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ABSTRACT The effects of supercritical CO2 bubbling (SC-CO2) treatment on the inactivation of microorganisms in water prior to treatment at a municipal water filtering plant (untreated water) were investigated as a way to produce safe drinking water. The coliform bacterial count decreased concomitantly with increasing CO2/sample flow rate in the SC-CO2 treatment. In particular, coliform bacteria could not be detected at a CO2/sample flow rate greater than 55%. Also, the total bacterial count dropped rapidly at first stage and slowly at second stage in the SC-CO2 treatment. Upon observation of Escherichia coli before and after the SC-CO2 treatment with scanning electron microscopy and transmission electron microscopy, it was observed that the cells treated with SC-CO2 were shorter than untreated cells and that cytoplasm with low electronic density in the treated cells disappeared. In addition, four types of metabolic enzyme in E. coli cells were effectively inactivated by the SC-CO2 treatment. These results suggested that SC-CO2 treatment could effectively inactivate microorganisms in untreated water, and induce morphological changes and inactivate metabolic enzymes of E. coli cells
Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 241 - Inactivation of microorganisms in untreated water by a continuous flow system with supercritical CO 2 bubbling Fumiyuki KOBAYASHI*, Futoshi YAZAMA**, Hiromi IKEURA*, Yasuyoshi HAYATA* Norio MUTO** and Yutaka OSAJIMA*** * School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan ** Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562, Nanatsuka, Shobara, Hiroshima 727-0023, Japan *** Faculty of Agriculture, Kyusyu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka, 807-8586, Japan ABSTRACT The effects of supercritical CO 2 bubbling (SC-CO 2 ) treatment on the inactivation of microorganisms in water prior to treatment at a municipal water filtering plant (untreated water) were investigated as a way to produce safe drinking water. The coliform bacterial count decreased concomitantly with increasing CO 2 /sample flow rate in the SC-CO 2 treatment. In particular, coliform bacteria could not be detected at a CO 2 /sample flow rate greater than 55%. Also, the total bacterial count dropped rapidly at first stage and slowly at second stage in the SC-CO 2 treatment. Upon observation of Escherichia coli before and after the SC-CO 2 treatment with scanning electron microscopy and transmission electron microscopy, it was observed that the cells treated with SC-CO 2 were shorter than untreated cells and that cytoplasm with low electronic density in the treated cells disappeared. In addition, four types of metabolic enzyme in E. coli cells were effectively inactivated by the SC-CO 2 treatment. These results suggested that SC-CO 2 treatment could effectively inactivate microorganisms in untreated water, and induce morphological changes and inactivate metabolic enzymes of E. coli cells. Keywords: supercritical carbon dioxide bubbling, total and coliform bacteria, electron microscopy INTRODUCTION Chlorine inactivation has generally been used to inactivate microorganisms in tap water. However, it has been suggested that chlorine is toxic to the human body even at low concentrations. In particular, there is a possibility that trihalomethanes, which are carcinogenic agents, are produced by the chemical reaction between chlorine and organic compounds in water (Graham et al., 1989). Thus, bottled water consumption has rapidly increased, as many consumers prefer to drink non-chlorinated water. However, imported bottled water might not be sanitary, because it is not subjected to the water quality standards used for drinking water (Satsuta et al., 2001; Warburton, 1993). Therefore, many scientists have proposed alternative methods to chlorine inactivation to inactivate microorganisms in drinking water, such as titanium dioxide photocatalytic reaction (Wist et al., 2002), iodine (Backer and Hollowell, 2000) and ozone (Cho et al., 2003) treatments. Pasteurization using pressurized carbon dioxide has been widely studied in the field of food science (Lin et al., 1992, 1993; Ballestra et al., 1996; Louka et al., 1999; Hong and Pyun, 1999, 2001; Gunes et al., 2006; Garcia-Gonzalez et al., 2009). Ishikawa et al. (1995a) developed supercritical carbon dioxide bubbling (SC-CO 2 treatment), which Address correspondence to Yasuyoshi HAYATA, School of Agriculture, Meiji University, Email: yhayata@isc.meiji.ac.jp Received March 11, 2009, Accepted July 29, 2009. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 242 - can increase the dissolved CO 2 concentration in a solution with the use of a filter, and Shimoda et al. (1998, 2001, 2002) reported that some bacteria could be inactivated by SC-CO 2 treatment. In our previous papers, we reported that certain compounds that give tap water a musty odor could be removed by a continuous flow treatment with SC-CO 2 (Kobayashi et al., 2006) and suggested that inactivation of Escherichia coli in sterile distilled water and coliform bacteria in water prior to treatment at a municipal water filtering plant (untreated water) by the SC-CO 2 treatment depended on the dissolved CO 2 concentration (Kobayashi et al., 2007a). In this study, we aim to determine the effects of the CO 2 /sample flow rate and exposure time in the SC-CO 2 treatment on the inactivation of coliform and total bacteria in untreated water and to propose the SC-CO 2 treatment as a novel method for inactivating microorganisms in drinking water. In addition, we aim to investigate the effect of the SC-CO 2 treatment with regard to structural damage and enzyme inactivation in E. coli cells, which we previously demonstrated could be reduced to nondetectable levels by the SC-CO 2 treatment (Kobayashi et al., 2007a). MATERIALS AND METHODS Untreated water As described in our previous paper (Kobayashi et al., 2007a), water was taken from a municipal water filtering plant (Miyoshi, Hiroshima, Japan) prior to being treated for use as tap water. The coliform bacteria and total bacterial counts in this untreated water were approximately 4.0×10 2 MPN/100ml and 9.0×10 2 ～1.2×10 3 CFU/ml, respectively. Preparation of E. coli suspensions Strains of E. coli (ATCC 11775) were inoculated to each test tube containing 10 ml of nutrient broth (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and incubated at 37°C for 16 hr. The cultures were then transferred to 300 ml flasks containing 190 ml of nutrient broth and incubated at 37°C for 24 hr. Microorganisms were collected by centrifuge (4°C, 8000 rpm, 10 min) and suspended in sterile distilled water at approximately 5.0×10 8 CFU/ml. Continuous flow treatment with SC-CO 2 bubbling The schematic diagram of the device used for continuous flow treatment with SC-CO 2 bubbling is shown in Figure 1. CO 2 compressed by a pump was bubbled through a filter (with 10 μm pore size) in a mixing vessel (about 10 ml in volume) and mixed with the sample water. The mixed sample and SC-CO 2 were fully agitated in a coil (about 30 ml in volume) and delivered to a gas-liquid separation vessel (about 160 ml in volume), and the foul-odor components in the sample were transferred to SC-CO 2 , discharged from valve I and collected in a knockout drum. When the surface of the sample in the gas-liquid separation vessel touches thermocouple I, the sample is discharged from valve II and decompressed to atmospheric pressure. When the surface of the sample loses contact with thermocouple II, the discharge of the sample is stopped. Before this device was used, a 0.1% sodium hypochlorite solution was made to flow into the vessel for 30 min, and the device was sufficiently washed with sterile distilled water. The SC-CO 2 treatment conditions were as follows: temperatures were 35, 45 and 55°C, pressure was 10 MPa, sample flow rates were 2.5, 5.0 and 15 ml/min, CO 2 flow rates were 4.0, 6.0, 8.0 and 10 g/min and exposure times were 13, 40 and 80 min. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 243 - CO 2 /sample flow rate was calculated as follows: CO 2 /sample flow rate = CO 2 flow rate/sample flow rate ×100 valve I pump heating coil pump heater cooler agitation coil knockout drum treatment sample microfilter (pore size 10 μm) thermocouple II mixing vessel valve II thermocouple I gas-liquid separation vessel CO 2 CO 2 sample CO 2 drain Figure 1 - Schematic diagram of the experimental apparatus for a continuous flow treatment with SC-CO 2 bubbling. Measurement of surviving bacteria The coliform and total bacterial counts in the untreated water were determined using the method of Japan Water Works Association (JWWA, 2001). For this measurement, 1 ml of each sample diluted with physiological saline solution was added to each of five test tubes (φ18 mm × 180 mm), each of which contained 10 ml of lactose bile (LB) broth (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and a small glass tube (φ8 mm × 30 mm), and samples were incubated at 37°C for 48 hr. The cultured LB broth was then transferred to test tubes (φ18 mm × 180 mm) that contained 10 ml of brilliant green lactose bile (BGLB) broth (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and a small glass tube (φ8 mm × 30 mm), with a platinum loop, and incubated at 37°C for 48 hr. Based on the presence of gas in the small glass tube, the most probable number (MPN) was calculated and the coliform bacterial count was determined. The detection limit of the coliform bacterial count by the detection method was 0 MPN/100ml (JWWA, 2001). We also determined the total bacterial count using the standard plate counting method. One ml of appropriate dilutions of samples was inoculated in standard plate count agar (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and aerobically incubated at 37°C for 48 hr, and the colony-forming units (CFU) were counted. The coliform and total bacterial counts were expressed in MPN/100ml and CFU/ml, respectively (JWWA, 2001). The data presented are the means with standard errors of the results of experiments performed in triplicate. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 244 - Electron microscopy E. coli cells before and after SC-CO 2 treatment were prefixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.2) at room temperature for 2 hr. For scanning electron microscopy (SEM), the fixed samples were then rinsed with PB and post-fixed with 1% osmium in 0.1M PB (pH 7.2) at 4°C for 2 hr. After dehydration in a graded concentration of ethanol, immersion in t-butyl alcohol and drying using a freeze drier (ES-2030, Hitachi, Tokyo, Japan), the samples were sputter-coated with gold-palladium and examined in a Hitachi S-2460N scanning electron microscope operated at 10 kV. For transmission electron microscopy (TEM), the pre-fixed samples were fixed with 1% osmium in 0.1M PB (pH 7.2) at 4°C for 2 hr. After being rinsed with distilled water, the pellets of the fixed cells were embedded in 1% agar, dehydrated using an ethanol series and embedded in Epon 812. Ultra-thin sections were doubly stained with uranyl acetate and lead nitrate and then examined with a JEM-1200 EX transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV. Five SEM photographs were taken for each E. coli cells before and after SC-CO 2 treatment, ten cells were randomly selected from SEM photographs, and the length and width of cells were measured. The data presented was the means with standard deviation of five replications and subjected to the least significant differences (LSD) test (P<0.01). Measurement of enzyme activity Enzyme activities in E. coli cells before and after the SC-CO 2 treatment were measured with an APIZYM kit (BioMerieux, Marcy-l’Etoile, France). This kit permits monitoring of 20 different constitutive enzyme activities from a complex sample that had not been purified (Ballestra et al., 1996). The number of E. coli cells to measure the enzyme activities was approximately 5.0×10 8 CFU/ml. All enzyme activities were visually measured based on color changes, which fit with the increasing quality of hydrolyzed substrate (0-40 nmoles). The data presented were expressed as activity marks (0-5) which corresponded to hydrolyzed substrate of 0-40 nmoles and are the means with standard errors of the results of triplicate experiments. RESULTS AND DISCUSSION Inactivation of microorganisms in untreated water by SC-CO 2 treatment The effect of CO 2 /sample flow rate in SC-CO 2 treatment on the inactivation of coliform bacteria in untreated water is shown in Figure 2. The coliform bacterial count in the untreated water decreased almost linearly with increasing CO 2 /sample flow rate in the SC-CO 2 treatment. In particular, coliform bacteria could not be detected at a CO 2 /sample flow rate greater than 53%. In our previous papers, we reported that the ability of inactivating microorganisms by SC-CO 2 treatment depended on the dissolved CO 2 concentration, which increased concomitantly with increasing CO 2 /sample flow rate (Kobayashi et al., 2007a, b). Dissolved CO 2 can easily diffuse into the bacterial cell due to increased membrane permeability and accumulates in the cytoplasmic interior, decreasing the intracellular pH. Cell viability will seriously be impaired owing to a drop of intracellular pH (Garcia-Gonzales et al., 2009). Therefore, it was considered that the increase of the CO 2 /sample flow rate induced more cellular penetration of CO 2 . These results suggested that the SC-CO 2 treatment was very effective for inactivating coliform bacteria in untreated water due to the increase in the CO 2 /sample flow rate. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 245 - The effect of temperature during the SC-CO 2 treatment on the inactivation of total bacteria in untreated water is shown in Figure 3. The total bacterial count in the untreated water dropped rapidly at first stage and slowly at second stage in the SC-CO 2 treatment and the inactivation rate increased as the temperature increased. The survival curves described by a rapid-to-slow two stage kinetics were in agreement with the findings of some earlier studies (Ishikawa et al., 1995b; Louka et al., 1999; Liao et al., 2007). However, there were some previous reports in which the survival curves were described by a slow-to-rapid two stage kinetics (Ballestra et al., 1996; Oulé et al., 2006). The reported differences in inactivation kinetics may be due to the difference in the efficiency of contact between CO 2 and the microorganisms (Zhang et al., 2006). Therefore, it was considered that our present result was obtained by the difference of the penetration of CO 2 into the bacterial cells. Since it is stated by law that the number of coliform and total bacteria must be kept less than 0 and 100 CFU/ml, respectively (JWWA, 2001), SC-CO 2 treatment at the CO 2 /sample flow rate higher than 67% and exposure time longer than 13 min might become a sterilization method to produce drinking water. 3 1 2 0 Log of viable cells (MPN/100 ml) CO 2 /sample flow rate (%) 20 30 10 0 40 70 60 50 * * Figure 2 - Effect of CO 2 /sample flow rate in SC-CO 2 treatment on the inactivation of coliform bacteria in untreated water. SC-CO 2 treatment conditions: temperature 35°C, pressure 10 MPa, CO 2 flow rate 4.0, 6.0, 8.0 and 10 g/min, sample flow rate 15 ml/min, CO 2 /sample flow rate 27, 40, 53 and 67%, exposure time 13 min. * not detected. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 246 - 4 1 3 2 0 Exposure time (min) Log of viable cells (CFU/ml) 0 20 40 80 60 100 Figure 3 - Effect of temperature during the SC-CO 2 treatment on the inactivation of total bacteria in untreated water. SC-CO 2 treatment conditions: temperature 35(◆), 45(■) and 55°C(▲), pressure 10 MPa, CO 2 flow rate 10 g/min, sample flow rate 2.5, 5.0 and 15 ml/min, CO 2 /sample flow rate 67, 200 and 400%, exposure time 13, 40 and 80 min. Electron micrographs of E. coli cells before and after SC-CO 2 treatment SEM photographs of E. coli cells before and after SC-CO 2 treatment are shown in Figure 4. In an earlier study we confirmed that surviving E. coli cells could not be detected after SC-CO 2 treatment (Kobayashi et al., 2007a). The damage in E. coli cells by SC-CO 2 treatment could not be observed by SEM. Ballestra et al., (1996) found that 25% of E. coli cells treated by pressurized CO 2 were intact, but the cell viability was only 1%. Since their reports agreed with our present result, we suspect that the E. coli cells treated with SC-CO 2 were not necessarily killed by cellular membrane damages. However, the lengths of E. coli cells treated with SC-CO 2 were shortened by 18% (from 1.067 to 0.876 μm), while the widths were hardly changed from 0.583 to 0.561 μm (Figure 5). TEM photographs of E. coli cells before and after SC-CO 2 treatment are shown in Figure 6. On the observation with TEM, the cell membranes might be slightly damaged by the SC-CO 2 treatment. We also found during TEM observation that cytoplasm with low electronic density in E. coli cells subjected to the SC-CO 2 treatment had disappeared. Hong and Pyun (1999, 2001) found that a possible leakage of cytoplasm was caused by the modification of the cell membrane of L. plantarum by pressurized CO 2 . In our present results, the leakage of cytoplasm could not be confirmed, although the leakage is somehow conveyed by our observations. Thus, it was considered that the shortening of E. coli cell indicated by SEM photograph might show the leakage of cytoplasm caused by damaging the cell membrane, when E. coli was killed by the SC-CO 2 treatment. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 247 - Figure 4 - SEM photographs of E. coli cells before (A) and after (B) SC-CO 2 treatment. SC-CO 2 treatment conditions: temperature 35°C, pressure 10 MPa, CO 2 flow rate 10 g/min, sample flow rate 15 ml/min, CO 2 /sample flow rate 67%, exposure time 13 min. Length Width 0 0.2 0.4 0.6 0.8 1.0 1.2 * Size (μm) Figure 5 - The size of E. coli cells before (■) and after (■) SC-CO 2 treatment. SC-CO 2 treatment conditions: temperature 35°C, pressure 10 MPa, CO 2 flow rate 10 g/min, sample flow rate 15 ml/min, CO 2 /sample flow rate 67%, exposure time 13 min. Five SEM photographs were taken for each E. coli cells before and after SC-CO 2 treatment, ten cells were randomly selected from SEM photographs, and the length and width of cells were measured. The data presented was the means with standard deviation of five replications. Asterisk (*) indicated significant difference between the size of E. coli cells before and after SC-CO 2 treatment by the least significant differences (LSD) test (P<0.01). Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 248 - Figure 6 - TEM photographs of E. coli cells before (A) and after (B) SC-CO 2 treatment. SC-CO 2 treatment conditions: temperature 35°C, pressure 10 MPa, CO 2 flow rate 10 g/min, sample flow rate 15 ml/min, CO 2 /sample flow rate 67%, exposure time 13 min. Enzyme activities in E. coli cells before and after the SC-CO 2 treatment To investigate the effect of the SC-CO 2 treatment on the cytoplasm in E. coli cells (Figure 7), we measured seven types of metabolic enzyme activities in E. coli cells before and after the SC-CO 2 treatment with an APIZYM kit. Four types of metabolic enzymes, Leucine arylamidase, β-galactosidase, β-glucuronidase and α-glucosidase, were completely inactivated by the SC-CO 2 treatment, whereas phosphatase (alkaline and acid) or naphthol-AS-BI-phosphohydrolase was slightly or a little inactivated. Ishikawa et al. (1996) reported that enzyme inactivation by the SC-CO 2 treatment was caused by irreversible destruction of the α-helix structure of protein enzyme. Ballestra et al. (1996) suggested that selective inactivation of enzymes could result from a drop of internal pH of the microorganisms during the pressurized CO 2 treatment. The dissociation of dissolved CO 2 , present in large amounts in the cells, would lower the internal pH of the bacteria to a critical level which would induce inhibition of essential metabolic systems. Thus, we considered that these phenomena observed by SEM and TEM might have been caused by the denaturation of intracellular protein. Activity mark (0-5) 0 1 2 3 4 5 A B C D E F G Figure 7 - Enzyme activities in E. coli cells before (■) and after (■) SC-CO 2 treatment. Alkaline phosphatase (A), Leucine arylamidase (B), Acid phosphatase (C), Naphthol-AS-BI-phosphhydrolase (D), β-galactosidase (E), β-glucuronidase (F), α-glucosidase (G), SC-CO 2 treatment conditions: temperature 35°C, pressure 10 MPa, CO 2 flow rate 10 g/min, sample flow rate 15 ml/min, CO 2 /sample flow rate 67%, exposure time 13 min. Journal of Water and Environment Technology, Vol. 7, No. 4, 2009 - 249 - CONCLUSION In the present study, SC-CO 2 treatment was effective for inactivating microorganisms in untreated water. 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Supercritical Fluids, 38, 354-372. . Kanagawa, 214-8 571 , Japan ** Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562, Nanatsuka, Shobara, Hiroshima 72 7-0023, Japan. (ATCC 1 177 5) were inoculated to each test tube containing 10 ml of nutrient broth (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and incubated at 37 C for