Water for the experimental assays was collected from full-scale BDATs using recreated influent to mimic expected flush water, hand wash water, and personal hygiene water. The BDAT design and operation was previously described by Larsen et al. (2015); and the BAMBi was previously described by Künzle et al. (2015). The recreated influent contained 12.5 g feces, 25 mL urine, and 2.5 g hand soap (Consumerline Products, Kenya) in 1 L of tap water. This influent mixture was prepared every 3–4 days, refrigerated at 4°C and stirred at ~50 RPM. The feces and urine were collected from a urine-diverting dry toilet at Eawag. Feces were collected weekly, homogenized with a blender, and stored at −20°C for up to 60 days. Urine was stored at room temperature for up to 48 h. In the BAMBi tank, the recreated influent is mixed with recirculated, treated water from the storage tank at a ratio of 1:20.

The full-scale BDAT treatment included a 50-L BAMBi tank with nine panels of polyethersulfone ultrafiltration membrane UP150 (Microdyn Nadir, Wiesbaden, Germany) with a nominal cut-off of 150 kDa, seeded with 0.5 L of municipal wastewater sludge. Aeration was achieved by pumping air (0.2-m³ h⁻¹) through a perforated pipe underneath the membrane to maintain aerobic conditions on the exterior of the biofilm within the BAMBi tank. Water filtered through the BAMBI is then pumped into a 30-L clean water tank, which housed one or more of the post-treatment options: (1) GAC—to treat the remaining of organic matter and to reduce color (pore size of 0.6–1 mm, Norit Americas Inc., USA), (2) chlorination—to disinfect water using sodium hypochlorite (Fluka Chemie, Switzerland), and (3) electrolysis—to provide on-site chlorine for disinfection (Condias, Germany).

Water samples (20 L) were collected from the clean water tanks of two BDATs: one operating with only the BAMBi (after BAMBi), and one with the addition of a 6 L-GAC column after the BAMBi (after BAMBi+GAC). The samples were prepared followed a method modified from Vital et al. (2008) to remove indigenous microorganisms. Indigenous microorganisms were removed to preserve water quality at the time of collection so that a consistent source water could be used for all studies. Otherwise, storage of water with indigenous community intact would have led to water quality changes due to continued microbial activity and may have interfered with growth of target bacteria. Specifically, samples were filtered through a 1-μm pressurized filter (Geberit, Germany), pasteurized at 80°C for 60 min, and filtered again through a 0.2-μm membrane filter (Whatman, UK).

Notably, pretreatment did not substantially affect the concentration or characterization of DOC, as shown from chromatograms measured using an LC-OCD instrument for water samples before and after the procedure of filtration and pasteurization [Figure S1, Supporting Information (SI)]. All samples were processed within 6 h after collection and stored at 4°C until used. In this study, prepared water is referred to water after mentioned pasteurization and filtration steps. Characteristics of prepared water samples, including concentration of anions (NO2-, NO3-, PO43-, SO42-, Cl⁻) and cations (Ca²⁺, Mg²⁺, Na⁺, K⁺, Fe^3+/2+, Mn²⁺, Cu²⁺, Ba²⁺), were analyzed by the Engineering Analytical Laboratory at Eawag.

Four bacteria used in this study include two isolated indicators from wastewater [Escherichia coli (GenBank KU737538) and Enterococcus faecalis (GenBank KU737539)] and two pathogens [Pseudomonas aeruginosa PAO1 (ATCC 15692) and Salmonella typhimurium SB300 (provided by Dr. M. Suar from Institute of Microbiology, ETH Hönggerberg, Switzerland)]. The wastewater isolates were confirmed as E. coli and Ent. faecalis using 16sRNA sequencing (Microsynth, Switzerland). All experiments involved the four bacteria of interest were conducted under proper procedures in a Biosafety Level 2 laboratory at Eawag. Further details of sequencing and bacterial preparation are provided in the SI.

The experiments were set up following the pathogen growth potential assay of Vital et al. (2010). Target bacteria were grown overnight and seeded at an initial concentration of ~10³ cells mL⁻¹ with 20 ml prepared water samples (20 mL) into 40 mL carbon-free glass vials. Vials were then incubated at 30°C in 72 h in the dark to reach stationary phase. Samples (1 mL) were taken at the beginning, end and intermittently throughout to measure bacterial concentration using both flow cytometry and culture. Sterilized distilled water was included as negative controls. AOC concentration was defined by the growth of the indigenous community: 1 μg AOC L⁻¹ = 10⁷ cells L⁻¹ (Hammes and Egli, 2005).

Culture and flow cytometry (FCM) methods were used to measure concentrations of bacteria in water samples. Samples were analyzed immediately after collection. Further details are in SI.

GraphPad Prism 6.0.1 (GraphPad Software, USA) was used to perform statistical tests. Non-linear regression was performed to fit the sigmoid function to each bacterial growth curve and calculate growth coefficients. Comparison of net growth, growth coefficients, and water characteristics was conducted using either paired t-tests or two-way ANOVA. All statistical tests assumed an alpha of 0.05.

Water treated with the BAMBi had generally higher nutrient and metal ion concentrations than water treated with the BAMBi and GAC (Table 1). Specifically, DOC, PO43-, SO42-, and Cu²⁺ were statistically significantly lower in the BAMBi+GAC than the BAMBi (Student's t-test with Sidak correction, p < 0.05, Table 1). Although reductions were observed for pH, AOC, NO3-, Cl⁻, Na⁺, K⁺, Ca²⁺, and Mg²⁺, the reduction was not statistically significant (Student's t-test with Sidak correction, p > 0.05).

The indigenous microbial community, E. coli, and P. aeruginosa, grew in both waters during storage, whereas Ent. faecalis and S. typhimurium only grew in water after treated with BAMBi (Figure 1). The indigenous microbial community grew to a higher concentration in water after treatment with the BAMBi alone (Net growth_BAMBi = 1.3 × 10⁷ cells mL⁻¹) as compared to the BAMBi+GAC (4.0 × 10⁵ cells mL⁻¹, Figure 1). The indigenous community also grew faster in water after BAMBi (rBAMBi=0.1 h-1) than BAMBI+GAC (rGAC=0.08 h-1, Table S1). Similarly, the growth of both P. aeruginosa and E. coli in water treatment with BAMBi was higher than in water treated with BAMBi+GAC (56% more for P. aeruginosa, and 38% more for E. coli, Table S1). While P. aeruginosa had similar growth coefficient in both waters (Table S1), E. coli grew faster in water after BAMBi compared to in water after BAMBi+GAC (rBAMBiE. coli=10rGACE. coli).

There was a good agreement between TCC data and culture data for all four bacteria of interest in both water samples (two-way ANOVA, p > 0.05).

The reactivation/regrowth of E. coli and P. aeruginosa depended on the water and disinfectant characteristics (Figure 6). Bacterial reactivation occurred after exposed to low concentrations of chlorine without residual, as observed in previous studies (Jjemba et al., 2010; Li et al., 2013). E. coli and P. aeruginosa might enter the viable but non-culturable (VBNC) state under certain conditions during chlorination (Oliver, 2010), and therefore be able to reactivate.

No reactivation was observed after electrolysis, even in the presence of chlorine quencher. This result may be due, in part, to production of disinfectants (e.g., hydroxyl, nitrate radical, or phosphate radical besides chlorine that prevent formation of a reactivating VBNC state (Kerwick et al., 2005; Guitaya et al., 2015). We also suspect that the presence of reactive oxidants may change carbon structure and thereby remove available carbon required for regrowth.

As P. aeruginosa was shown to grow after being spiked into disinfected water without residual chlorine, pathogen contamination is potentially a high risk that can affect the system hygienic performance besides regrowth/reactivation. During storage, pathogen contamination can happen via multiple events, including malfunction of one or more treatment processes, access to storage tank by hosts that contain pathogens (e.g., aquatic organisms, insects, birds, rodents, contaminated human hands). Therefore, to ensure the safety of reusing treated fecal-contaminated water, it is important to manage the access of the storage tank carefully, maintain a cleaning procedure for the tank regularly, and have a proper residual of disinfectant constantly.

In this study, we investigated inactivation and reactivation/regrowth of E. coli and P. aeruginosa in the absence of an indigenous community. The indigenous microbiota were removed to prevent microbial degradation of source water during storage and to allow quantification of bacteria using culture-based methods and flow cytometry simultaneously. The indigenous community may influence inactivation and/or reactivation/regrowth rates of bacteria. For example, in studies of chlorination of both reclaimed and drinking water, inactivation using chlorine has been shown to result in tailing of intact cell counts of the indigenous microbial community (Ramseier et al., 2011; Li et al., 2013). This phenomenon has been attributed to a resistant sub-population and/or shielding through bacterial aggregation or particle adsorption (Ramseier et al., 2011). However, Li et al. (2013) demonstrated inactivation of in situ total coliforms, enterococci, and Salmonella spp. in reclaimed water at rates similar to those observed here. We therefore expect similar pathogen inactivation and reactivation/regrowth kinetics in studies including the indigenous communities, but further research is warranted to conclusively demonstrate this.

In general, conclusions from culture and FCM methods aligned for experiments measuring bacterial growth, but differed for inactivation experiments. The discrepancy between culture and FCM methods observed for inactivation was likely because ICC measures intact cell membranes for bacteria that may not be culturable. The ICC measurement relies on cell staining with Propidium Iodide which only stains cells with damaged membranes (Berney et al., 2007). Some dead or unculturable cells may have intact membranes, so ICC data is considered more conservative (i.e., overestimate concentrations of surviving cells) than culture (Joux and Lebaron, 2000; Bosshard et al., 2010).

Concerns related to pathogen growth in drinking water and reclaimed water have been raised in previous studies (van der Kooij, 2003; Oesterholt et al., 2007; Jjemba et al., 2010; Weinrich et al., 2010). In this study, we demonstrated that the growth of pathogens in water stored for reuse in an onsite sanitation technology (i.e., the BDAT) is a potential concern and, therefore, increases health risks. A bacterial indicator (E. coli) and an opportunistic pathogen (P. aeruginosa) were able to grow in water stored for reuse after multiple, different effective treatment processes. Water characteristics, including nutrient concentrations and indigenous bacterial communities, were shown to have strong effects on the bacterial growth. Additionally, we demonstrated the complex and non-uniform influence of indigenous communities on the growth of pathogens.

We also demonstrated that the choice of treatment processes was a key factor influencing bacterial growth. The presence of residual disinfectants (e.g., chlorine and/or other reactive active species) contributed to water biostability. Among three disinfection options, electrolysis appeared to be the most effective method to inhibit pathogen growth, followed by chlorination with high chlorine concentrations (e.g., >0.5 mg Cl₂ L⁻¹ for E. coli and >1.7 mg Cl₂ L⁻¹ for P. aeruginosa).

There were notable limitations to this study. First, we only tested growth, inactivation, and reactivation of four bacteria of interest. There is heterogeneity in both growth and inactivation coefficients of the four tested bacterial species, so future research is likely needed to investigate other pathogens (i.e., Legionalla spp.) to ensure safety of water reuse.

Second, the study focused on the growth and inactivation of bacteria in the planktonic phase. Biofilms in storage tanks, like the clean water tank where water is stored for reuse in the BDAT, likely also influence growth and inactivation of bacteria. As aggregates of microbial cells attach to surfaces, biofilm members have advantages of being protected from disinfectants and predators compared to planktonic cells (LeChevallier et al., 1988; Flemming and Wingender, 2010). In addition, the matrix of hydrated extracellular polymeric substances (EPS) helps biofilm members consume complex substrates (e.g., humic acids) as food, which are not bioavailable for planktonic bacteria (Fischer, 2003; Flemming and Wingender, 2010). More research is needed to understand the growth and inactivation of bacteria in biofilms in water stored for reuse, especially in the context of the BDAT.

Third, there is a discrepancy between conditions in the laboratory and in the real BDAT system. In our laboratory setup, all experiments were conducted in batch reactors, in which the concentration of nutrients, residual disinfectant, and bacteria drastically changes in the course of the experiments in 72 h. These conditions are different to those in the BDAT, where water after BAMBi is frequently added into the clean water tank and treated water is frequently removed upon every usage. The continuous flow conditions in the BDAT create a certain level of consistency in the bulk-phase concentration of nutrients and bacteria for prolonged periods of time. The concentration of residual disinfectant in the clean water tank of the BDAT is also kept constant with a regular dosing interval. Another difference between laboratory setup and real-life conditions is that the indigenous community is constantly present in the BDAT system whereas it was removed from the water samples for the subset of laboratory experiments on inactivation and reactivation/regrowth. The indigenous community in the BDAT may impact the inactivation and reactivation/regrowth of pathogens, for example by shielding and/or providing. Results learnt from inactivation experiments, for example the disinfectant demand for a sufficient pathogen removal, likely need to be adapted in the presence of the indigenous community in the BDAT conditions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.