Effects of substrate moisture content on physiological development, performance, and gut microbiome of black soldier fly larvae during swine waste treatment

Black soldier fly larvae (BSFL) composting is a sustainable approach for treating swine waste. BSFL's performance depends on its digestive enzyme activity, which is shaped and enhanced by the gut microbiome. Moisture content influences gut microbiome composition and function; however, it is often overlooked, and its role remains poorly understood. We evaluated the effects of three moisture levels (65%, 75%, and 85%) on the larval gut microbiome, digestive enzyme activity, larval growth, protein content, and contaminant removal. Microbial communities in larval guts and substrates were characterized using 16S rRNA sequencing and multivariate analyses. Larvae reared at 65% moisture had the highest protein content (40.8% DM) and achieved significantly greater reductions in total organic carbon (85.4%), total nitrogen (47.9%), and total phosphorus (47.7%) than those reared at higher moisture levels. However, protease activity was highest at 75% moisture and alpha-amylase activity was highest at 85% moisture. Linear discriminant analysis effect size identified a more functionally enriched gut microbiome at 65% moisture, characterized by taxa associated with organic matter degradation, including Bacillaceae, Paenibacillaceae, and Caryophanaceae, and genera such as Paenibacillus, Peribacillus, and Stenotrophomonas, whereas higher moisture levels favored anaerobic and fermentative taxa. Spearman correlation analysis further identified associations between specific bacterial genera and enzyme activities, including a strong positive association between Enterococcus (enriched at 85% moisture) and alpha-amylase activity (ρ = 0.90). This study contributes to optimizing BSFL-based waste management and highlights the need for microbial enhancement strategies and research on antimicrobial resistance.