Keywords: drinking water; water disinfection; hypochlorous acid;

sodium hypochlorite; BacTerminator; pH; alkalinity

Introduction

Chlorination of water has been used for more than a century to kill microorganisms in water [1].
Chlorine in water is present in different species depending on pH of the water; dissolved Cl2
gas, hypochlorite and hypochlorous acid, where hypochlorous acid has proven to be the most
bactericidal chlorine species [2]. At pH 1-2 chlorous gas is the predominant chlorine species, at
pH 3-6 it is hypochlorous acid and at pH > 8 sodium hypochlorite is the major species present
[3]. Hypochlorous acid is also produced by human immune cells for defense against bacteria
[4]. Opposed to hypochlorite, hypochlorous acid is able to penetrate the bacterial cell
membrane, and thus severely damage the bacterial cell [5]. The down side to hypochlorous
acid as a disinfectant is its instability, making it difficult to store and use [6].
BacTerminator is used for water-disinfection by electrolysis, which effectively produces
hypochlorous acid and hypochlorite from dilute salt in the water. This disinfection method only
needs power to disinfect water hence there is no need of handling or storage of chemicals.
Tap water can vary in pH from day to day in a range from about 6.5 to 8.2 (our own
observations). Thus it is interesting to investigate how much pH of the water can affect the
common methods for water-disinfection. This study will focus on pH 7, 8 and 9. Escherichia coli
(E. coli) has been used widely as a model organism for water treatment methods, however E.
coli is in general easily and quickly killed by most disinfectants, hence it is difficult to distinguish
the effect of different disinfectants. In order to prolong the killing time and thereby enlarge
differences in kill efficiency, a very low chlorine level, 0.055 ppm, was used in this study.

Aim of study
The scope of this study was to investigate the impact of pH in contaminated water on chlorine-
based disinfection at very low chlorine level, and compare three chlorine-based treatment
methods; BacTerminator (electrolysis), sodium hypochlorite and hypochlorous acid.
Methods
Three types of test water were prepared from tap water by adjusting pH to 7, 8, and 9,
respectively, using 1 N HCl or 1 N NaOH. E. coli strain ATTC 25922 cultured in tryptone soya
broth overnight was added to the test water resulting in concentrations about 5∙105 CFU/mL to
5∙106 CFU/mL.
BacTerminator: Current and voltage were adjusted in order to produce 0.055 ppm chlorine,
which was measured from the outlet. Water with E. coli was run through the electrode chamber
and samples were taken from the outlet. Sodium thiosulfate was added to samples to
neutralize chlorine at time 0 min, 0.5 min, 1 min, 2 min, 5 min and 15 min (for pH 8 free
chlorine was also neutralized at time 3 min. and 4 min.).
Sodium hypochlorite: sodium hypochlorite (household cleaning) was diluted in demineralised
water and added to samples of water with E. coli resulting in a concentration of 0.055 ppm
chlorine in the sample. Sodium thiosulfate was added to samples as described above.
Hypochlorous acid: Hypochlorous acid was made by adjusting pH to 5 with 1 N HCl, in a
sodium hypochlorite solution. The hypochlorous acid was added in water samples with E. coli
resulting in 0.055 ppm chlorine in the sample. Sodium thiosulfate was added to samples as
described above.
The three treatments were performed on the same test water with E. coli, and each test was
repeated three times. Dilution series were performed on all samples and were plated out in
duplicates at brilliance E.coli/coliform selective agar plates, and incubated at 37°C overnight.
Results and Discussion
Figure 1 shows that for water with pH 7, all (>99.9%) of the E. coli was killed after 1 min. when
treated with BacTerminator. Both sodium hypochlorite and hypochlorous acid did not succeed
in killing all bacteria, and even after 15 min. exposure only 58-66% of the E. coli was killed.
There is no significant difference on the kill effect for hypochlorite and hypochlorous acid.

Figure 1 Shows kill% of E. coli for three different chlorine-based treatments (BacTerminator,
sodium hypochlorite and hypochlorous acid) over time. Test water had pH 7. Kill% is calculated
from CFU/mL against a positive reference sample of the test water. Results are presented as a
weighted average of three replicates for each treatment, and standard deviations are shown as
bars on the curves.
At figure 2 results for test water with pH 8 are shown. At pH 8 it takes four minutes of exposure
before all bacteria are killed by BacTerminator, which is a 3 min. increase compared to results
for pH 7. Again both sodium hypochlorite and hypochlorous acid did not kill all bacteria.
Sodium hypochlorite only killed about 35% of the bacteria and hypochlorous acid killed about
25%. Thus killing efficiency for all three methods decreased considerably when pH was
increased from 7 to 8.
Figure 2 Shows kill% of E. coli over time obtained with test water with pH 8. See figure text
from figure 1 for further details.
Results of water with pH 9 are shown in figure 3. At this water pH BacTerminator was not able
to kill all bacteria within 15 minutes exposure time, but still had above 90% kill effect after 5
min. Thus the kill effect of BacTerminator decreases remarkably from pH 8 to pH 9. The kill
effect for hypochlorite and hypochlorous acid is not as affected by increasing water pH from 8
to 9. The kill effect obtained by sodium hypochlorite is about 35% and for hypochlorous acid
about 22%. For sodium hypochlorite it appears that it takes longer time to reach the max. kill-
effect in water with pH 9 compared to water with pH 8.
 Figure 3 Shows kill% of E. coli over time obtained with test water with pH 8. See figure text
from figure 1 for further details.
The chlorine level used in this study was very low, and at least 9-10 times lower than what is
usually used for disinfection. BacTerminator however, was still able to kill all bacteria within 4
min. at pH 7 and 8, and killed more than 90% within 5 min. at pH 9. Sodium hypochlorite and
hypochlorous acid were not near as effective a treatment method at any pH tested. It was
expected that hypochlorous acid would be more efficient -or at least significantly better than
sodium hypochlorite. The poor performance could be due to the instability of hypochlorous acid
when using it in a manner, where it is dosed from a solution to contaminated water.
Water pH appears to have a significant effect on killing efficiency on all three chlorine based
disinfection methods. The more alkaline the water is the less efficient are all three methods.
Hypochlorous acid is not more efficient than sodium hypochlorite when added to water in a
volume that will not lower the pH of the water.

Conclusion
Alkalinity of contaminated water decreases kill effect of BacTerminator, sodium hypochlorite,
and hypochlorous acid, however it has a greater negative impact on hypochlorite and
hypochlorous acid than on BacTerminator kill effect. At low chlorine level BacTerminator is by
far more efficient at killing E. coli in water than sodium hypochlorite and hypochlorous acid.

References
1. Rutala W A and Weber D J. Uses of inorganic hypochlorite (bleach) in health-care facilities.
Clin. Microbiol. 1997, 10(4):597
2. Le Dante C, Duguet J-P, Montiel A, Dumoutier N, Dubrou S, and Vincent C. Chlorine
disinfection of atypical Mycobacteria isolated from a water distribution system. Applied and
Environmental Microbiology, 2002, Vol. 68 No. 3, p. 1025-1035.
3. Shi L, Wang Wang Y, Wang Y, Qui X and Zhao G. Disinfection efficacy of Medilox Super-
oxidized water with neutral pH. Chinese Journal of Nosocomial Infections, 2009, 24: 3370-
3372.
4. Gray M J, Wholey W-Y, and Jakob U. Bacterial responses to reactive chlorine species.
Annu. Rev. Microbiol. 2013, 67:141-161
5. S O Vodop’yanov, Pavlovichy N V, Oleinikov I P, and Mishan’kin B N. The possible role of
transmembrane proton concentration gradient in developing Yersinia pestis sensitivity to the
bactericidal effect of active chlorine compounds at low pH. Biologicheskie Membrany
(Moscow), 1992, Vol. 9 Issue 6, pp. 581-587.
6. Kunigk L, Schraam J R and Kunigk C J. Hypochlorous acid loss from neutral electrolysed
water and sodium hypochlorite solutions upon storage. Braz. J. Food. Technol. 2008 V. 11 N. 2
p. 153-158.

 Home

 Our Green Technology

 BacTerminator® products

 Use BacTerminator® here

 Our references

Search