Desalination 248 (2009) 152159

A simple technique for water disinfection with hydrodynamic cavitation: Effect on survival of Escherichia coli
L. Mezulea , S. Tsyfanskyb, V. Yakushevichb, T. Juhnaa
a

Department of Water Engineering and Technology, Riga Technical University, Riga LV-1048, Latvia Tel. 37129692491; Fax 37167089085; email: linda.m@inbox.lv b Research laboratory Nonlinear Phenomena of Vibrating Systems, Riga Technical University, Riga LV-1006, Latvia
Received 31 January 2008; revised accepted 15 May 2008

Abstract Effect of hydrodynamic cavitation on disinfection of Escherichia coli was investigated in laboratory scale device. The cavitation was generated using a rotor, driven by a simple milling cutter, in the thin layer of water which was circulated from and to a reservoir. Disinfection efficacy was analyzed by measuring respiratory activity using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and the ability to multiply with direct viable count (DVC) method. Experiments showed that hydrodynamic cavitation was very effective in reducing bacterial ability to divide. Exposure of 3 min using energy input of 490 W/L stopped the division of 75% of E. coli cells. However, analyses with CTC showed that most of the cells sustained respiratory ability, indicating that bacteria enter active but not culturable (ABNC) state. Thus, the potential of bacteria to resuscitate from ABNC state should be further investigated. The study showed that hydrodynamic cavitation is a simple technique and could potentially be used for drinking water disinfection in rural communities. Keywords: Water disinfection; Hydrodynamic cavitation; E coli; Viability

1. Introduction Chlorination is one of the most frequently applied disinfection method for drinking water in developing countries. However, it has several disadvantages including the formation of carcinogenic disinfection by-products (reaction of chlorine with natural organic

Corresponding author.

matter), appearance of taste and odor problems in processed water [1]. Besides, chlorine is not effective against bacteria which are hidden in loose deposits or in biofilm which are present in most of water distribution networks. Chlorination does not always induce physiological injury to microorganisms and thus many of them have adapted to relatively high doses of chlorine [2,3]. Although chlorination is considered relatively cheap and easy to use this disinfection method

Presented at the Water and Sanitation in International Development and Disaster Relief (WSIDDR) International Workshop Edinburgh, Scotland, UK, 2830 May 2008.
0011-9164/09/$ See front matter 2009 Published by Elsevier B.V. doi:10.1016/j.desal.2008.05.051

L. Mezule et al. / Desalination 248 (2009) 152159 requires careful control of dosing which increases the maintenance costs. There still is a need for new approaches of water disinfection which could be effective, safe, easy to perform and less labor intensive. The methods which induce physiological injury or destroy microorganism have a potential for application in disinfection of drinking water, especially in areas where highly qualified operators are not always available. Ultrasonication has been used decades in laboratories for disintegration of microbial cells. This method applied in water produces acoustic cavitation effects viz. formation of artificial gas bubbles in liquid that collapse resulting in generation of a shock waves and fast (up to 400 m/s) microjects in fluid [4]. This generates enough energy to mechanically weaken or disrupt bacteria [5]. Additional effect is the generation of hydrogen radicals, which have disinfectant properties. Similar effects to ultrasonic radiation could be achieved by subjecting liquid to high pressure drops and shear stress, thus inducing so called hydrodynamic cavitation. Hydrodynamic cavitation normally occurs whenever the pressure at a point in a liquid is momentarily reduced below its vapor pressure. The potential of application of cavitation (both acoustic and hydrodynamic) for drinking water disinfection has been investigated. However, until now the studies were limited to analyses on culturability of bacteria [68] whereas other metabolical states in which pathogenic bacteria may be occurring in drinking water were not investigated. It has been described that many microorganisms when subjected to stresses such as disinfection can enter active but nonculturable (ANBC) state [9], sometimes referred to as viable but nonculturable (VNBC) [10], where cells show no potential to divide, they cannot be grown to detectable levels in vitro on traditionally used agars but with certain vitality assays show activity [9]. Thus, in addition to traditionally used culture methods several molecular techniques such as direct viable count (DVC) [11] in combination with fluorescence in situ hybridization (FISH) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining [12,13] could provide more detailed information about changes of viability of microorganisms during disinfection. DVC method is based on the incubation of samples with antimicrobial agents (nalidixic acid for Escherichia coli) and nutrients, where

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nalidixic acid acts as a specific inhibitor of DNA synthesis and prevents cell division without affecting other cellular metabolic activities and causes the formation of long filamentous cells [14]. CTC method is based on incubation of cells with redox dye CTC which in the presence of functioning electron transport chain act as artificial electron acceptor, resulting in formation of fluorescent insoluble formazan crystals inside metabolically active cells [12,13,15]. The objective of this study was to determine the effect of hydrodynamic cavitation on viability of bacteria in drinking water. The experiments were carried in laboratory scale using E. coli as a model organism for disinfection experiments.

2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli ATCC 25922 was inoculated into prefiltered liquid LuriaBertrani (LB) media (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) and incubated with constant shaking (150 rpm) overnight at 37 C. 2.2. Sample preparation Overnight culture of E. coli was centrifuged at 3000 rpm (Nu vefuge CN090, Turkey) for 10 min. Then the pellet was washed twice with sterile phosphate buffered saline (7 mM Na2HPO4, 3 mM NaH2PO4, 130 mM NaCl, pH 7.2) and resuspended in sterile distilled water. In order to determine number of cells in suspension a small volume (0.11 ml) was filtered through 25mm-diameter 0.2-mm-pore-size filters (Anodisc; Whatman plc, UK) and fixed with 34% formaldehyde for 15 min, washed with sterile distilled water, air dried and stained with 10 mg/mL DAPI (40 ,6-diamidino-2phenylindole, Merck, Germany) for 5 min. Cell numbers were determined by epifluorescence microscopy by counting 20 random fields of view (Ex: 545 + 30 nm; Em. 610 + 75 nm, dichromatic mirror 565 nm, Leica DMLB, Germany). Then a known concentration of cells was added to 2 L of artificially prepared water (1 mL/L of AOC salt solution, containing 4.55 g/L (NH4)2SO4; 0.2 g/L KH2PO4; 0.1 g/L MgSO4 7H2O; 0.1 g/L CaCl2 2H2O; 0.2 g/L NaCl, distilled water, pH 7 + 0.5) which after preparation (before

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C a v ita tio n d e v ic e M

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R e s e r v o ir
0.14 W/cm3 0.35 W/cm3 0.35 W/cm3 -2 0.49 W/cm3 0.49 W/cm3 -2 Critical temperature

2 4 3

2 l

Fig. 1. Experimental setup of the cavitation system used in the study. It consisted of cavitation device and water reservoir. Cavitation took place in the liquid layer (4) within the space between rotor (2) and the plate (5). The rotation (900023,000 min1) of the rotor was driven by a motor (1). The circulation of water between the reservoir and cavitation device was controlled by a valve (3) placed on the connecting pipes.

Temperature, C

65 60 55 50 45 40 35 30 25 20 15 10

4 6 Time, minutes

10

Fig. 2. Increase in temperature of samples processed with hydrodynamic cavitation, showing selected treatment regimes and time chosen for treatment (in legend).

inoculation of E. coli) was filtered through 47-mmdiameter 0.2-mm-pore-size nitrocellulose filters (Millipore, USA). All experiments were done in triplicates.

2.3. Sample treatment All samples in no more than 2 h after preparation were processed with hydrodynamic cavitation in a laboratory scale system. It consisted of reservoir (V 2 L) and cavitation device which was made of a plate in which the rotating disk was located. The disk was driven by the motor (*89 A, 1850 W, plunge capacity 020 mm, load speed 9000 23,000 min1, Makita 3612C) so that the angular speed created highly turbulent flow (Re > 3 105) and cavitation (Fig. 1) in the liquid layer between the surface of the disk and the plate. Due to shear force induced upon the liquid layer, water was circulating between the reservoir and the cavitation device. Before treatment the device was washed with disinfectant (Carela Bio-Des, R. Spa ne GmbH, Germany) for 10 min at 0.14 W/cm3 (8.7 A, 220 V), then rinsed with sterile distilled water for 2 min (0.14 W/cm3, 8.7 A, 220 V). The sample was poured inside the apparatus (leaving approx. 10 ml of sample as untreated control), initial sample temperature was recorded and previously sterilized (cleaned with 96 ethanol and rinsed with distilled water) liquid ice pack was inserted

into sample holder in order to control the temperature of the apparatus. Treatment regimes differed for each experiment ranging from 0.14 W/cm3 (8.7 A, 220 V) to 0.49 W/cm3 (8.79.3 A, 220 V) and lasted from 3 till 9 min (depending on the moment when temperature in sample holder exceeded or reached 38 C (Fig. 2)). After treatment 10 ml of sample were collected in sterile bottles and processed further. 2.4. Staining of samples with CTC Within 2 h 1 ml of either treated or untreated sample was stained with CTC in accordance with a modification of the procedure described by Rodriguez et al. [12]. Final concentration of 4 mM CTC (Fluka, BioChemika, Germany) was used (previously determined as being optimal). After incubation the sample was filtered through a 25-mm-diameter 0.2-mm-pore-size filter, air dried, fixed with 34% formaldehyde for 15 min, rinsed with sterile distilled water and stained with 10 mg/mL DAPI. Actively respiring and non-respiring cell numbers were determined with epifluorescence microscope (Leica DMLB) equipped with a 50-W power supply, mercury lamp, filter sets for DAPI (Ex: 340/380 nm;. Em: >425 nm) and for fluorescent formazan crystals (Ex: 545 + 30 nm; Em: 610 + 75 nm or the same channel as for DAPI in order to avoid the counting of any extracellulary fluorescing units) and a camera (CoolSNAP Pro, Media Cybernetics Inc., USA). For image processing Image Pro Plus 4.5.1.

L. Mezule et al. / Desalination 248 (2009) 152159 software (Media Cybernetic Inc., Silver Spring, MD) was used. 2.5. DVCFISH procedure Cell potential of dividing was determined by modified DVC method by Kogure et al. [16] and combined with FISH. Sample (1 ml) was filtered through a 25-mm-diameter 0.2-mm-pore-size filter and placed on cellulose pads (Millipore) previously soaked in 2 mL of Tryptone Soya broth (Oxoid Ltd., UK) and 10 mg/mL nalidixic acid mix and incubated in humidified chambers for 14 h at 30 C. After incubation the filters were removed, air dried and fixed with 34% formaldehyde solution. After fixation the filter was rinsed with sterile distilled water and air dried. Then, 2030 mL of PNA hybridization mix consisting of hybridization buffer (50 mM TrisHCl, 10% w/v 50% dextran sulphate, 0.1 mM of NaCl, 30% v/v formamide, 30% v/v tetra-sodium pyrophosphate, 0.2% w/v polyvinylpyrrolidone, 0.2% w/v Ficoll 400, 5 mM Na2EDTA, 0.1% v/v Triton X-100) and 200 nM of fluorescently labeled PNA probe (TCA ATG AGC AAA GGT [17] labeled with CY3 (Ex: 550, Em: 570)) was applied to the filter and covered with a cover glass. The samples were incubated in a tight humidified vessel for 60 min at 57 C. After hybridization samples were washed with plenty of sterile distilled water and air dried. For counterstaining 10 mg/mL DAPI was applied for 5 min, rinsed with sterile distilled water and air dried. Samples were visualized with epifluorescence microscope. For detection of E. coli a narrow range Y3 filter (Ex: 545 + 30; Em: 610 + 75, dichromatic mirror 565 nm) was used. For image processing Image Pro Plus 4.5.1 software was used. Cells were considered DVC positive if they were 1.5 times longer than the average length of cells with no DVC treatment (DAPI stained). 2.6. Statistical analysis For CTC assay the enumeration of bacteria was done with direct microscopic counting of 20 random fields of view for each sample. Both respiring and nonrespiring bacteria were enumerated in each field of view. For DVC assay at least 20 random pictures were taken from each sample and length measurements of at least 300 cells were done with Image Pro Plus 3. Results and discussion

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4.5.1 software. For non-elongated cell length determination DAPI stained samples were analyzed as described above.

Preliminary experiments showed that cavitation rapidly increases the temperature of water. To exclude thermal effect on disinfection efficiency of bacteria, all experiments were continued until temperature reached 38 C and then stopped (Fig. 2). As a result the maximal length of experiments was 5 (for highest rotation speed and energy input of 0.49 W/cm3) to 9 min (for lowest rotation speed and energy input of 0.14 W/cm3) depending on rotation speed. The analyses of water samples with DAPI staining before and after experiments showed that cavitation did not increase the total number of bacteria (data not shown), confirming effectiveness of disinfection and cleaning of the system between the experiments. Analyses of E. coli suspension before the experiments with CTC method showed that untreated samples contain approximately 5% of non-respiring cells, whereas the rest 95% were actively respiring bacteria. These results agreed with data reported previously, where CTC positive cells in exponential and stationary phases ranged from 90% to 100% [1820]. In our experiments cavitation, depending on the dose and time applied (Fig. 3) increased the proportion of nonactive cells to 10% till 20%, implying that effect of cavitation on respiratory activity was minor. Due to increase of the temperature above 38 C with increase of the dose and exposure time it was not possible to test higher cavitation intensities. For this a modification of the cavitation device is needed by equipping it with cooling or other energy co-generation equipment. The highest non-respiring cell fraction was obtained when samples were treated for 5 min at 0.49 W/cm3, which also was the most intensive treatment used. The least effective treatment regime was 0.14 W/cm3 for 9 min. This indicated that energy input of cavitation is more important than the exposure time for reducing metabolic activity of E. coli. According to the description of CTC method, the formation of formazan crystals within the bacterial cells is indication of the metabolic activity of the bacteria. Microscopic analyses revealed that formazan

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Non-respiring cells (%)

20 15 10 5 0
3 0,14 W/cm 1 9 min

0,35 W/cm 2 6 min

0,35 W/cm 3 8 min

3 0,49 W/cm 4 3 min

3 0,49 W/cm 5 5 min

Fig. 3. Percent of non-respiring cells formed after treatment with different regimes of hydrodynamic cavitation (data shown after subtraction of initially non-respiring cells). Standard deviation represents the dispersion of the results of three separate experiments. Hundred percent represents the total population.

crystals formed in control samples were large in size and often more than one crystal was found in a single cell (Fig. 4a), whereas, the cells treated with hydrodynamic cavitation mostly contained a single crystal with variable size ranging from very small to large ones (Fig. 4b). This indicates a slower respiration rate of the treated cells and lower ability to form crystals, but still functioning Electron Transport Chain [21]. However, all cells containing visible crystals were counted as metabolically active.

DVC analysis of control samples showed reproduction potential for approximately 90% of total population, which corresponds to CTC results. However, even the mildest treatment with hydrodynamic cavitation resulted in large numbers of non-elongated cells (Fig. 5). Liquid treatment with hydrodynamic cavitation caused about 75% cell decrease in reproduction potential, resulting in large metabolically active, but nondividing population of cells. This condition already

Fig. 4. Formazan crystals (bright spots inside cells) formed after CTC reduction in a control sample (a), sample treated for 5 min at energy input of 0.49 W/cm3 (b). Bar 3 mm.

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120 100

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Elongated cells(%)

80 60 40 20 0 0.14 W/cm3 9 min 0.35 W/cm3 6 min 0.35 W/cm3 8 min 0.49 W/cm3 3 min 0.49 W/cm3 5 min

Fig. 5. Percent of elongated (DVC positive) cells in control samples (dark) and samples treated with different regimes of hydrodynamic cavitation (light). Standard deviation represents the dispersion of the results for three separate experiments. Hundred percent represents the total population.

discussed in different studies [22], might be caused by rapid setting of stress of the bacterial cells. In control samples average cell length after DVC analysis was approximately 6 mm, however, the lengths of cells varied starting from non-elongated cells (below 2 mm) to 20 mm or longer cells (Fig. 6a). Treated samples did not show such length variation usually more than 2/3 of the elongated cells were just above the same as elongation limit (1.5 times than DAPI stained cell length) (Fig. 6b). Results showed that cavitation was very effective in stopping multiplication of E. coli. A treatment as

short as 3 min with energy input of 490 W/L was sufficient to reduce E. coli ability to divide by 75%. If we assume that cessation of the ability to divide is an indication of bacteria death, the cavitation appears an effective method of disinfection. Even though bacteria were not dividing it still possessed the ability to continue to carry out respiratory process. It appears the E. coli enter active but not culturable (ABNC) state. Thus, it should be further investigated whether these bacteria are able to resuscitate after cavitation effect and start to divide thus cause potential risk to drinking water consumers.

Fig. 6. Elongated E. coli cells in control sample (a) and sample treated with 0.49 W/cm3 for 5 min (b). Bar 3 mm.

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To solve hygienic problems in developing countries nowadays several novel disinfection techniques are being developed including solar disinfection [23] and Mixed Oxidant Gases Systems [24]. This study showed hydrodynamic cavitation could be another potentially applicable non-reagent technique. This technique employs cavitation reactor, which could be manufactured from a simple milling cutter or another electrical appliance with a motor. The cutter can be supplemented with specially constructed rotor which generates cavitations in the volume of water. The contact time in such a reactor should be in order of a few minutes. During this time the microorganisms are subjected to multiple effects of cavitation and as a result are damaged or disintegrated thus are not able to divide and cause diseases. Although relatively high electricity consumption is required, hydrodynamic cavitation is very simple and provides fast disinfection without application of reagents. The device is inexpensive and could be used in households or in water treatment plants of small communities.

[3]

[4]

[5]

[6]

[7]

[8]

4. Conclusions Treatment with hydrodynamic cavitation for several minutes decreased E. coli ability to divide with more than 75%, however, further investigations must be made in order to determine resuscitation potential for ABNC population obtained during treatment. Hydrodynamic cavitation is a promising technique for water disinfection.

[9]

[10] [11]

[12]

Acknowledgments
This work was funded by the IZM-RTU Project No. R7242, Effect of pressure induced cavitation on survival of pathogenic bacteria in drinking water. We thank Dr. Simona Larsson for helpful discussions.
[13]

[14]

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