Investigation on microbial inactivation and urea decomposition in human urine during thermal storage

The World Health Organization suggests storing human urine for at least 6 months at 20°C prior to application as fertilizer to reduce the potential health risks from pathogenic organisms. Such a storage condition for human urine,however,not only requires along period of time and large space but also ignores the risk of nitrogen losses. In this study, human urine underwent thermal treatment during storage to improve disinfection and to inhibit urea hydrolysis. Microbial indicators such as Escherichia coli and fecal coliforms and the concentration of ammonia/ammonium were investigated in urine samples that were stored at 60°C and 70°C. Both the inactivation of indicators and decomposition of urea improved under storage temperatures of 60°C and 70°C compared with storage under ambient temperature. Therefore, human urine is recommended to be stored at 70°C for 7 days for hygienic and stabilization purposes. Under this storage condition, pH is maintained below 8.0 and ammonia/ammonium content is maintained at approximately 800 mg/L.


INTRODUCTION
The concept of source-separated human feces and urine collection to promote sustainable sanitation solutions from the local to the global level has gained increased attention in recent decades (Boh et al. ; O'Neal & Boyer ). Given that urine contains available nutrients for plant growth, including nitrogen, phosphorus, and potassium (Lind et al. ; Karak & Bhattacharyya ), urine is a good source of green (Akpan-Idiok et al. ) and multinutrient fertilizer (Karak & Bhattacharyya ), as well as providing a range of environmental benefits (Tidåker et al. ). Several technologies on the laboratory or industrial level have been utilized to recover nutrients from urine for agricultural purposes (Pronk & Koné process of human urine. Moreover, temperature is a crucial parameter that influences microbial inactivation rates (Maurer et al. ). High pH, high temperature, and long storage periods are required to produce safe and hygienic liquid fertilizer (Magri et al. ; Hu et al. ). Therefore, guidelines from the World Health Organization (WHO ) recommend a storage period of 6 months at 20 W C or higher for the safe application of human urine on unrestricted crops. However, nitrogen loss via ammonia volatilization as a result of urea hydrolysis is another issue that should be addressed for the collection and transport of stored urine (Udert et al. ). Ammonia volatilization not only decreases the efficiency of nitrogen recovery but also adversely affects environmental and human health (Galloway & Cowling ). In addition, the storage time of 6 months requires a large volume of storage tanks and is not cost-effective.
Hence, it is necessary to develop an efficient method that minimizes the required volume of storage tanks for human urine storage, as well as promoting the disinfection and stability of stored human urine. Although freezing concentrates almost 80% of the nutrients in urine to 25% of the original volume (Lind et al. ), the utilization of frozen urine is problematic.

Experimental set-up
Fresh urine samples were stored in 30 sealed 50-mL glass bottles to avoid cross-contamination during sampling. All the bottles were disinfected and dried prior to filling. The storage temperatures of 60 W C and 70 W C were controlled with a water bath. The urine samples were then cooled down to ambient temperature after a reasonable storage time. Urine that was stored at ambient temperature was used as control. The actual ambient temperature was measured daily. Each experimental scenario was maintained for several days until the end of the experiment. The physical, chemical, and biological parameters of the urine samples were tested. Figure 1 presents the installation set-up of the experiment.

Urine samples
Fresh urine was collected from toilets at the School of Civil and Resource Engineering, University of Science and Technology, Beijing, China. To simulate a low-flushing urinal, the collected urine samples were diluted at a urine: distilled water ratio of 2:1 prior to distribution into experimental bottles. Given that urine was randomly collected in a plastic bucket from urinals without flushing water, the initial composition of fresh human urine varied from time to time. In this research, three experimental scenarios were investigated; thus, the initial characteristics of urine differed, as presented in Table 1.

Sample analysis
Chemical analysis pH was measured with a hand-held pH meter (HACH HQ30d, USA) and a corresponding electrode (pHC10101).
Urine in glass bottles was measured immediately after sampling. Ammonia/ammonium content was analyzed

Inactivation of bacteria
The overall storage time-dependence of the fecal coliform and E. coli concentrations in the three stored diluted urine samples is presented in Figures 2 and 3 coliforms and E. coli decreased rapidly within the first week in urine that was stored at 60 W C or 70 W C compared with urine that was stored at ambient temperature.   Under ambient storage conditions, the main process for disinfection is urea hydrolysis and high pH together with high ammonia/ammonium concentration, which are lethal to fecal coliforms and E. coli. In this experiment, the variation in pH and ammonia/ammonium concentration was correlated with the removal of fecal coliforms and E. coli. The thermal effect is responsible for pathogen inactivation in the thermally stored urine. Moreover, the high storage temperature caused the rapid removal of fecal coliforms and E. coli (Figures 2 and 3). Thermal storage has been hypothesized not only to enhance disinfection efficiency but also to prevent urea from hydrolyzing into ammonia/ammonium. Urea hydrolysis is catalyzed by urease from UPB. The thermal effect, however, inactivates UPB, thus inhibiting urease production during storage. In this study, the inactivation of bacteria was slower at 60 W C than at 70 W C; thus, the remaining bacterial concentration in the urine that was stored at 60 W C was higher than that of the urine that was stored at and UPB is less active. The reduction of urease concentration via UPB inactivation likely contributes more to the inhibition of urea hydrolysis. Therefore, considering disinfection effect and urea hydrolysis, storage at 70 W C for 7 days produces the optimal pathogenic bacteria inactivation and urea stabilization. The storage process is as described in Figure 6.

CONCLUSIONS
In this study, the thermal treatment of urine at 60 W C and 70 W C was conducted and evaluated in terms of disinfection Further research should be conducted before this storage system can be used for practical applications. Future studies should: (1) use more indicators for sanitation monitoring to broaden the spectrum of cross-sectional pathogenic inactivation efficiency to find an optimal storage temperature; (2) investigate the mechanism of microorganism inactivation during high-temperature storage; (3) compare the loss of different gaseous nitrogen forms from thermal storage with that from conventional storage; and (4) analyze energy balance and minimize energy consumption.