Sawdust-cow manure compost and non-sterile soil casing synergistically enhance Stropharia rugosoannulata yield: Carbon-use dynamics, enzyme activities and microbial drivers

This study explores the utilization of agroforestry biowastes as compost substrates for edible mushroom cultivation, a practice that mitigates environmental burdens in agroecosystems. Here we performed a fully replicated, factorial experiment to quantify how (i) substrate type (sawdust–cow-manure vs. straw–cow-manure compost), (ii) casing material (non-sterile soil, sterile soil, or perlite + vermiculite) and (iii) fungal inoculation duration (0, 30, 90, 210 d) control S. rugosoannulata productivity. By integrating yield data with high-resolution enzyme assays and 16S amplicon sequencing, we aimed to identify the carbon-compound and microbial drivers that underpin high biological efficiency and to benchmark the protein conversion efficiency of the system against insect- and livestock-based platforms. Our findings identify primary determinants for improving mushroom yield and compost quality: i) Application of unsterilized soil as a casing material induced substantial shifts in substrate nutrient composition, particularly in total carbon and nitrogen fractions; ii) Selection of sawdust-cow manure blends as initial composting substrates enhanced cellulase activity, facilitating decomposition of hemicellulose, cellulose, and lignin—key C-compounds critical for fungal nutrition. Cellvibrio and Stenotrophomonas emerged as dominant bacterial genera, with bacterial Shannon diversity increasing progressively with inoculation duration. Throughout cultivation, substrate moisture and cellulose content were identified as primary drivers of bacterial diversity, while total sugar, cellulose, and lignin levels significantly shaped community structure. These results provide actionable guidelines for optimizing substrate formulations, casing material selection, and environmental parameters in S. rugosoannulata cultivation. Their implementation would enable efficient conversion of agroforestry biowaste composts into edible mushroom production systems, thereby advancing sustainable agricultural practices and circular bioeconomy objectives. • Efficient utilization of agroforestry biowaste for edible mushroom production. • 43 % increase in the biological efficiency by combined use of soil casing and sawdust • Mushroom mycelium inoculation significantly enhanced N-metabolism in substrates. • Mushroom cultivation promoted cellulase activity, shaped bacteria community. • An efficient conversion of agroforestry biowastes into high-quality protein sources.