Isolated lignin loading elicits hydrolysis-centered cross-scale responses during anaerobic cellulose digestion with a mesophilic inoculum at two temperatures.

Lignin constrains cellulose hydrolysis during anaerobic digestion of lignocellulosic biomass, but its effects on downstream conversion kinetics and microbial community states remain unclear. Here, microcrystalline cellulose was co-digested with isolated lignin using a mesophilic inoculum at 35 and 55℃ conditions to characterize loading-dependent changes in methane conversion, kinetic parameters derived from Anaerobic Digestion Model No. 1 (ADM1), and microbial community states. Increasing lignin loading progressively delayed methane conversion, prolonged the times required to reach 50% and 90% of the final cumulative methane yield, and reduced methane-production intensity, with stronger retardation observed under mesophilic conditions. ADM1-derived parameter estimates indicated coordinated declines in the apparent cellulose hydrolysis rate constant (21% under mesophilic and 40% under thermophilic conditions) and apparent maximum specific monosaccharide uptake rate (56% and 49%, respectively). At the reference sampling times, high-lignin treatments exhibited distinct bacterial and archaeal community states. Predicted bacterial functional profiles showed lower relative representation of glycolysis/gluconeogenesis under both temperature regimes, whereas pyruvate metabolism was comparatively maintained under thermophilic conditions. These coordinated responses were consistent with downstream kinetic and microbial changes accompanying a lignin-associated hydrolysis constraint. Under the tested inoculum preparation conditions, thermophilic digestion retained faster methane conversion and comparatively maintained predicted pyruvate-metabolism potential under lignin loading.