Hydrogen nanobubble water coordinates functional microbes and metabolic genes in high-solid co-digestion of swine manure and Paulownia leaf litter for enhanced methanogenesis.

High-solid anaerobic digestion (HSAD) is frequently constrained by a kinetic mismatch between the release of soluble substrates and their downstream conversion to methane. This study aimed to determine whether hydrogen nanobubble water (H2-NBW) could regulate the coordination of hydrolysis, acidogenesis, acetogenesis, and methanogenesis during the mono- and co-digestion of swine manure (SM) and Paulownia fortunei leaf litter (PFL) under different total solids (TS) conditions. At 10% TS, the strongest response occurred in SM-PFL co-digestion, where cumulative biogas and methane yields reached 289 ± 8 and 196 ± 7 mL/g VS, representing increases of 25.1% and 26.8% relative to the deionized-water control, respectively. In contrast, no comparable enhancement was observed at 15% TS. H2-NBW induced an early volatile fatty acid (VFA) pulse followed by accelerated VFA depletion and recovery of the volatile fatty acid-to-total alkalinity (VFA/TA) and intermediate alkalinity-to-partial alkalinity (IA/PA) ratios, indicating improved process stability and coupling between acidogenesis and methanogenesis. This response was accompanied by higher hydrolytic activity, enrichment of key hydrolytic-fermentative and methanogenic microorganisms, and strengthened functional potentials related to substrate conversion and terminal methanogenesis. These changes supported complementary acetoclastic and hydrogenotrophic methanogenic pathways. Although H2-NBW cannot overcome the constraints imposed by excessive solids loading, it acts as a condition-dependent regulator within a suitable HSAD operating window, promoting intermediate conversion and methane production.