Doxycycline-induced toxic perturbations on manure GHG potential: Phage-microbe interactions and BSF pretreatment mitigation

The large-scale production of poultry manure poses significant environmental safety challenges, and Black Soldier Fly (BSF) has emerged as a potential resource utilization solution. However, the toxicological effects of doxycycline (DOX) contamination in poultry feces on methane (CH 4 ) and nitrous oxide (N 2 O) emission potential throughout the BSF manure recycling process remains unclear, especially from a phage-microbe interaction perspective. This study investigates the roles of bacterial and phage-related functional genes associated with CH 4 and N 2 O metabolism in the “chicken manure–organic fertilizer” and “chicken manure–BSF–chicken” pathways. The results reveal that DOX in poultry manure causes toxicological perturbations of the microbial community, significantly elevating the levels of key functional genes ( mcrA/pmoA and (nirS+nirK)/nosZ ) linked to GHG emission potential. These DOX-induced elevated gene levels persisted in BSF-derived organic fertilizer and in the feces of laying hens fed BSF reared on contaminated manure. However, 24-hour starvation pretreatment combined with 8-hour drying at 65 °C can effectively alleviate the negative toxicological effects induced by DOX, and the emission potentials of CH₄ and N₂O in the feces of laying hens fed BSF treated in this way were reduced by 91.1% and 82.4%, respectively. Importantly, phage-mediated horizontal gene transfer (HGT) plays a significant regulatory role in these gene changes, further amplifying the DOX-induced GHG emission risk. This study highlights the potential of BSF pretreatment to reduce the environmental risk of antibiotic-contaminated poultry manure while addressing climate concerns from an environmental toxicology perspective. It also provides a scalable toxicological risk mitigation strategy to reduce greenhouse gas emission potential in poultry farming systems, which is of great significance for the environmental safety of intensive poultry production.