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

Liquid manure storage is one of the major global sources of agricultural methane (CH₄) 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 CH₄ 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 CH₄ emissions much earlier (day 31) than WOI tanks (day 79), and exhibited 17.9% higher cumulative CH₄ emissions. Modified Gompertz modelling showed that inoculation shortened the apparent methanogenic lag phase by 20.64 days, confirming its accelerating effect on system-level CH₄ emission dynamics under continuous filling. This study provides the first investigation of inoculation effects in daily filled manure storage, linking microbial community dynamics with CH₄ emission patterns and offering a framework for identifying CH₄ emission trajectories and key control windows.