Environ. Sci. Technol. 2005, 39, 7294-7298
Inactivation of Cryptosporidium parvum by Ultrasonic Irradiation I . O Y A N E , * ,† M . F U R U T A , ‡ C. E. STAVARACHE,† K. HASHIBA,§ S. MUKAI,⊥ M. NAKANISHI,# I. KIMATA,¶ AND Y. MAEDA† Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan, Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan, Hitachi, Ltd. Higashi-Koigakubo 1-280, Kokubunji-shi, Tokyo, 185-8601, Japan, Water Quality Inspection and Control Section, Osaka Prefectural Water Quality Control Center, 7-2, Murano-Takamidai, Hirakata, Osaka, 573-0014 Japan, The Water Works Service Foundation of Osaka Prefecture, 7-2, Murano-Takamidai, Hirakata, Osaka, 573-0014 Japan, and Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585 Japan
The inactivation of Cryptosporidium parvum was investigated by the use of three different sonicators utilizing the squeezefilm effect, which may occur when ultrasound is irradiated into an extremely thin space and generate intensified pressure in the sample suspension. To expand from the smallscale horn-type sonicator to large-scale cylindrical or cleaning bath sonicators, the inactivation effect was improved. In the case of the cylindrical sonicator (26.6 kHz, 30 W), 97% of the initial concentration of 2260 oocysts mL-1 was inactivated at 33 mL min-1 (residence time of approximately 5.2 min). Hundreds of cubic meters of water can be treated per day at several kW using this sonicator. In addition, the simultaneous use of sonication and chlorination showed a beneficial effect on inactivation for C. parvum based on the evaluation of infectivity testing and morphological observation.
Introduction Disinfection of hazardous microorganisms is an important subject for environmental safety. Especially, the water supply is facing a serious problem in the appearance of an emergent water pathogen that cannot be inactivated by conventional water treatments. The contamination from various pollution sources can expand immediately through the water supply and bring about various diseases. Cryptosporidium parvum and Giardia are two of the most apprehensive pathogenic protozoa because their hard cell wall, called the “oocyst”, shows high resistance to chemicals. At present, chlorination is the most common and cost-effective technique in the purification process of tap water utilities. However, it is impossible to use an increasing amount of chlorine because * Corresponding author phone/fax: +81-722-54-9321; e-mail:
[email protected]. † Graduate School of Engineering, Osaka Prefecture University. ‡ Research Institute for Advanced Science and Technology, Osaka Prefecture University. § Hitachi, Ltd. ⊥ Osaka Prefectural Water Quality Control Center. # The Water Works Service Foundation of Osaka Prefecture. ¶ Osaka City University Medical School. 7294
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it has been pointed out that carcinogenic byproducts are formed by the residual chlorine. C. parvum has been noticed since the first outbreak was reported in the 1980s. One of the most extensive epidemics occurred in Milwaukee, WI in 1993. In this case, approximately 400 000 people were infected with Cryptosporidiosis, accompanied by terrible diarrhea without any specific remedy (1). Similar accidents have also been reported in Japan. For example, several thousands of people in Saitama, Japan were infected in 1996. Due to such large-scale outbreaks of Cryptosporidiosis, many substitutable techniques for chlorination, such as filtration (2), ozonation (3, 4), ultraviolet (5), and their synergetic disinfection (6-8) have been developed for inactivation of C. parvum. Ozonation is one of the promising methods for inactivation of C. parvum. It has already been introduced into some tap water purification plants. However, the operation cost is high and the leaking of ozone into the air must be prevented because of its toxicity. Therefore, the use of alternative techniques has been strongly encouraged for disinfection of chemical-resistant microorganisms. Ultrasonic irradiation is a well-known method for microbial decontamination due to a physical effect (9). The ultrasonic procedure is handy and does not lead to the formation of hazardous byproducts compared to other methods. The irradiation by ultrasound of an aqueous solution generates cavitation, which leads to various chemical and physical phenomena, i.e., the occurrence of reactive free radicals (e.g., •OH, HO2•, and O•) and shock waves (or liquid jets) accompanied by the violent collapse of the cavitation bubble. Particularly, microscopic turbulence caused by shock waves, which is estimated at approximately 100 m s-1, acts effectively on the disruption of microorganism cell walls (10). Thus, ultrasonic irradiation was used for inactivation of various microorganisms (11), and some research has examined the ultrasonic inactivation of C. parvum as well (12, 13). However, it seems that the results are still insufficient for practical use because a high-intensity ultrasound is needed to achieve sufficient inactivation. One reason is the microbial agglomeration around the nodal region of the ultrasound standing wave. This may weaken the inactivation effect because cavitation occurs at the opposite side, i.e., the antinodal region. To prevent the agglomeration and improve the conflict of the reaction sites, we attempted to use the squeeze-film effect for sonication. The squeeze-film effect causes an increase in the interspace pressure when one of two close parallel plates is vertically vibrated with high frequency (14). This effect may occur when ultrasound is irradiated in an extremely thin space and generates cavitation in the entire sample suspension. Our previous study demonstrated that stable cavitation was generated by the squeezefilm effect, and Saccharomyces cerevisiae and Escherichia coli were effectively inactivated (15, 16). In this study, we used three different sonicators to investigate the squeeze-film effect for a batch and/or a flow system. To assess the practical use of ultrasound for tap water treatment, we attempted to simultaneously use sodium hypochlorite (NaOCl, 1 ppm) and ultrasound. After the ultrasonic irradiation, the damages to the oocysts were observed morphologically by use of a fluorescence microscope. In addition, we performed an infectivity test using mice, because C. parvum cannot germinate in artificial nutrition but can germinate in the intestines. 10.1021/es0502977 CCC: $30.25
2005 American Chemical Society Published on Web 08/16/2005
The reactor was open to the atmosphere, and the probe was inserted in the center of the reactor. (2) Cylindrical Sonicator. A 26.6 kHz cylindrical device (Hitachi, Ltd.) utilizing the squeeze-film effect was used for a larger-scale flow-through sonication (see Figure 1b). A cylinder connected to the transducer oscillated inside the device and transferred intensified pressure to the liquid within the squeeze-film. The thickness of the squeeze-film was 3 mm, the electrical power was 30 W, and the total volume of the squeeze-film part was 170 mL. The suspension of C. parvum was introduced into the sonicator at a constant flow rate by a pump and was withdrawn from an outlet after all of the suspension was sufficiently replaced. Little temperature rise was observed without cooling during the operation because the squeeze-film was surrounded by thick aluminum base alloy body. (3) Cleaning Bath Sonicator. As another large-scale device, a cleaning bath sonicator (Honda Electronics Co., Ltd.) was used. The dimensions of the reactor were W (68) × L (760) × H (e85) mm3. As shown in Figure 1c, the water surface was covered with a stainless steel plate as a reflector of the ultrasonic wave in order to create a squeeze-film. The volume of the reactor was 300 mL, and the height of the reflector was 5 mm, which was the optimal thickness to lead to the squeezefilm effect. The ultrasound was operated at an output power of 300 W and a frequency of 22.6 kHz. During sonication, cooling water was circulated around the reactor. The suspension was introduced to the sonicator at a flow rate of 10 mL min-1 by a pump and withdrawn from an outlet at the reverse side of the inlet after all of the suspension was sufficiently replaced. In addition, a combination treatment of sonication and chlorination was carried out by adding NaOCl (Wako)sto achieve an available concentration of chlorine of 1 ppmsto the sample container just prior to sonication.
FIGURE 1. Experimental setup for ultrasonic irradiation: (a) horntype sonicator, (b) cylindrical sonicator, (c) cleaning bath sonicator.
Experimental Section Source of Oocysts. The Cryptosporidium parvum HNJ-1 oocysts used in this study were isolated from an immunocompetent patient and maintained by subinoculation into severe combined immuno deficiency (SCID) mice at the Department of Medical Zoology, Osaka City University Medical School. The oocysts were collected from the feces of the infected mice by the sucrose centrifugal flotation method. The purified oocysts were maintained in the solution containing the antibodies (100 units mL-1 of penicillin and 100 µg mL-1 of streptomycin) at 4 °C and were used for sonication within one month. Then, test solutions were prepared by dilution of this solution to 103 oocysts mL-1. Sonication Apparatus. Sonication was carried out by using three apparatus designed to utilize the squeeze-film effect. All of the systems were equipped with a frequency generator (NF Electric Instrument), a broadband power amplifier (ENI), and a transducer. Details of each apparatus are given below. (1) Horn-Type Sonicator. The horn-type sonicator (27.5 kHz) was selected as a small-scale device for the fundamental experiment of the squeeze-film effect. Figure 1a shows the setup of this sonicator. Detailed information of this system was reported previously (15). The electrical power of the ultrasound was 52 or 126 W. The thickness between the tip of the probe and the bottom of the reactor was fixed (mainly 2 mm) to lead to the squeeze-film effect. A sample solution of 3 mL was sonicated in a cylindrical reactor, and ultrasound was transmitted into the solution through the tip of the probe.
Observation of Oocyst Morphology. After the sonication, Tween 80 was added to the sample to reach a concentration of 0.1% (v/v). Then, 50 mL oocyst suspensions were centrifuged at 1700g for 10 min and at 5300g for 3 min, respectively, and the supernatant was decanted to concentrate to one-tenth in volume. A 5 µL portion of concentrated suspension was placed into a tube and incubated in a water bath at 100 °C for 5 min. The oocyst wall was visualized by the addition of 95 µL of fluorescein isothiocyanate (FITC) labeled monoclonal antibody (cat. No. A400FL, Waterborne, Inc., New Orleans, LA) and incubated for 30 min at 37 °C. And then 10 µL of solution containing a 4 µg mL-1 concentration of 4′,6-diamino-2-phenylindole (DAPI) was added in order to detect the nuclei, which were contained in sporozoites of the oocyst, and the mixture was left at room temperature for 5 min. A 5 µL portion of the mixture was placed on a well slide and covered with a coverglass. The slides were examined by epi-fluorescence microscopy for oocyst walls labeled with FITC and nuclei labeled with DAPI. Five portions of each sample were observed in this way, and the results were expressed as means. Infectivity in Mice. Two SCID mice were inoculated with a known number (103, 104, 105 oocysts) of untreated oocysts through gastric gavage. Their feces were collected every 24 h to draw standard curves for the increasing number of oocysts excreted daily for 28 days. The number of days required to reach the concentration of 105 excreted oocysts is plotted against the gavaged untreated oocyst in Figure 2. Sonicated samples were concentrated to 1.5 mL, and two SCID mice were inoculated with 0.1 mL of the concentrated sample through gastric gavage as well. The number of infectious oocysts was calculated from the day taken to reach 1 × 105 oocysts in feces using the equation that resulted from the calibration curve presented in Figure 2. VOL. 39, NO. 18, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Calibration curve of the infectivity test between the number of days required to reach the concentration of 105 excreted oocysts and the gavaged untreated oocyst.
FIGURE 4. Time profile of the inactivation by the horn-type sonicator. 4 oocysts retaining the wall, b oocysts containing nuclei. Ultrasound power ) 52 W, volume ) 3 mL, cell number ) 103 mL-1, film thickness ) 2 mm.
FIGURE 3. Inactivation of C. parvum by the horn-type sonicator as a function of the thickness of the squeeze-film. Irradiation time ) 1 min, ultrasound power ) 52 W, oocyst number ) 103 mL-1.
FIGURE 5. Pictures of the oocyst observed by fluorescence microscopy (a) before sonication and (b) after sonication; (left) stained with FITC and (right) stained with DAPI.
Results and Discussions
with DAPI and FITC in Figure 5. Before the ultrasonic irradiation, the oocyst had a round shape and four nuclei (Figure 5a). After the ultrasonic irradiation the cell wall of the oocysts burst and the nuclei protruded (Figure 5b). Due to the shape change, we assume that the oocysts were disrupted by the shock waves rather than by the OH radicals that were generated by the squeeze-film sonicator with the violent collapse of the cavitation bubble. With the use of mice, infectivity tests were also examined for some samples. In the case of sonication at 52 W, 72.5% of the oocysts were inactivated after 60 s of sonication, while at 126 W, 94.9% were inactivated. The inactivated ratio estimated by the infectivity test was higher than that estimated by morphological observation. This means that some oocysts lost the ability to germinate although they still retained their wall and nuclei. These oocysts might be damaged by OH radicals without morphological changes. To simulate the effect of ultrasonic decontamination in natural environments, a suspension of C. parvum in raw (river) water was sonicated. Morphological observation and infectivity testing showed that the inactivation rate was almost
The horn type sonicator was employed for sonication of a small volume of suspension as a fundamental experiment of the squeeze-film. The very short distance between the top of the horn and the bottom of the reactor, which forms the squeeze-film, was varied from 1 to 4 mm. As shown in Figure 3, it was found that the inactivation ratio of C. parvum was highest when the thickness of the squeeze-film was 2 mm. Thus, the subsequent experiment using this sonicator was performed with the constant distance of 2 mm. A time profile of sonication was examined up to 300 s. The result obtained from morphological observation by florescence microscopy is shown in Figure 4. The reduction of oocysts stained with FITC was due to the complete disruption of the oocyst wall by ultrasonic cavitation. Some oocysts have no nuclei although their wall seemed to be intact. These “empty” oocysts were also regarded as nonviable ones. As shown in this figure, C. parvum oocyst was inactivated with increasing duration of sonication, and 80% of the oocysts were denuclearized in 300 s. We illustrate one example of the morphological observation of oocysts stained 7296
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TABLE 1. Inactivation Ratio of Flow-Through Sonication with Different Flow Rates by the Cylindrical Sonicator flow rate (mL min-1)
cell no. before treatment (cell/mL)
cell no. after treatment (cell/mL)
inactivation ratio (%)
33 240 1500 3000 4000
2260 2260 3330 3330 3330
69 252 752 1769 1785
97 89 78 46.9 46.4
the same as that of sterile pure water (date not presented), i.e., the inactivation effect of C. parvum did not depend on the purity of the water. Thus, the sonication process may be also introduced for backwash water to prevent oocyst to decontaminate river water. To improve the efficiency, we attempted to use a cylindrical sonicator with a larger capacity than that of the horn-type sonicator. Table 1 shows the result of the oncethrough inactivation by the cylindrical sonicator. At the flow rate of 33 mL min-1, 97% inactivation of 2260 oocysts mL-1 was achieved (the residence time was approximately 5.2 min), indicating much higher efficiency compared with that of the small-scale horn-type sonicator. With an increase of the flow rate, the inactivation ratio decreased due to the reduction of the residence time (θ ) V/Q) of the oocysts in the reactor. The advantages of the flow system for multistage sonication are the possibility of coupling the process with the conventional treatment plant and the controlling the temperature. In a continuous flow system, the reactor is regarded as a continuously stirred tank reactor, and the mass balance for a series of continuous flow reactors can be written as the following:
Qc0 - Qc - rV ) 0
(1)
where Q is the flow rate (mL min-1), c0 is the initial oocyst concentration (oocyst mL-1), c is the oocyst concentration after sonication, r is the inactivation rate (oocyst ml-1 min-1), and V is the volume of the reactor (mL-1). This equation can be applied to obtain the oocyst concentration in a series of n number of reactors since the inactivation of the C. parvum progresses by the pseudo first-order reaction
Cn )
C0 (1 + kθ)n
or
x)
kθ (1 + kθ)n
FIGURE 6. Reciprocal plot of the residence time against the inactivation ratio.
(2)
where Cn is the concentration of the oocyst in the nth reactor (oocyst mL-1) and x ) (c0 - c)/c0. From the plot between 1/x and 1/θ shown in Figure 6, the inactivation rate constant k was calculated and found to be 20.0 (min-1). By estimation using eq 2, for seven cylindrical sonicators connected in series at a flow rate of 1.7 L min-1, 99.96% of the C. parvum can be inactivated. To treat hundreds of cubic meters of water per day, six systems must be connected in parallel. This 7-series and 6-parallel lines system of sonicators at a required total electric power of 1.3 kW is readily available for practical use for the inactivation of C. parvum. In addition, this inactivation procedure was more efficient in terms of power consumption. In the present study the power consumption was only 210 W, while Biwater treatment Ltd. (12) achieved a 99.9% inactivation of the initial concentration of 240 oocysts mL-1 under a 6-times lower flow rate. A cleaning bath sonicator with a large volume was also tested since it is a popular apparatus in the majority of chemical laboratories. To enhance the efficiency of cavitation, we fixed a reflector on the water surface, by which the squeeze-film was developed as shown in Figure 1c. The result
FIGURE 7. Survival of oocysts of C. parvum determined by morphological counting stained with FITC and DAPI: (a) control, (b) sonication, (c) sonication and NaOCl (1 ppm).
TABLE 2. Mice Infectivity by Sonicated Oocysts in the Cleaning Bath Sonicator survival rate case
flow
flow (+NaClO)
1 2 3
0.058 0.067 0.047
0.019 0.013 0.011
obtained by morphology observation is shown in Figure 7. The optimal thickness of the squeeze-film was 5 mm. For flow treatment at 10 mL min-1, 95% of the oocysts were inactivated in 30 min. Although the efficiency of inactivation by the cleaning bath was insufficient for practical use in water treatment, it was confirmed that inactivation of C. parvum can be achieved even by a cleaning bath sonicator by applying the squeeze-film effect. Moreover, low-concentration sodium hypochlorite (1 ppm of available chlorine), which is commonly used for tap water purification, was added simultaneously with ultrasound. From the results of the morphological observation and infectivity testing using mice as described in Table 2, it became evident that the simultaneous use of sonication and VOL. 39, NO. 18, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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chlorination showed a beneficial effect on inactivation of C. parvum. However, C. parvum has a high resistance to chlorination and is usually impossible to inactivate by 1 ppm of NaOCl alone. It is obvious that the penetration of chlorine on the oocyst wall was increased due to the shock wave induced by the ultrasonic cavitation. The synergetic effect leads to cost reduction, and also the risk of carcinogenic byproducts formation is lowered. These results showed that the squeeze-film effect is highly applicable to larger sonication systems for practical use in water purification. Moreover, ultrasonic treatment is useful for the relatively high concentration of oocysts that existed in filter backwash water. The running cost of the sonicator is much lower than that of a conventional ultrasonic sterilizer, although it seems to still be higher than that of ozonation. It was demonstrated that the use of a cylindrical sonicator for the water purification process following chlorination was most effective.
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(6) Rennecker, J. L.; Kim, J.-H.; Vasquez, B. C.; Marinas, B. J. Role of disinfectant concentration and pH in the inactivation kinetics of Cryptosporidium parvum oocysts with ozone and monochloramine. Environ. Sci. Technol. 2001, 35, 2752. (7) Cho, M.; Chung, H.; Yoon, J. Quantitative evaluation of the synergistic sequential inactivation of Bacillus subtilis spores with ozone followed by chlorine. Environ. Sci. Technol. 2003, 37, 2134. (8) Kanjo, Y.; Kimata, I.; Isaki, M.; Miyanaga, S.; Okada, H.; Banno, C.; Matsumoto, M.; Shimada, Y. Inactivation of Cryptosporidium spp. oocysts with ozone and ultraviolet irradiation evaluated by in vitro excystation and animal infectivity. Water Sci. Technol. 2000, 41, 119. (9) Sinisterra, J. V. Application of Ultrasound to Biotechnologysan overview. Ultrasonics 1992, 30, 180. (10) Phull, S. S.; Mason, T. J. Advances in Sonochemistry; JAI Press, 2001; Vol. 6, pp 1-23. (11) Scherba, G.; Weigel R. M.; Obrien, W. D. Quantitative assessment of the germicidal efficacy of ultrasonic energy. Appl. Environ. Microbiol. 1991, 57, 2079. (12) A1 Biwater Treatment Ltd. European Patent 0 567 225, 1993. (13) Ashokkumar, M.; Vu, T.; Grieser, F.; Weerawardena, A.; Anderson, N. Pilkington, N.; Dixon, D. R. Ultrasonic treatment of Cryptosporidium oocysts. Water Sci. Technol. 2003, 47, 173. (14) Hashiba, K.; Kawabata, K.; Masuzawa, H.; Kimata, I.; Maeda, Y. Stable Cavitation Fields by Using Squeeze-Film Effect, Proceeding of 9th Symposium of the Japan Society of Sonochemistry; 2000; 41. (15) Tsukamoto, I.; Yim, B.; Stavarache, C. E.; Furuta, M.; Hashiba, K.; Maeda, Y. Inactivation of Saccharomyces cerevisiae by ultrasonic irradiation. Ultrason. Sonochem. 2004, 11, 61. (16) Furuta, M.; Yamaguchi, M.; Tsukamoto, I.; Yim, B.; Stavarache, C. E.; Hasiba, K.; Maeda, Y. Inactivation of Escherichia coli by ultrasonic irradiation. Ultrason. Sonochem. 2004, 11, 57.
Received for review February 14, 2005. Revised manuscript received June 7, 2005. Accepted July 18, 2005. ES0502977
