Earthworm-associated microbial community reassembly accompanies higher inorganic-N concentrations in spent mushroom substrate.

INTRODUCTION: Spent mushroom substrate (SMS) is a major residual stream from edible mushroom production and a potential feedstock for organic fertilizer. Although earthworm composting can alter SMS transformation, the microbial processes associated with nitrogen transformation remain poorly resolved. METHODS: Seven mixtures of black fungus SMS (AhSMS) and golden needle mushroom SMS (FfSMS) were subjected to conventional composting or Eisenia fetida-mediated composting and sampled after 0, 30, and 60 d. RESULTS: Earthworm composting was associated with higher bacterial alpha-diversity values and a transition from early Bacteroidota-dominated communities to later communities enriched in Pseudomonadota and Actinomycetota. At the treatment level, mean NH4+-N in Group E increased from 2.34 to 44.17 mg kg-1 between day 0 and day 60 (1788%), while NO3--N increased from 10.33 to 26.10 mg kg-1 (153%). These changes coincided with candidate taxa associated with predicted nitrogen-cycling functions, including Arenimonas, Pseudoxanthomonas, Croceibacterium and Kineobacterium. PICRUSt2-based functional prediction and network analysis further identified coordinated changes in predicted nitrogen-cycle EC categories and candidate hub taxa, including Mesorhizobium and Flavobacterium. DISCUSSION: Earthworm composting was associated with higher inorganic-N concentrations and bacterial community reassembly. However, these concentration changes do not by themselves demonstrate greater total nitrogen retention because dry-matter loss was not measured. Moreover, as the functional profiles were inferred from 16S rRNA gene data, metagenomic and transcriptomic analyses are required to verify the underlying mechanisms. These findings provide a framework for optimizing earthworm-assisted SMS valorization and prioritizing microbial taxa and nitrogen-cycling pathways for functional validation.