Developing and validating recipes of simple synthetic solutions for fresh and anaerobically stored urine

Synthetic urine solutions are essential for advancing nutrient recovery and treatment technologies, yet existing formulations often fail to replicate the organic and buffering dynamics of real urine. As these parameters shift during anaerobic storage, synthetic designs must account for both states separately. In this study we developed and validated synthetic formulations for fresh (pH 6.2) and anaerobically stored (pH 9.1) urine, tailored to 24-hour and daytime collection profiles. For varying research needs, fresh urine recipes were designed using a four-organic (urea, creatinine, hippurate, and citrate) and ten-organic matrix (additional glycine, lactate, creatine, alanine, trigonelline, and formate). Stored urine was represented by a five-compound organic matrix (acetate, benzoate, methylamine, propionate, and butyrate). Organic concentrations were increased by factors of 1.3 to 5.3 to match the measured chemical oxygen demand (COD) of the respective real urine. By integrating alkalinity into stoichiometric design, target pH levels were achieved without external titration. Validation with 43 days of anaerobic storage confirmed an accurate representation of urea hydrolysis and the shift toward an organic profile dominated by acetate, methylamine, and benzoate. The latter two compounds were over-represented due to the necessary upscaling of dosed organics. Notably, an observed 10-25% decline in measured COD during storage was identified as an analytical artefact of the standard dichromate method, which recovered only 17% of the theoretical COD for methylamine. The proposed synthetic solutions provide robust, practical proxies for real fresh and anaerobically stored urine, enabling more accurate investigations into technology performance and metabolic pathways during urine treatment.