Systematic optimization of paper mulberry silage: Engineered Lacticaseibacillus paracasei-Lactiplantibacillus plantarum consortium with enzymes and industrial by-products for stable biomass preservation

Unpredictable natural microbial communities drive suboptimal fermentation in paper mulberry (Broussonetia papyrifera) silage, resulting in protein degradation and elevated ammonia emissions, limiting its utility as a stable biomass resource. A formulated lactic acid bacteria (LAB) consortium (Lactiplantibacillus plantarum and Lacticaseibacillus paracasei at 2:1 ratio) was engineered to replace spontaneous fermentation. This strategy is further optimized by comparing the combination effects of industrial by-products (molasses, M; rice husk powder, RHP; corn cob powder, CCP) and enzymes (fibrolytic enzymes, CE; tannase, DE) with LAB on the fermentation quality, chemical composition, and bacterial community of paper mulberry silage. Results showed that all additives lowered pH values, reduced ammonia nitrogen/total nitrogen ratio (AN/TN) and anti-nutritional tannins, while increasing the beneficial flavonoid contents compared to untreated silage (CON). Bacterial community analysis revealed that the LAB was successfully established, significantly increasing the relative abundance of Lacticaseibacillus within the silage microbiome compared to CON. This microbial community engineering correlated with superior fermentation: a significantly lower pH and reduced AN/TN ratio. Functional prediction using PICRUSt2 indicated LAB treatment, particularly with molasses (LAB_M), specifically upregulated carbohydrate metabolism pathways (‘Fructose and mannose metabolism’, ‘Galactose metabolism’). Synergistic integration of the LAB with CE, DE, or M significantly enhanced fiber degradation (reduced neutral detergent fiber, except for M), and preserved more crude protein and starch than RHP and CCP. This study establishes that targeted LAB engineering constitutes an effective bioengineering strategy for stabilizing paper mulberry biomass. Furthermore, the LAB has broader applicability, potentially enhancing biomass conversion efficiency across diverse feedstock sources.