the first known strain of B. subtilis capable of simultaneous lignin depolymerization and direct hydrogen production-a dual metabolic capability not previously reported in this species. B. subtilis T7 demonstrated 63.38% lignin degradation and 56.53% Azure B decolorization over seven days, with HPLC detection of aromatic intermediates-ferulic acid (0.85 mg/L) and vanillin (0.666 mg/L)-confirming active lignin catabolism. Agar-based assays revealed robust hydrolytic enzyme activities, including proteases (23.3 mm), cellulases (24.5 mm), amylases (14.2 mm), and xylanases (11.6 mm), surpassing those of many reported Bacillus strains. Whole-genome analysis confirmed a cascade of carbohydrate-active enzymes (CAZymes) including AA10 (lytic polysaccharide monooxygenases), AA3 (oxidoreductases), AA6 (quinone reductases), and CE1 (acetyl xylan esterases). These enzymes are associated with enhanced cellulolytic and xylanolytic activities, as well as increased lignin degradation. Batch fermentation experiments demonstrated that B. subtilis T7 produced hydrogen yields ranging from 0.53 to 1.41 mol H₂/mol substrate across various feedstocks, including xylose, glucose, carboxymethyl cellulose (CMC), starch, and untreated food waste. Xylose exhibited the highest volumetric productivity, achieving 274 mL H₂/g volatile solids, along with the most rapid production kinetics, indicating efficient metabolic utilization of this pentose sugar. In contrast, untreated food waste yielded the maximum molar hydrogen output of 1.41 mol H₂/mol substrate, attributable to its heterogeneous carbohydrate composition and lower average molecular weight, which likely enhanced enzymatic accessibility and substrate solubilization. These findings indicate that B. subtilis T7 encodes a functional [FeFe]-hydrogenase operon, along with an expanded repertoire of oxidative CAZymes, enabling it to bioprocess waste biomass into hydrogen without the need for syntrophic partners. KEYPOINTS: • B. subtilis T7 show dual potential for lignin degradation and hydrogen production. • The genome contains CAZymes (AA10, AA3, AA6, CE1) responsible for lignocellulose deconstruction. • The strain T7 produced a hydrogen yield of 1.411 mol H₂/mol substrate from xylose.