Inoculation effects on microbial and methane dynamics in daily filled dairy manure storage

• First system-level evaluation of inoculation under daily filled manure storage. • Inoculation accelerated early enrichment of hydrolytic bacteria and methanogens. • Inoculation shortened the apparent CH 4 emission lag phase by 20.64 days. • Early storage identified as critical phase controlling CH 4 emission trajectories. Liquid manure storage is one of the major global sources of agricultural methane (CH 4 ) emissions, and storage tanks on livestock farms are rarely emptied completely. Although the effects of residual slurry acting as an inoculum on subsequent microbial activity and CH 4 generation have been examined previously, most studies have used batch or static storage systems that do not reflect the continuously filled conditions typical of dairy farms. To address this, this study for the first time simulated farm-like daily continuous filling by incubating dairy cattle manure for 92 days in laboratory-scale storage tanks with inoculum (WI) and without inoculum (WOI). Microbial community dynamics were assessed using 16S rRNA gene sequencing, while physicochemical properties and methane emissions were monitored simultaneously. Within this continuously fed system, inoculation reshaped microbial succession, leading to the early enrichment (day 8) of hydrolytic bacteria and methanogens in WI tanks. These shifts corresponded to distinct physicochemical trajectories: WI tanks maintained stable pH (7.0–7.6), accumulated fewer volatile fatty acids, reached peak daily CH 4 emissions much earlier (day 31) than WOI tanks (day 79), and exhibited 17.9% higher cumulative CH 4 emissions. Modified Gompertz modelling showed that inoculation shortened the apparent methanogenic lag phase by 20.64 days, confirming its accelerating effect on system-level CH 4 emission dynamics under continuous filling. This study provides the first investigation of inoculation effects in daily filled manure storage, linking microbial community dynamics with CH 4 emission patterns and offering a framework for identifying CH 4 emission trajectories and key control windows.