Comparative evaluation of disinfection mechanism of sodium hypochlorite, chlorine dioxide and electroactivated water on Enterococcus faecalis
Introduction
Disinfection is a process of deactivation of pathogenic or non-pathogenic microorganisms and chemical disinfection is mainly used for this purpose. Chlorine-based technologies are favorable disinfection methods; chlorine gas, hypochlorite solution and other chlorine compounds in liquid and solid form are available as chemical agents (EPA, 1999). Sodium hypochlorite (NaOCl), which is the major compound of bleach, is an effective antiseptic substance against microorganisms (McDonnell & Russell, 1999). Chlorine dioxide (ClO2) includes both oxygen and chlorine as oxidizing agents; thus, it is highly effective against bacteria, virus (Morino, Fukuda, Miura, & Shibata, 2011), fungi (Burton, Adhikari, Iossifova, Grinshpun, & Reponen, 2008) and even against spores (Young & Setlow, 2003). Chlorine dioxide has a wide range of application areas such as wastewater and/or drinking water treatment, food and beverage processing (Jonnalagadda & Nadupalli, 2014).
Electro activated water (EAW) is produced by electrolysis of a saline solution passing through the electrolysis chamber. When electric current is applied to the solution, electron exchange takes place between electrodes and water by means of a unipolar electrochemical process. Physical-chemical character of water changes due to the formation of polarization energy on electrodes during the electrolysis process. Inside the electrolysis chamber, negative and positive ions are separated; positive charges are accumulated at the anode surface while negative charges gather at the cathode surface. Consequently, two new products called catholyte and anolyte (namely EAW) are formed in the aqueous solution. Electro activated solution of anolyte has pH values from 7 to 1 and ORP from 0 to +1250 mV, while the catholyte solution has pH values from 7 units to 14 units and ORP from 0 to −950 mV. Anolyte is an aqueous solution with anomalously enhanced electro-acceptor properties. Thus, under unipolar electroprocessing of liquid, reduction products with alkaline reaction are being formed in a diaphragm cell in the cathode zone, and in the anodic zone, there are highly oxidative products with acid reaction.
Electro activated water is composed of oxygen gas and many active species such as chlorine, chlorine dioxide and free radicals, while catholyte contains hydrogen gas and sodium hydroxide (Koseki & Itoh, 2000; Kumon, 1997). Due to the presence of a sufficient number of strong oxidants and free radicals, EAW is a solution that has strongly biocidal properties (Hao et al., 2012). Several studies have presented inhibition capacity of EAW on vegetative and spore forms of various bacteria (Hao, Wu, Li, & Liu, 2016; Huang, Hung, Hsu, Huang, & Hwang, 2008; Rahman, Ding, & Oh, 2010; Udompijitkul, Daeschel, & Zhao, 2007). The mechanism of inhibition by EAW has also been evaluated on different bacterial cells so far (Table 1).
As stated before, the EAW contains more than one active specie, unlike chlorine and chlorine dioxide. The reason for these three disinfectants to cause different effects may be the diversity of components in the EAW content and their mode of action in the bacterial inactivation process. Therefore, the cell inactivation mechanism of EAW is thought to be different from other disinfectants. Although HOCl is the most abundant in the content of EAW, there are also ozone and free radicals increasing its ORP, which are responsible for the cell membrane degradation (Ding et al., 2016). Hypochlorous acid is present in both NaOCl and EAW as an oxidant, and it is responsible for the protein deterioration. Hypochlorous acid destroys certain enzymes participating in carbohydrate metabolism by oxidizing their sulfhydryl groups (Kim, Hung, & Brackett, 2000; Rahman et al., 2010).
The fact that the EAW has different physicochemical properties than chlorine, an interest has arisen in determining the differences in the inactivation mechanism. E. faecalis was selected as a test organism in this study to assess the disinfection mechanisms of three chlorine-based disinfectants since E. faecalis is an opportunistic pathogen and it can easily become dangerous and infectious for the human body (Rashid et al., 2017). Our previous study (Turkay et al., 2017) on E. faecalis inactivation presented only preliminarily results of inactivation mechanism of EAW. Indeed, E. faecalis was inactivated by the release of protein and intracellular components following cell membrane degradation. However, in order to propose the use of EAW as an alternative to presently used NaOCl and ClO2, it is more appropriate to assess the inactivation effects comparatively and to discuss the results based on the differences in mechanisms of action. Therefore, in this study, sublethal concentrations of chlorine-based disinfectants (i.e. EAW, NaOCl and ClO2) were applied to the pure culture of E. faecalis and a deeper investigation on the inactivation mechanism of EAW was conducted by using various methods. Cell viability determination by flow cytometry and TTC-dehydrogenase enzyme activity assay, investigation of cell membrane integrity by measurement of DNA and protein leakage, conductivity and lipid peroxidation levels, and examination of cellular components by the SDS-PAGE and FTIR analysis were conducted to clarify this effect.
Section snippets
Preparation of disinfectants
NaOCl (6–14% active chlorine) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution was prepared with an estimated concentration and diluted to final concentration maximum 10 mg of free chlorine (i.e. sum of hypochlorous acid and hypochlorite ion concentrations) content. A back flow calculation was made to prepare desired concentration of free chlorine. Electroactivated water was produced in a divided electrochemical reactor that contains a graphite anode, steel cathode and
Effect of chlorine-based disinfectants on bacterial survival
To calculate the dead cell ratio of the samples, flow cytometry analysis was conducted. Whereas EAW achieved almost complete degradation of bacteria (95.4%) within 1 min (Fig. 1a), other disinfectant agents provided almost 70% of bacteria inactivation after 20 min. Bacterial inactivation capacity of EAW treatment in all tested times was significantly higher than that of NaOCl and ClO2 (p < 0.001). Only 5 min treatment of ClO2 inactivated significantly more E. faecalis cells than that of NaOCl (p
Conclusion
In this study, chlorine-based disinfectant agents were applied to the pure culture of E. faecalis and the inactivation mechanisms of disinfectants were evaluated upon various test methods. Typically, all disinfectants caused cell death in 20 min with different inactivation efficiencies. However, EAW was determined as the most robust chlorine-based disinfectant, since it inactivated the bacteria in a shorter time with greater damages to bacterial cells. Because of that the molecular structure of
Declarations of interest
None.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We would like to thank the referees for their constructive comments on the draft document.
References (35)
- et al.
A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins
International Journal of Food Microbiology
(2001) - et al.
Interactions between dendrimer biocides and bacterial membranes
Biomaterials
(2002) - et al.
Mechanisms of Escherichia coli inactivation by several disinfectants
Water Research
(2010) - et al.
A Review of the Mechanisms and Modeling of Photocatalytic Disinfection
Applied Catalysis B: Environmental
(2010) - et al.
Disinfection efficacy and mechanism of slightly acidic electrolyzed water on Staphylococcus aureus in pure culture
Food Control
(2016) - et al.
Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes
Free Radical Biology and Medicine
(1991) - et al.
Roles of hydroxyl radicals in electrolyzed oxidizing water (EOW) for the inactivation of Escherichia coli
International Journal of Food Microbiology
(2012) - et al.
Application of electrolyzed water in the food industry
Food Control
(2008) - et al.
Efficacy of electrolyzed oxidizing (EO) and chemically modified water on different types of foodborne pathogens
International Journal of Food Microbiology
(2000) - et al.
Modeling of the inactivation of Salmonella typhimurium by supercritical carbon dioxide in physiological saline and phosphate-buffered saline
Journal of Microbiological Methods
(2007)
Effectiveness of low concentration electrolyzed water to inactivate foodborne pathogens under different environmental conditions
International Journal of Food Microbiology
Kinetics of membrane damage to high (HNA) and low (LNA) nucleic acid bacterial clusters in drinking water by ozone, chlorine, chlorine dioxide, monochloramine, ferrate (VI), and permanganate
Water Research
Disinfection of E. coli by the Ag-TiO2/UV system: lipid peroxidation
Journal of Photochemistry and Photobiology A: Chemistry
Effect of Gaseous Chlorine Dioxide on Indoor Microbial Contaminants
Journal of the Air & Waste Management Association
Oxidative stress in bacteria and protein damage by reactive oxygen species
International Microbiology
Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria
Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
Response surface modeling of Listeria monocytogenes inactivation on lettuce treated with electrolyzed oxidizing water
Journal of Food Process Engineering
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