From Destruction to Reconstruction: Substrate-Driven Biosynthesis of Antioxidants from Antibiotic Residues in Hyperthermophilic Composting

Abstract The accumulation of tetracycline (TC) in organic fertilizers poses severe risks by fostering antibiotic resistance in soil ecosystems. Hyperthermophilic composting effectively mitigates these threats, achieving nearly complete TC removal via thermal selection. Although previous studies have widely characterized the catabolic degradation routes of TC, we propose a novel transformation pathway in which TC-derived intermediates are redirected into secondary metabolism to synthesize antioxidant alkaloids and flavonoids. Integrated abiotic–biotic controls first demonstrated that while heat initiates breakdown, hyperthermophilic microbiota drive the subsequent deep transformation and diversification of products. FT-ICR-MS revealed that this process is underpinned by substrate restructuring, where recalcitrant compounds are depolymerized into bioenergetically favorable low-molecular-weight organic matter (LMW-OM). Cross-feeding assays confirmed that this accessible substrate pool significantly enhances cometabolic efficiency compared to conventional composting-derived substrates. Mechanistically, multiomics indicated that LMW-OM elevates intracellular NADH and ATP within the Thermus-dominated community, shifting metabolism from maintenance to active biosynthesis. Consequently, TC intermediates acting as structural mimics of endogenous aromatic precursors are recruited into these energized pathways to generate antioxidants. Crucially, diverting residues into anabolic networks eliminates the selective pressure exerted by intermediates, explaining the extensive attenuation of the antibiotic resistance genes. This work highlights pivotal substrate-microbe interplay, offering a new paradigm for converting antibiotic-laden waste into functional bio-organic fertilizers.