Efficient anaerobic metformin biodegradation driven by a Cross-Feeding Consortium: Novel Pathways, Enzymes, and...

Metformin is one of the most widely prescribed antidiabetic drugs worldwide and is now ubiquitously detected in aquatic environments, yet its anaerobic biodegradation remains largely unexplored and mechanistically unresolved. Here, an anaerobic membrane bioreactor (AnMBR) was operated and near-complete metformin removal (∼98%) at influent concentrations up to 5.3 mg/L was achieved, corresponding to a maximum degradation rate of 7.2 mg/L/d, approximately sixfold higher than previously reported anaerobic systems degrading metformin. High-resolution mass spectrometry identified three concurrent anaerobic metformin transformation pathways. Notably, a previously unreported biological -C-N bond cleavage route yielding dimethylguanidine was discovered, expanding the known anaerobic metabolic repertoire of metformin. In silico toxicity prediction revealed a non-monotonic toxicity trajectory during metformin transformation, with transiently elevated toxicity at intermediates (particularly 2,4-AMT) followed by an overall attenuation at the terminal product guanidine. Metagenomic and metatranscriptomic analyses uncovered a cooperative, cross-feeding microbial network dominated by Ignavibacterium album and Denitrolinea symbiosum, and identified HypAB (metformin-to-guanylurea), YafV and AmiA/B/C/E (guanylurea-to-guanidine), and, critically, SpeB as the key enzyme initiating the newly proposed -C-N bond cleavage pathway. Molecular dynamics simulations further suggested stable binding of metformin to SpeB with strong affinity, providing mechanistic support for SpeB-mediated initiation of the novel pathway. Overall, this study establishes the first mechanistic framework for anaerobic metformin biodegradation, reveals an unprecedented -C-N scission pathway, and demonstrates that high-rate, low-carbon pharmaceutical removal can be achieved through intrinsic microbial metabolism, offering new conceptual and practical foundations for energy-efficient treatment of emerging pharmaceutical contaminants.