Characterization of Urea Hydrolysis in Fresh Human Urine and Inhibition by Chemical Addition

Urea hydrolysis is a chemical reaction that occurs in soils, the human body, and in wastewater urine diversion systems. The reaction, which transforms the urea in urine into ammonia and bicarbonate, results in ammonia volatilization and mineral scaling in bathroom fixtures, piping, and storage tanks. Urea hydrolysis is inhibited through different chemical additions that affect the function of the urease enzyme. Bench-scale batch experiments were performed where urea hydrolysis was simulated by adding Jack bean urease to both synthetic and real, fresh human urine. Urea hydrolysis was characterized by measurements of urea concentration, ammonia concentration, conductivity, and pH over time. Conductivity was positively correlated with ammonia concentration and negatively correlated with urea concentration making conductivity a simple, surrogate measurement for tracking the extent of urea hydrolysis. Acetic acid, citric acid, and vinegar were effective at inhibiting urea hydrolysis at concentrations varying from 3.2 × 101 to 1.6 × 102 meq L−1 in both synthetic and real, fresh urine as indicated by the conductivity and pH remaining constant throughout the experiments. Fluoride did not inhibit urea hydrolysis in real, fresh urine at concentrations of 3.2 × 10−2, 3.2 × 10−1, and 3.2 meq L−1. Ionic zinc and ionic silver were ineffective inhibitors of urea hydrolysis due to interactions with phosphate and chloride in urine, respectively, which caused precipitative loss of the metals from solution.


Batch chemical addition experiments
Fourteen separate beakers each containing a magnetic stir bar were filled with 75 mL of fresh urine and placed on the multistir plate in four rows of three and one row of two. The pH and conductivity of the 14 samples were measured before the start of the experiment for t = 0 min reading. A timer was set for 15 min and the stirplate was set to 350 rpm speed. At the start of the timer, 2.5 mL of the highest concentration of inhibitor was added to each beaker in the first row, next 2.5 mL of the middle concentration of inhibitor was added to each beaker in the second row, and 2.5 mL of the lowest concentration of inhibitor was added to each beaker in the third row.
The fourth row contained only urine and inhibitor and thus the highest concentration inhibitor was added to the first beaker, the middle concentration inhibitor was added to the second beaker, and the lowest concentration inhibitor was added to the third beaker in the row. This row did not receive any urease. The fifth row of two beakers did not receive any inhibitor as one beaker serves as a urine only control and the other beaker served as a urine and urease control. At t = 15 min, the pH and conductivity of each sample was recorded and the pre-weighed urease batches were added to the first three rows and to the first sample in the fifth row. The first three rows containing urease were the samples simulating hydrolysis and the inhibition of the inhibitor was being observed for the three different concentrations. The fourth row did not contain any urease and were urine and inhibitor only controls. The first sample in the fifth row containing urine and urease only served as a positive control to track uninhibited hydrolysis. The second sample in the fifth row was urine only, which served as a negative control. The pH and conductivity of each sample was recorded every 15 min for 240 min and each inhibitor dose was tested in triplicate as described above.

Analytical methods
For the ammonia monitoring during urea hydrolysis and urea monitoring during hydrolysis experiments, 1 L of synthetic fresh urine was prepared. For the pH monitoring during urea hydrolysis, conductivity monitoring during urea hydrolysis, and chemical addition inhibition experiments, 2 L of synthetic urine was prepared. For each experiment, about 1400 mL of fresh urine was obtained.
The ammonia probe was calibrated to six calibration points, 5, 425, 825, 1700, 5000, and 8700 mg/L as NH3, following the detailed instructions found in the probe manual. The 5, 425, 825, and 1700 mg/L calibration standards were prepared using the 0.1 M ammonium chloride standard (CAS 13-641-923, Fisher Scientific) and the 5000 and 8700 mg/L calibration standards were prepared using ammonium chloride (CAS A649-500, Fisher Scientific). 0.0346 (±0.0002) g of sodium chloride (CAS S641-500, Fisher Scientific) was added to the 5, 425, 825, and 1700 mg/L calibration standards. 0.8646 (±0.0002) g of sodium chloride was added to the 5000 mg/L calibration standard and 0.8638 (±0.0002) g was added to 8700 mg/L calibration standard. The sodium chloride was added to the calibrations standards following the instructions in the ammonia probe manual. The calibration standards should have an ionic strength similar to solution being measured from, which in this case was urine. After the probe was calibrated, the accuracy was checked by measuring a 10 mg/L sample made from the 1000 mg/L ammonia as N standard (CAS 13-641-924C). The ammonia concentration was measured following the detailed instructions in the ammonia probe manual. Table S2 were entered into the software. For the ionic zinc and ionic silver, the component concentrations were altered to match the altered synthetic urine used in certain experiments. The chemical inhibitor concentrations were then entered into the software. The software was run at both a fixed pH of 6 as well as with the pH being calculated from the mass balance. The software produced saturation indices for different minerals where a negative number represents an undersaturation and a positive number represents an oversaturation of the mineral.

Compound
Concentration (      For the zinc nitrate experiment, the additional sodium chloride concentration was determined by adding the corresponding chloride molar concentration of phosphate that was removed. For the silver nitrate experiment, all chloride compounds were replaced with corresponding nitrate compounds (i.e., sodium nitrate for sodium chloride). Data are mean ± one standard deviation for triplicate samples. Corresponding conductivity plots in Fig.  3.