Construction of a synthetic bacterial-fungal consortium for cephalexin biodegradation: transformation pathways and ecotoxicological assessment

Cephalexin (CPX), a widely used β-lactam antibiotic, is frequently detected in aquatic environments and poses risks to ecological and human health, yet the biodegradation pathways underlying its microbial removal remain incompletely characterized. In this study, a synthetic bacterial-fungal consortium (CPXS), comprising Achromobacter aegrifaciens, Citrobacter amalonaticus, Elizabethkingia anophelis, Bacillus wiedmannii, and Geotrichum pandrosion, was constructed and evaluated for CPX biodegradation. Following systematic single-factor and multivariable optimization (pH 8, 30 °C, 85 mg/L CPX), the consortium achieved 98.5% CPX removal within 48 h, and degradation was further accelerated to 30 h with sodium acetate as a co-substrate. LC-MS-based accurate mass profiling was used to identify 19 transformation products, from which five plausible degradation pathways were predicted, proposed to be initiated by ring-opening hydrolysis of the β-lactam moiety and proceeding through key intermediates, including 2-amino-2-phenylacetamide, toward simple terminal metabolites such as N-methylacetamide and (Z)-but-2-enal, consistent with substantial progression toward mineralization. The consortium also degraded amoxicillin and ampicillin in synthetic and real municipal wastewater, indicating broad-spectrum activity and applicability under complex matrix conditions. Toxicity assays using Escherichia coli, Staphylococcus aureus, and HEK-293 cells showed that degraded samples retained negligible antibacterial activity and exhibited low cytotoxicity, confirming effective detoxification of CPX and its byproducts. Overall, this study proposes a detailed, multi-pathway degradation network for CPX mediated by a bacterial-fungal consortium, culminating in simple bioassimilable end products, and identifies CPXS as a promising candidate for the bioremediation of β-lactam antibiotic-contaminated wastewater.