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

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. 2013; O'Neal & Boyer 2013). Given that urine contains available nutrients for plant growth, including nitrogen, phosphorus, and potassium (Lind et al. 2001; Karak & Bhattacharyya 2011), urine is a good source of green (Akpan-Idiok et al. 2012) and multinutrient fertilizer (Karak & Bhattacharyya 2011), as well as providing a range of environmental benefits (Tidåker et al. 2007). Several technologies on the laboratory or industrial level have been utilized to recover nutrients from urine for agricultural purposes (Pronk & Koné 2009). These technologies include struvite recovery via precipitation or electricity generation (Sakthivel et al. 2012; You et al. 2014), nitrification and distillation (Udert & Wachter 2012), and stripping and absorption (Başakçilardan-Kabakci et al. 2007; Zhang et al. 2015). The direct application of urine as fertilizer after proper storage is favored in rural and suburban areas.

Urea is biochemically hydrolyzed into ammonium ⁠, bicarbonate ⁠, and hydroxyl (OH⁻), thus increasing pH and ammonium concentration (Vinnerås et al. 2006; Jonsson & Vinnerås 2007) and exerting lethal effects on microorganisms. Therefore, pH, storage duration, and temperature are the major factors that influence the storage process of human urine. Moreover, temperature is a crucial parameter that influences microbial inactivation rates (Maurer et al. 2006). High pH, high temperature, and long storage periods are required to produce safe and hygienic liquid fertilizer (Magri et al. 2015; Hu et al. 2016). Therefore, guidelines from the World Health Organization (WHO 2006) recommend a storage period of 6 months at 20 °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. 2006). Ammonia volatilization not only decreases the efficiency of nitrogen recovery but also adversely affects environmental and human health (Galloway & Cowling 2002). 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. 2001), the utilization of frozen urine is problematic. Acidification or chemical oxidation (Hellström et al. 1999; Zhang et al. 2013) can inhibit urea decomposition; however, adding reagents to the urine is not an ideal solution. Temperature considerably affects urine storage and benefits pathogen elimination. For example, increasing storage temperature from 24 °C to 34 °C considerably decreases the number of enteric pathogens in diluted urine (Höglund et al. 1998; Vinnerås et al. 2008). In addition, bacteria, such as Salmonella typhimurium, Streptococcus faecalis, and Escherichia coli, are inactivated at 65 °C within 4 min (Fjendbo et al. 1998). Meanwhile, Enterococcus faecalis and E. coli are eliminated within 20 s under 65 °C (Spinks et al. 2006). Moreover, solar pasteurization at 55 °C decreases E. coli counts below the detectable limit (Dobrowsky et al. 2015). Thus, utilizing the thermal effect on pathogen inactivation is an interesting alternative for human urine storage. Similar works on the disinfection of gray water, rainwater, and secondary effluent disinfection by solar energy showed that thermal disinfection is efficient (Amin et al. 2014; Giannakis et al. 2014; Lee et al. 2016). Temperatures above 50 °C significantly affect the chemical hydrolysis of urea (Frankenberger & Tabatabai 1982). Urea hydrolysis is catalyzed by the urease produced by urease-producing bacteria (UPB); the optimal temperature of urease activity is normally 65 °C (Hagenkamp-Korth et al. 2015). High temperatures, however, can reduce urea hydrolysis by inhibiting UPB growth. Therefore, the thermal storage of urine has potentially high sanitizing efficiency while minimizing urea hydrolysis.

Given that these advantageous properties have not been discussed in the presented literature, this study aims to investigate the thermal storage of human urine at 60 °C and 70 °C. It also aims to determine the effects of storage temperature on pathogen inactivation and urea decomposition. E. coli and fecal coliforms were used as the indicators of bacterial inactivation, whereas pH and ammonia were used as the main indicators of urea hydrolysis.

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.

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 calorimetrically with a DR/600 spectrophotometer (HACH, Co., USA) at 420 nm. All assays were performed in triplicate. Results were presented as averaged values.

There is currently no consensus on which organism(s) is the most useful hygiene indicator. Furthermore, there is no national regulation that mandates a single standard for urine reuse. According to the WHO Guidelines for the Safe Use of Wastewater, Excreta, and Greywater (WHO 2006), one or more of the total coliform, fecal coliform, and E. coli bacteria are used as wastewater effluent or reuse standards. Fecal coliforms originate from feces and are indicators of possible disease transmission (Fuhrmeister et al. 2015); thus, fecal coliforms are often used as primary bacterial indicators. Moreover, the United States Environmental Protection Agency recommended E. coli and Enterococci as health risk indicators for water. Thus, fecal coliforms and E. coli were used as the core indicators in this study.

Initially, fecal coliform and E. coli in the urine samples were analyzed daily at the same sampling time. The sampling interval was adjusted during the later periods of storage. All the samples were diluted to a suitable concentration for parameter measurement.

The standard membrane filter method was used to quantify fecal coliform and E. coli (APHA 2012). An appropriate volume of urine sample was filtered through a 0.45-μm acetate cellulose filter. The filtered sample was placed in MFC broth and incubated at (44.5 ± 0.2) °C for 24 h. Blue colonies were counted to estimate the population of fecal coliforms. The number of fecal coliforms was presented as colony-forming unit per liter (CFU/L). To quantify E. coli, the filtered membrane was placed on Fuchsin basic sodium sulfite agar at 37 °C for 24 h and then transferred to NA-MUG agar for further incubation at 36 ± 1 °C for 4 h. Then, the bacteria were counted under a 366-nm ultraviolet lamp. Bacteria with blue fluorescence were counted to estimate the population of E. coli.

Figure 2 demonstrates that the inactivation of fecal coliforms in urine that was stored at ambient temperature was considerably slower than that in urine that was stored at high temperatures. In the former, the concentration of fecal coliforms decreased to undetectable levels on the 16th day of storage. The population of fecal coliforms in the urine samples that were stored at 60 °C and 70 °C decreased rapidly from 6 log to approximately 3 log on the 1st day of storage. The rate of population decline gradually stabilized and fecal coliform concentrations decreased to undetectable levels on the 8th and 6th days of storage. Similar results were obtained for E. coli. As shown in Figure 3, E. coli reached undetectable levels on the 3rd day of storage at 60 °C and 70 °C. By contrast, E. coli reached undetectable levels on the 5th day of storage at ambient temperature.

The thermally stored urine was cooled down to ambient temperature. Then, the microbial indicators were continuously tested to evaluate the long-lasting disinfecting effect of high temperature. The three bacterial indicators were not reactivated in the two urine samples, which indicated that thermal storage has a stable disinfection effect. The experimental results showed that compared with the ambient temperature, high temperatures accelerate disinfection.

Fecal coliforms and E. coli in the two urine samples were completely inactivated within 2 days of storage. Fecal coliforms and E. coli decreased to undetectable levels after the first storage day, particularly in duplicate-1. For urea hydrolysis, little variation was found in the pH and ammonia/ammonium concentration of the two urine samples during the 7 days of storage. These results further supported the efficiency and reproducibility of the previous experimental results. The pH values of the two urine samples on the 7th day of storage were 7.84 and 7.89, whereas the ammonia concentrations were 749.39 and 778.46 mg/L, i.e., only a slight increase was observed. Moreover, disinfection and stability were retained even after storing the urine under ambient temperature, as evidenced by the lack of significant changes in the microbial, physical, and chemical characteristics of the samples.

Thermal treatment is a potential alternative technology for community toilets with source-separated systems. In fecal sludge composting, applying high temperatures for a short duration may be as effective as applying low temperatures for a longer duration (Day et al. 2001). Given that similar operational parameters are suitable for urine storage, increasing temperature from the ambient to 70 °C during the thermal storage of urine can minimize storage tank volume. Thus, the thermal storage of urine is suitable for communities with limited space and is a potential, inexpensive solution for some solar energy-rich regions. Moreover, this system can be heated with renewable energy, such as biogas and ground source heat. The present study proved that the thermal treatment of human urine at 70 °C for 7 days for disinfection and stabilization can be achieved technically. However, a more comprehensive study on the tradeoff between energy costs, environmental impact/resource use from heating with traditional energy sources (compared with renewable energies), and efficient nutrient recovery from a waste stream should be discussed before the practical application of thermal storage for human urine.

In this study, the thermal treatment of urine at 60 °C and 70 °C was conducted and evaluated in terms of disinfection and stabilization. Storage at 70 °C for 7 days realized optimal pathogenic bacteria inactivation and urea stabilization. High storage temperature had multiple advantages for human urine treatment, including: (1) acceleration of pathogen inactivation; (2) inhibition of urea decomposition; and (3) minimization of storage tank volume. Urea hydrolysis was likely inhibited by the decreased UPB count because urease production became limited.

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.

The authors would like to acknowledge the financial support provided by the Bill and Melinda Gates Foundation. The authors would also like to thank the National Environment and Energy International Cooperation Base for their support. The first two authors contributed equally to the work.