Hydrogen nanobubbles overcome metabolic bottlenecks and remodel transcriptional networks for high-rate methanogenesis in the anaerobic digestion of Paulownia leaf litter.

Abstract

Volatile fatty acid (VFA) accumulation limits anaerobic digestion (AD) of lignocellulosic biomass by causing acidification and microbial metabolic imbalance. Paulownia leaf litter (PLL), an underutilized lignocellulosic residue with potential for renewable energy recovery, was selected as the substrate. This study evaluated hydrogen nanobubble water (H2-NBW) as a strategy to alleviate VFA inhibition and investigated associated microbial responses using integrated multi-omics analyses. H2-NBW increased cumulative methane yields by 26.4-34.5% compared with controls, accelerated VFA turnover, improved acid-base stability and buffering capacity, and enhanced extracellular hydrolytic enzyme activities by 12.7-42.4%. Coenzyme F420 fluorescence intensity increased by 11.5-30.7%, indicating enhanced methanogenic activity. Amplicon sequencing suggested that H2-NBW enriched microorganisms associated with carbohydrate degradation and fermentation, while overall community restructuring remained limited. Metagenomic analysis revealed increased functional potential for lignocellulose degradation, reflected in carbohydrate-active enzyme and H2 metabolism genes. Meta-transcriptomic analysis further showed increased transcription of genes associated with hydrolysis, acetate-forming metabolism, the Frh/Mvh-Hdr hydrogenase system, and hydrogenotrophic methanogenesis. These responses suggest that H2-NBW may support carbon conversion and hydrogenotrophic methanogenesis, accompanied by enhanced transcription of genes involved in substrate degradation, H2 metabolism, and methane formation. The observed improvements may be associated with exogenous H2 availability, microbial metabolic responses, and potential nanobubble-associated physicochemical effects. Overall, this study links improved AD performance with coordinated microbial functional and transcriptional responses, highlighting the potential of H2-NBW to improve lignocellulosic biomass conversion and methane recovery.