In this paper, the disinfection of water is investigated using plasma spark treatment and the results are compared with conventional techniques. Inactivation of the Enterococcus faecalis and Escherichia coli bacteria is considered in the treatment process of water by the plasma spark. For this purpose, many physical and chemical parameters of water are measured and the obtained results demonstrate a reduction of 8-log in colony forming units of E. coli and E. faecalis at 15 minutes and 12 minutes, respectively. The results of this research show that no ozone is produced during the plasma spark treatment. Moreover, inactivation of a large number of bacteria without any change of pH shows that pH is not the cause of the bacterial inactivation. It is concluded that the main causes of the inactivation of bacteria in the treated water are H2O2 molecules and the electrical fields generated by plasma.

INTRODUCTION

The quality of water has a substantial role in public health. Contaminated water can cause disease outbreaks and serve as a mechanism to transmit communicable diseases. The World Health Organization (WHO) estimated that diarrhoeal disease claimed the lives of 2.5 million people annually (WHO 2013). The conventional method of water disinfection is using chlorine. In recent years, due to the side effects and the environmental hazards of chlorine, the use of ozone has replaced it and become more common (Glaze et al. 1987). However, due to the absence of residual ozone in water, it is unable to eliminate secondary pollution (Liu et al. 2010; Grellier et al. 2015).

The use of plasma is one of the new techniques that has recently attracted attention among researchers into water disinfection (Fridman et al. 2007; Oehmigen et al. 2010). Since in this method no additional substance is employed in the water, it is highly interesting to scientists (Mededovic & Locke 2007; Kim et al. 2014). Plasma can be produced by a variety of electrical discharges. All plasma systems, in terms of electronic density or temperature, are divided into two major categories, namely thermal and non-thermal (Joshi et al. 1995; Babaeva et al. 2014; Jiang et al. 2014).

One of the new applications of plasma is the inactivation of microorganisms (Gibson et al. 2011; Akan & Çabuk 2014). The inactivation effect has been attributed to the presence of active plasma species (OH, O, O3, H2O2, UV, and NO), decreased pH and electric fields generated by the discharge of plasma (Julák et al. 2012; Johnson et al. 2015). Each of these factors plays a significant role in the inactivation of microorganisms (Guo et al. 2015). Various types of plasma discharges, such as dielectric barrier discharge, corona discharge and spark discharge plasma, were reported to be able to produce active plasma species (Li et al. 2014; Kim et al. 2015).

Recently, Kim et al. (2013) used gliding arc discharge for inactivation of Escherichia coli in water. They observed 5-log reductions in the colony forming unit (CFU) number of E. coli in 20 min. They also stated that the main cause of microorganism inactivation is the creation of hydrogen peroxide molecules. The inactivation of bacteria in water using atmospheric plasma was studied by Liu et al. (2010). They achieved a 100% inactivation rate of Staphylococcus aureus in 16 min. In their study, the decrease in pH with the increase in oxidation of perhydroxyl radicals (HOȮ) was introduced as the main cause of bacterial death.

In the present study, there are three main objectives. First, the inactivation of two main bacteria in water pollution (i.e., E. coli, Enterococcus faecalis) using spark plasma is under investigation. Inactivation of E. faecalis in water by plasma is studied for the first time in the world. The bacteria E. coli and E. faecalis are important indicators of water pollution. At the second step, the principal cause of water disinfection treated by plasma is studied. The final aim is to assess the side effects of plasma in water, to which less attention has been devoted in the literature until now.

METHODS

In this section, the three main objectives mentioned above are studied in the following subsections. In the first section, water disinfection with a short description of the experimental procedure is presented, and in the two following sections the detection of the cause of disinfection and its side effects are explained.

Disinfection of water

The spark discharge plasma used for the disinfection of water consists of two separated needle electrodes and a pulsed power supply (10 kV @ 30 Hz). The non-thermal plasma (NTP) is generated by a simple apparatus of an open-air type. The two needle electrodes are separated from each other by 5 cm, and the distance of the electrodes from the water surface is 4 mm.

Our experiments were conducted by plasma injection. In this system, the bacteria are suspended in the water poured in a shallow container (whose volume is typically 10 mL) and the NTP is applied to the surface of the liquid. Two different bacterial cell wall structures were used in the present study. Gram-positive bacteria E. faecalis and Gram-negative E. coli, isolated from water and by microbiological and immunological methods, were identified and were used as the test organism.

E. coli and E. faecalis were cultured by PCA (plate count agar). Fresh cultures of bacteria colonies were used for making the bacterial suspension in water and PBS (phosphate buffered saline, 2.7 mM KCL, 137 mM NaCl, 1.5 Mm KH2PO4 and 8.1 mM Na2PO4). In order to make the initial concentration of the bacteria suspension, 0.5 McFarland standard was used as well. The initial cell concentration was controlled at 108 CFU/mL. The initial cell concentration and cell viability were measured by culture and by a dilution of 10−6. The CFU count of bacteria with different dilutions after plasma treatment for various time durations in a range of 1, 3, 5, 7, 10, 12 and 15 min was evaluated. The PCA samples were incubated for 24 hr at 35 °C and then the number of colonies was counted.

Detection of disinfection factor

The main possible causes of disinfection in the water treated by plasma (increasing temperature, decreasing pH, hydrogen peroxide, ozone, and nitric oxide) were investigated. The physical and chemical parameters were measured in untreated water (t = 0 min) and the plasma-treated water (t = 1, 3, 5, 7, 10, 12, 15 min).

pH

pH was measured using a Metrohm pH-meter (Model 744) apparatus with a glass microelectrode.

H2O2

The concentration of H2O2 in water was detected based on the colorimetric method. It was measured using a peroxide test strip (EMD Chemicals, Germany) and by observing the colour change in the strip.

Temperature

The changes in temperature were measured using a digital thermometer (Testo- T1).

O3

The concentration of O3 in the total samples was evaluated using the CHECKIT Comparator kit, based on the colorimetric measurement of colour product of the ozone reaction with N,N-diethyl-1,4-phenylenediamine (DPD)/potassium iodide.

Plasma side effects

The side effects of plasma in water were considered in previous studies by the investigation of nitrate and nitrite anions alone, while in the present paper many more substantial parameters of the quality of drinking water were considered.

NO−3/NO−2/SO4−2

The concentrations of nitrate anions (NO3), nitrite anions (NO−2) and sulfate anions (SO42) in untreated and plasma-treated water were measured based on the spectroscopic method and using a spectrophotometer (HACH, DR-5000) and nitrate (Cat: 21061-69), nitrite (Cat: 21071-69) and sulfate (Cat: 21067-69) kits.

Dissolved oxygen

Dissolved oxygen (DO) was determined using the DO-Meter (JENCONS, 970) apparatus with the a membrane electrode.

Turbidity measurements

Turbidity in the water was evaluated by a turbidity meter (WTW, TURB 355 IR).

Total hardness

Total hardness (CaCo3) was detected based on the reaction of ethylene diamine tetraacetic acid.

Conductivity and total dissolved solids

Water conductivity and total dissolved solids (TDS) were measured by a conductivity meter (Metrohm – 744) with a measuring range of 1 μS/cm to 100 mS/cm.

RESULTS

Disinfection of the water

The bactericidal effect of the water exposed to spark discharge for 1, 3, 5, 7, 10, 12 and 15 min and then stored for 24 hr in a refrigerator is shown in Figures 1 and 2.
Figure 1

Results of E. coli inactivation in water exposed to spark discharge and stored for 24 hr in a refrigerator.

Figure 1

Results of E. coli inactivation in water exposed to spark discharge and stored for 24 hr in a refrigerator.

Figure 2

Results of E. faecalis inactivation in water exposed to spark discharge and stored for 24 hr in a refrigerator.

Figure 2

Results of E. faecalis inactivation in water exposed to spark discharge and stored for 24 hr in a refrigerator.

In the case of water (i.e., with an initial concentration of 108 CFU/mL), there is an 8-log reduction in the CFU count of E. coli during the 15 min plasma treatment and there is an additional 1-log reduction during the storage period of 12 min in Figure 1. After a 12 min treatment of water, there is an 8-log reduction in the CFU count of E. faecalis. In addition, a 3-log reduction during the storage period (24 hr) of 10 min was achieved (Figure 2).

The complete inactivation of E. faecalis was observed after 12 min of incubation in 10 mL of exposed water. In contrast, the complete inactivation of E. coli needed 15 min of incubation. In the tests with plasma treatment, the E. coli and E. faecalis concentrations consistently dropped during the period of treatment in water. With the same initial concentration as water, there is an 8-log reduction in the CFU count during plasma treatment of 12 min in the PBS case and also there is an additional 8-log reduction of the CFU count during 3 min of plasma treatment with a storage period of 24 hr. It can be concluded that the PBS significantly improved the bacterial inactivation during the storage period (Figure 3).
Figure 3

Results of E. coli inactivation in PBS exposed to spark discharge and stored for 24 hr in a refrigerator.

Figure 3

Results of E. coli inactivation in PBS exposed to spark discharge and stored for 24 hr in a refrigerator.

Similar graphs are also plotted for other experimental conditions (stored and fresh water and PBS, exposed to spark discharge). PBS exposed to spark discharge exhibits complete inactivation in the 10 mL of exposed water. A similar difference between buffered and water cases can be found in regard to the inactivation of microorganisms by the spark plasma action. E. coli in water is completely inactivated after 15 min of incubation in 10 mL of exposed water.

Investigation of the disinfection factor

Temperature

Changes in water temperature before and after the plasma radiation and PBS show that the temperature increases to 37 °C and 45 °C.

pH measurements

A sudden drop in pH is observed in the water at short time exposure. The pH of the water decreases from an initial mean value of 8.43 ± 0.10 to 4.15 ± 0.1 at the end of plasma treatment. In addition, a significant change of pH is observed as well in all the exposed samples after 24 hr storage (Figure 4). No major change of pH was observed in PBS. The pH under plasma spark remains almost unchanged, with the minimal pH 7.14 in 10 mL (Figure 5).
Figure 4

Decrease in pH, when plasma was exposed to water and change of pH after storage for 24 hr.

Figure 4

Decrease in pH, when plasma was exposed to water and change of pH after storage for 24 hr.

Figure 5

Buffer pH remained unchanged.

Figure 5

Buffer pH remained unchanged.

Hydrogen peroxide determination

The concentration of H2O2 in water was measured over 15 min so as to determine the effective residual time. The results of H2O2 concentrations in water in the form of colour changes in peroxide test strips show the increasing of H2O2 content in all samples with the growth of exposure time up to 100 mg/L under the plasma spark in 10 mL of water. In all samples, for exposed plasma sparks stored for 24 hr in a refrigerator, the H2O2 content did not decrease. The maximum H2O2 concentration with the peroxide test strips is 100 mg/L, as shown in Figure 6.
Figure 6

Increase in H2O2 concentration, when plasma was exposed to water.

Figure 6

Increase in H2O2 concentration, when plasma was exposed to water.

O3

After plasma irradiation, the initial concentration (0 mg/L) of ozone remained unchanged, therefore plasma does not have the capability of producing ozone.

Plasma side effects

The side effects of plasma in water were also studied. The results show that the plasma has little effect on parameters including total hardness, turbidity, sulfate, TDS, conductivity and DO. As a result of plasma radiation there was an increase of nitrate and nitrite concentrations in water (Figure 7).
Figure 7

Increased nitrate and nitrite concentrations in water exposed to plasma.

Figure 7

Increased nitrate and nitrite concentrations in water exposed to plasma.

DISCUSSION

Contaminated water is the main cause of many infectious diseases. According to the latest report from the World Health Organization, each year about 2.5 million people die due to bloody diarrhea caused by contaminated water (WHO 2013). Disinfection of microorganisms is an important step in the water treatment processes (Shaw et al. 2014). Disinfection by chlorination is a common technique (Moore 2014). The main concern in chlorine treatment methods is THM (trihalomethane) compounds, due to the increase in the risk of cancer (Dore 2015; Teo et al. 2015).

Ozone is a remarkably versatile and powerful disinfectant, although the need to produce it on site makes it expensive and there is a lack of residual disinfection capability in comparison to alternatives such as chlorination or UV (Rajab et al. 2015). While previous researchers have explored the effect on bacterial inactivation by active species plasma (Sugiarto et al. 2002), this study is mainly focused on the inactivation of microorganisms, the investigation of disinfection factors and the side effects in plasma-treated water with spark discharge.

In particular, the study investigates the efficiency of plasma in the inactivation of indicator bacteria in water pollution (E. coli, E. faecalis). The results show that plasma has the capability to destroy high concentrations of E. coli and E. faecalis in water. E. faecalis compared with E. coli bacteria is inactivated in less time. This can be due to the different structure of the cell wall of the two bacteria (Figure 8).
Figure 8

Comparison of inactivation of E. coli with E. faecalis in water exposed by plasma.

Figure 8

Comparison of inactivation of E. coli with E. faecalis in water exposed by plasma.

The cell walls of Gram-negative bacteria (E. coli) are much more complicated than the walls of Gram-positive bacteria (E. faecalis). Gram negatives contain the major components of lipopolysaccharide, lipoprotein, and relatively little peptidoglycan (<10% of the total cell wall) in their cell walls, whereas the walls of Gram positives are mainly composed of peptidoglycan (usually 30–70% of the total cell wall), polysaccharides, teichoic acid or teichuronic acid (Schleifer & Kandler 1972). As mentioned, the polymers making up the cell walls are chemically extremely different in these two groups of bacteria, which can have an effect on the entry capability and the influence of plasma active species. The inactivation effect of bacteria was observed for the exposed water stored for 24 hr as well. In addition, 1 and 3-log reduction was observed during the storage period for E. coli and E. faecalis, respectively, due to the stability of the active plasma species.

In this study, the production of disinfection in water treated by plasma is measured. The results of measurement during plasma radiation show that the small temperature rise, up to 45 °C, cannot be the cause of the inactivation of microorganisms. In previous studies, the pH of water during exposure to plasma decreased due to the presence of H+ (Chen et al. 2009; Julák et al. 2012). These findings roughly correspond with our results. Also, in our study, the value of pH in PBS exposure to plasma was measured. The pH of the buffer is not reduced while the inactivation of bacteria in the buffer is greater than in water. Although hydrogen peroxide in acidic conditions is a strong oxidizer and can have a greater impact on the bacterial cell membrane, the present results demonstrate that it cannot be attributed to the acidification alone.

The inactivation rate (Figures 9 and 10) is a non-dimensional parameter, and is the difference of the concentrations (CFU/mL) of bacteria before and after plasma radiation in each duration time to the initial concentrations of the bacteria.
Figure 9

Inactivation of E. coli as compared with variations of pH in exposed water and PBS.

Figure 9

Inactivation of E. coli as compared with variations of pH in exposed water and PBS.

Figure 10

Inactivation of E. coli as compared with increased H2O2 concentrations in exposed water.

Figure 10

Inactivation of E. coli as compared with increased H2O2 concentrations in exposed water.

It is calculated by the following formula: 
formula
where N1 is the initial concentration (CFU/mL) of the bacteria before plasma radiation and N2 is the concentration of bacteria (CFU/mL) after plasma radiation.

Ozone, as one of the active plasma species, is not generated only due to the plasma radiation on the water surface. Based on the results of this paper, the concentration of nitrate and nitrite increased steadily with the exposure time in water. These results were consistent with previous studies (Liu et al. 2010). Investigation of other parameters did not show any significant changes in the water exposed to plasma.

Experimental results obtained in this study indicate that the spark discharge reacting with water can effectively generate H2O2. When the water is directly exposed to a spark discharge, the reactions can occur with the dissociation of water molecules, and then H2O2 can be produced from the recombination of hydroxyl radicals (Kim et al. 2014). Therefore, the formation of H2O2 molecules confirms the existence of hydroxide radicals in the water. Hydrogen peroxide (H2O2) demonstrates broad-spectrum efficacy against viruses, bacteria, yeasts and bacterial spores. Thus it is widely used as a biocide, particularly in medical, food and industrial applications (Linley et al. 2012). Hydroxide radicals and H2O2 with the electrical potentials of 2.8V and 1.78V are powerful oxidizers which attack essential cell components, including lipids, proteins and DNA. In general, greater activity is seen against Gram-positive than Gram-negative bacteria (McDonnell & Russell 1999; Block 2001). Hence it can be said that E. faecalis is destroyed faster than E. coli due to the presence of hydrogen peroxide in the water exposed to plasma.

CONCLUSIONS

In the present paper, the disinfection of water using plasma spark treatment is investigated. The inactivation of bacterial indicators in water pollution, the major cause of inactivation of bacteria and its side effects are also studied. The results show that the plasma has a strong capability to kill bacteria. 8-log CFU reductions in the concentration of both species are observed, and a few seconds of radiation lead to the death of millions of bacteria. It is demonstrated that the plasma destroying ability for Gram-positive bacteria is greater than for Gram-negative bacteria.

The main factors in plasma bactericidal activity are due to the consequence of electric field generation and the appearance of H2O2 molecules created by the combination of hydroxyl radicals. H2O2 molecules and hydroxyl radicals are very strong oxidizers that have the ability to inactivate bacteria efficiently. It is also observed that pH reduction and other factors have no effect on the inactivation of bacteria. The study of plasma side effects shows that the physical and chemical parameters (total hardness, turbidity, sulfate, TDS, conductivity and DO) change a little. An increase in the concentration of nitrate and nitrite in water treated by plasma is an important side effect of this method. In order to prevent the generation of nitrate and nitrite, it is suggested to provide a new experimental procedure taking into account considerations such as vacuum conditions, lack of nitrogen gas or the use of different electrodes.

REFERENCES

REFERENCES
Block
S. S.
2001
Peroxygen compounds
. In:
Disinfection, Sterilization, and Preservation
(
Block
S. S.
, ed.).
Lippincott Williams & Wilkins
,
Philadelphia, PA
,
USA
, pp.
185
204
.
Dore
M. H.
2015
Threats to human health: Use of chlorine, an obsolete treatment technology
. In:
Global Drinking Water Management and Conservation
(
Dore
M. H.
, ed.).
Springer
,
Heidelberg
,
Germany
, pp.
197
212
.
Fridman
G.
Brooks
A. D.
Balasubramanian
M.
Fridman
A.
Gutsol
A.
Vasilets
V. N.
Ayan
H.
Friedman
G.
2007
Comparison of direct and indirect effects of non-thermal atmospheric-pressure plasma on bacteria
.
Plasma Processes and Polymers
4
(
4
),
370
375
.
Gibson
K.
Haas
C.
Güçeri
S.
Fridman
A.
2011
Plasma Assisted Decontamination of Biological and Chemical Agents
.
Springer Science & Business Media
,
Dordrecht
,
The Netherlands
.
Glaze
W. H.
Kang
J.-W.
Chapin
D. H.
1987
The chemistry of water treatment processes involving ozone, hydrogen peroxide and ultraviolet radiation
.
Ozone Science and Engineering Journal
9
(
4
),
335
352
.
Jiang
B.
Zheng
J.
Qiu
S.
Wu
M.
Zhang
Q.
Yan
Z.
Xue
Q.
2014
Review on electrical discharge plasma technology for wastewater remediation
.
Chemical Engineering Journal
236
,
348
368
.
Johnson
D. C.
Bzdek
J. P.
Fahrenbruck
C. R.
Chandler
J. C.
Bisha
B.
Goodridge
L. D.
Hybertson
B. M.
2015
An innovative non-thermal plasma reactor to eliminate microorganisms in water
.
Desalination and Water Treatment
57
(
18
),
8097
8108
.
Julák
J.
Scholtz
V.
Kotúčová
S.
Janoušková
O.
2012
The persistent microbicidal effect in water exposed to the corona discharge
.
Physica Medica
28
(
3
),
230
239
.
Kim
H.-S.
Wright
K. C.
Hwang
I.-W.
Lee
D.-H.
Rabinovich
A.
Fridman
A.
Cho
Y.
2013
Concentration of hydrogen peroxide generated by gliding arc discharge and inactivation of E. coli in water
.
International Communications in Heat and Mass Transfer
42
,
5
10
.
Kim
H.-S.
Lee
D.
Fridman
A.
Cho
Y. I.
2014
Residual effects and energy cost of gliding arc discharge treatment on the inactivation of Escherichia coli in water
.
International Journal of Heat and Mass Transfer
77
,
1075
1083
.
Kim
J.-H.
Lee
M.-A.
Han
G.-J.
Cho
B.-H.
2015
Plasma in dentistry: a review of basic concepts and applications in dentistry
.
Acta Odontologica Scandinavica
72
(
1
),
1
12
.
Li
Y.
Kojtari
A.
Friedman
G.
Brooks
A. D.
Fridman
A.
Ji
H.-F.
2014
Decomposition of L-valine under nonthermal dielectric barrier discharge plasma
.
The Journal of Physical Chemistry B
118
(
6
),
1612
1620
.
Linley
E.
Denyer
S. P.
McDonnell
G.
Simons
C.
Maillard
J.-Y.
2012
Use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action
.
Journal of Antimicrobial Chemotherapy
67
(
7
),
1589
1596
.
Liu
F.
Sun
P.
Bai
N.
Tian
Y.
Zhou
H.
Wei
S.
Zhou
Y.
Zhang
J.
Zhu
W.
Becker
K.
Fang
J.
2010
Inactivation of bacteria in an aqueous environment by a direct-current, cold-atmospheric-pressure air plasma microjet
.
Plasma Processes and Polymers
7
(
3–4
),
231
236
.
McDonnell
G.
Russell
A. D.
1999
Antiseptics and disinfectants: activity, action, and resistance
.
Clinical Microbiology Reviews
12
(
1
),
147
179
.
Mededovic
S.
Locke
B.
2007
Primary chemical reactions in pulsed electrical discharge channels in water
.
Journal of Physics D: Applied Physics
40
(
24
),
7734
7746
.
Moore
B.
2014
Modeling Chlorine Residual and Disinfection Byproduct Formation in Circulating Distribution Systems
.
MS Dissertation
,
University of Alaska
,
Anchorage
.
Oehmigen
K.
Hähnel
M.
Brandenburg
R.
Wilke
C.
Weltmann
K. D.
Von Woedtke
T.
2010
The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids
.
Plasma Processes and Polymers
7
(
3–4
),
250
257
.
Schleifer
K. H.
Kandler
O.
1972
Peptidoglycan types of bacterial cell walls and their taxonomic implications
.
Bacteriological Reviews
36
(
4
),
407
477
.
Sugiarto
A. T.
Ohshima
T.
Sato
M.
2002
Advanced oxidation processes using pulsed streamer corona discharge in water
.
Thin Solid Films
407
(
1
),
174
178
.
Teo
T. L.
Coleman
H. M.
Khan
S. J.
2015
Chemical contaminants in swimming pools: occurrence, implications and control
.
Environment International
76
,
16
31
.
WHO
2013
Water Quality and Health Strategy 2013–2020
.
World Health Organization
,
Geneva
,
Switzerland
. .