The temperature-dependent kinetics and bacteria regrowth by performic acid and sodium hypochlorite disinfection

Sodium hypochlorite (NaOCl) has been the most widely used disinfectant in water and wastewater, as well as in food industries, due to its convenient handling and storage (compared to chlorine gas), high efficiency, and low cost (U.S. EPA 1999; Fukuzaki 2006). However, there are several disadvantages associated with NaOCl disinfection, such as the possibilities of producing toxic chlorine gas, decreased efficiency in the presence of organic loads, and deleterious effects on some metals. One of the most severe drawbacks is the formation of mutagenic and carcinogenic halogenated disinfection byproducts (DBPs), which may result in high toxicity to humans and animals (Monarca et al. 2000; Crebelli et al. 2005).

Recently, the long-term presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on cold-chain food packaging has been shown (Chi et al. 2021), and the concern of disinfection at low temperatures has thus raised great research interest. NaOCl was once believed to be a very effective disinfectant against Bacillus subtilis (B. subtilis) spores at low temperatures, even at subzero with the addition of ethylene glycol to prevent freezing (Jones et al. 1968). However, a slight effect on HOCl dissociation was related to temperature reduction (Gray 2014). In the aqueous solution, HOCl, OCl⁻, and a small portion of Cl₂ were in equilibrium. Both HOCl and OCl⁻ have disinfection abilities, but the former has a much higher ability to penetrate cell walls and membranes (McDonnell & Russell 1999). It has also been reported that the log reduction of Escherichia coli (E. coli) and Enterococcus spp. inactivated by NaOCl was lower at 4 than at 20 °C, and the log reduction could not be increased by extending the contact time to 4 °C (Hassaballah et al. 2020).

Performic acid (PFA) belongs to the family of aliphatic peracids. It has the highest oxidation potential among the peracids, thus it is industrially relevant, such as in food processing and fine chemical production industries (Luukkonen & Pehkonen 2017). PFA can be prepared by mixing formic acid and hydrogen peroxide, with or without a catalyst. It has always been prepared on-site due to its unstable characteristics and safety concerns, and the temperature for storage was recommended below 20 °C (Gehr et al. 2009). PFA has been reported to efficiently inactivate some enteric bacteria, such as E. coli, Campylobacter jejuni, Listeria monocytogenes, Salmonella typhimurium, and Salmonella enteritidis at 2.5 °C, thus it could be used as a disinfectant in low-temperature food processing and storage rooms (Heinonen-Tanski & Miettinen 2010).

A study has shown that no brominated DBPs were formed in a full-scale PFA disinfection experiment, other than a stoichiometric increase of formic acid (Ragazzo et al. 2013). Therefore, the lower likelihood of producing DBPs than chlorine has made PFA a disinfectant of great interest in water and wastewater industries (Luukkonen & Pehkonen 2017). PFA was able to successfully inactivate pathogens including E. coli, fecal coliforms, Enterococci, intestinal Enterococci, Aeromonas spp., Pseudomonas aeruginosa, Staphylococcus aureus (S. aureus), somatic coliphages, and murine norovirus in combined sewer overflows and treated wastewater (Chhetri et al. 2014; Tondera et al. 2016; Maffettone et al. 2020; Ding et al. 2023), whereas the inactivation of resistant microorganisms such as Bacillus subtilis (B. subtilis), Clostridium, and Giardia in the secondary effluents required much higher doses than the above-mentioned pathogens (Karpova et al. 2013; Ragazzo et al. 2013; Luukkonen et al. 2015; Ding et al. 2023). Although showing effectiveness, most laboratory-scale PFA disinfection experiments were conducted at room temperature, and the effect of temperature on pathogen inactivation and regrowth has never been analyzed. The significance of this study was to evaluate PFA as a potential alternate disinfectant for water and wastewater industries, especially in a cold environment. In this study, the common pathogenic bacteria E. coli and S. aureus were selected as representatives of Gram-negative and Gram-positive bacteria, respectively, and B. subtilis as a resistant microorganism, to evaluate the disinfection efficacy of PFA.

The objectives of this study were to (a) establish disinfection kinetics of NaOCl and PFA at 4 and 20 °C, and (b) analyze the effect of temperature on bacteria regrowth after disinfection.

NaOCl solution (10%) was purchased from Fuchen Tianjin Chemical Reagent Factory. The residual chlorine after disinfection was tested using a residual chlorine detector (DR 300, Hach, USA). PFA was synthesized before each experiment by mixing formic acid and hydrogen peroxide (H₂O₂), with the addition of sulfuric acid as the catalyst. The concentration of PFA was detected by titration. Detailed procedures refer to Ding et al. (2023).

The lyophilized powder of E. coli (BNCC 133264), S. aureus (BNCC 186335), and B. subtilis (BNCC 109047) was dissolved in beef peptone solution medium in an ultra-clean bench to form the bacterial broth. E. coli and S. aureus were incubated at 37 °C for 24 h, and B. subtilis for 48 h. The bacteria were passed for at least three generations before the disinfection experiment. B. subtilis spores were detected under a microscope via staining, and a percentage of 20–40% sporulation was observed. The cultured bacterial suspension was centrifuged at 10,000 r/min for 10 min, and the precipitate was re-suspended in the sterilized phosphate-buffered saline (PBS) by a vortexer (VM-300, Qunan, China), resulting in approximately 10⁸–10⁹ colony forming unit (CFU)/mL.

The disinfection experiment was conducted by adding 1 mL bacterial suspension into 500 mL deionized water (pH 7.09 ± 0.01) in a 1-L conical flask. The flask was either submerged in ice water (4 ± 1 °C) or directly mounted on top of a magnetic stirrer at room temperature (controlled at 20 ± 1 °C). Magnets were put into each reaction flask to ensure even mixing during the reaction.

The disinfection experiment was carried out for 1 h at 4 and 20 °C, followed by adding Na₂S₂O₃ to quench NaOCl, and Na₂S₂O₃ and peroxidase to neutralize PFA, as described in Section 2.3. The water samples disinfected at 20 °C were stored at 20 °C, and those disinfected at 4 °C were stored at 4 and 20 °C, both in the dark and under ambient light. Water samples were withdrawn at 12, 24, and 72 h during the storage, and sent for cell culture for bacterial regrowth analysis, as described in Section 2.2. All analyses were conducted in triplicate.

The calculated CTs based on the optimal model at each condition are listed in Table 1. At an initial concentration of 0.3 mg/L, NaOCl was extremely efficient at inactivating E. coli at both 4 and 20 °C, with CTs of 0.1372 and 0.1588 mg/L·min to achieve a 4-log inactivation, respectively. PFA required much higher CTs than NaOCl to achieve the same inactivation of E. coli and S. aureus. However, with a higher initial concentration of 7.5 mg/L, PFA was more efficient on B. subtilis than NaOCl, and required 2–4 times lower CTs to achieve 4-log inactivation. A slightly lower effectiveness of PFA toward E. coli and S. aureus in the secondary effluent was also observed in one of our previous studies (Ding et al. 2023). Ragazzo et al. (2013) also reported that at a CT of 10–15 mg·L⁻¹·min, NaOCl disinfection resulted in higher removal of enterococci in the secondary effluent than PFA at an initial dose of 1 mg/L. Nevertheless, by increasing the contact time or initial disinfection dose, the effectiveness of PFA against enterococci reached the same level as NaOCl, and that against E. coli exceeded that of NaOCl (Ragazzo et al. 2013; Ragazzo et al. 2020).

According to the literature, there are basically three mechanisms involved in bacteria regrowth after disinfection, which are reproduction, repair, and reactivation (Wang et al. 2021). Reproduction refers to the viable bacteria that maintain the inherent reproducibility with intact bacteria cells, which can be interpreted as non-inactivated bacteria. Repair includes dark-repair and photo-reactivation of damaged DNA by photo-irradiation (Sinha & Häder 2002; Kraft et al. 2011). The mechanism of reactivation mostly refers to the reactivation of viable but non-culturable (VBNC) state bacteria (Chen et al. 2018). VBNC state is an adaptive strategy for the survival of bacteria under stress. They may retain the ability to reactivate and regrow when external stress disappears (Ayrapetyan et al. 2018). The chemical disinfectants used in this study aimed at attacking or permeating cell walls and membranes, but not specifically damaging the DNA of the bacteria, thus the bacteria repair mechanism involving photo-reactivation and dark-repair might not apply, as shown in this study that there was no specific trend showing bacteria regrowth under light or in dark (Figures 3–5). Possibly, the regrowth was induced by the reactivation of VBNC bacteria.

Previous studies have reported that the regrowth of bacteria after disinfection was caused by the bacteria in the VBNC state. Over 99.95% E. coli was in the VBNC state when chlorinated at 0.2 mg/L for 30 min (Wang et al. 2022), and a slightly higher dose (0.5 mg/L) also could not fully inactivate E. coli but reduced its culturability to the VBNC state (Lin et al. 2017). Similarly in this study, inactivation by an initial NaOCl concentration of 0.3 mg/L for 1 h, the regrowth of E. coli was prominent. Other than chlorination, disinfection treatments using chloramination or UV irradiation also induced the VBNC state of bacteria (Zhang et al. 2015; Chen et al. 2018). In our previous study, we found that the inactivation of E. coli, S. aureus, and B. subtilis detected by cell culture-based plate counting and flow cytometry were significantly different, indicating the presence of VBNC state bacteria after disinfection (Ding et al. 2023). However, the regrowth of PFA-inactivated bacteria was not as evident as NaOCl disinfected bacteria, as shown in this study, and the reason might be related to the inactivation mechanisms of the disinfectants.

HOCl is the predominant reactant accounting for NaOCl disinfection. It has strong permeability to cell walls and membranes (McDonnell & Russell 1999); however, it shows a lower redox potential than PFA (Zhang et al. 2018). Therefore, although showing a much higher inactivation by cell culture-based detection, the regrowth of bacteria after NaOCl disinfection was more prominent than that after PFA disinfection (Figures 3–5). Peracetic acid (PAA), another widely used peracid, has also shown more effective inactivation of E. coli than NaOCl, with lower induction of VBNC state bacteria (Teixeira et al. 2020). In this study, the inactivation rate of NaOCl and PFA on the three tested bacteria was higher at 20 °C than at 4 °C (Figures 1 and 2). Temperature has been proved to positively correlate with bacteria inactivation by chlorine and its derivatives, and PAA (Stampi et al. 2001; Fukuzaki 2006).

However, there have been controversies on the effect of temperature on bacteria regrowth. In this study, the regrowth of the bacteria inactivated by NaOCl at 4 °C and stored at 4 °C was more prominent than those stored at 20 °C. It was believed that adverse conditions such as low temperature are inducers of bacteria entering the VBNC state (Arana et al. 2010). The low temperature (<10 °C) and low nutrient conditions may induce a set of specific proteins to tune cell metabolism and readjust to the new conditions (Barria et al. 2013). While storing at low temperatures, the metabolism of bacteria has been restricted to the lowest level, along with the delayed cell damage (Orruno et al. 2017). On the other hand, when maintained at 20 °C, E. coli populations were more prone to damage (Arana et al. 2010). Wu et al. also demonstrated that in an artificial seawater sample, live Vibrio cholerae decreased from 10⁸ CFU/mL to 10⁶ and 10⁵ when stored at 22 and 37 °C, respectively, while all of those maintained at 4 °C entered into the VBNC state (Wu et al. 2016) and remained alive. In contrast, other studies reported that low temperatures hindered bacteria regrowth after disinfection (Giannakis et al. 2014). However, this finding was primarily substantiated by bacterial regrowth after UV disinfection (Wang et al. 2021); and the temperature was considered an influential factor of photo-reactivation (Lindenauer & Darby 1994).

Compared with NaOCl, PFA showed a superior ability in the suppression of bacteria growth. Previous studies also reported that bacteria regrowth happened less frequently when treated by peracids. Hassaballah et al. (2020) inactivated E. coli and Enterococcus spp. in the secondary effluent by PAA at 2 mg/L for 24 h; no bacteria regrowth was observed thereafter. In addition, the authors also reported that for E. coli inactivation at 4 °C, increasing the contact time from 10 to 30 min had no significant effect on NaOCl but enhanced PAA disinfection. With a higher redox potential than PAA, PFA at 0.8 mg/L and a contact time of 10 min allowed for stable disinfection of E. coli and intestinal Enterococci, with no growth in the dark after 24 h (Pigot 2021), suggesting PFA irreversibly reacted with cell membrane components, most likely by means of chemical reaction with the disulfide and sulfide components in the protein residuals and unsaturated fatty acids (Voet & Voet 1995).

According to the optimal disinfection models of E. coli, S. aureus, and B. subtilis inactivated by NaOCl and PFA, respectively, E. coli and S. aureus were extremely susceptible to NaOCl, whereas PFA seems to be more effective on B. subtilis. For both disinfectants, inactivation was more effective at 20 than 4 °C, detected by cell culture-based plate counting. Although NaOCl showed a higher efficacy on E. coli and S. aureus inactivation, bacteria regrowth was prominent, especially for those inactivated at 4 °C and stored at 4 °C, possibly through the mechanism of reactivation of VBNC bacteria. PFA was superior on suppressing bacteria regrowth, which suggested that it could be used as a wastewater disinfectant in a cold environment.

This work was supported by the National Natural Science Foundation of China (52100071).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.