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Anthropogenic biguanide compounds are widely used in agriculture, industry, and medicine, making them prevalent in the environment. The extensive use of compounds like cyanoguanidine, guanylurea, and metformin has led to their accumulation as pollutants in waterways. This research focused on the microbial degradation of cyanoguanidine, a common biguanide compound used as an additive in agricultural products and frequently present as an impurity in the production of pharmaceuticals such as metformin. Due to its extensive use, cyanoguanidine is classified as a persistent and mobile pollutant commonly detected in wastewater. The presence of cyanoguanidine in wastewater is hypothesized to exert selective pressure on microbial communities, driving the evolution of bacteria capable of metabolizing this compound as their sole nitrogen source. We hypothesized that sludge-derived microbial cultures could serve as a reservoir for isolating cyanoguanidine-degrading bacteria. To test this, enrichment cultures were established using thick and return-activated sludge from a municipal wastewater treatment plant. Microbial media containing cyanoguanidine as the sole nitrogen source was used to support bacterial growth. Aliquots of enrichment cultures were plated on minimal media containing cyanoguanidine to isolate bacteria capable of its metabolism. HPLC analysis was employed to monitor and quantify cyanoguanidine degradation, revealing its conversion to guanylurea, CO2, and NH3 by two bacterial isolates. Genome sequencing identified these cyanoguanidine degraders as Pseudomonas stutzeri and Pseudomonas mendocina. Bioinformatic analyses identified candidate genes involved in the degradation pathway, including nitrile hydratase and guanylurea hydrolase enzymes in both bacterial genomes. Although further investigation is needed to confirm the role of nitrile hydratase in cyanoguanidine metabolism, this study advances our understanding of microbial biguanide degradation. These findings contribute to the development of biotechnological strategies for removing biguanide pollutants from the environment.