Correlation coefficients among various operating parameters associated with pressure cycling and the
Advanced water disinfection technologies that do not produce harmful by-products would be highly desirable. This study presents results for the use of pressurized carbon dioxide (CO2) and a liquid-film-forming apparatus for disinfection of seawater. The sensitivity of Escherichia coli to the pressurized CO2 was examined for various conditions of pressure, temperature, working volume ratios (WVRs), flow rates, and pressure cycling. Morphology of E. coli was observed by using scanning electron microscopy (SEM). A strong correlation between the E. coli inactivation efficiency and pressure cycling was detected (p < 0.001). The frequency and magnitude of pressure cycling were the key factors responsible for high rates of E. coli inactivation during the pressurized CO2 treatment. The results from linear regression analyses suggest that the model can explain about 91% of the E. coli inactivation efficiency (p < 0.001). The pressurized CO2 treatment (at 0.7 MPa, 20°C, 50% WVR) in the process involving pressure cycling (∆P = 0.12 MPa, 15 cycles) resulted in complete inactivation (5.2 log reduction) of E. coli within 3 min. These findings suggest that pressurized CO2 could be a potentially useful disinfection method for water treatment.
- bactericidal performance
- Escherichia coli
- inactivation effect
- pressurized carbon dioxide
- water disinfection
For more than a century, chlorination has been the most common method used worldwide for drinking water disinfection. Chlorine and chlorine-based compounds are widely used for the control of waterborne pathogens because of their high oxidizing potential, low cost, and residual disinfectant properties that prevent microbial recontamination. Unfortunately, the chemical reaction between chlorine and organic compounds in water generates carcinogenic agents such as trihalomethanes and halogenic acetic acids [1, 2]. Furthermore, some resistant microorganisms may only be inactivated with very high chlorine doses, which can exacerbate the formation of disinfection by-products (DBPs) . Presently, growing concerns about the potential hazards associated with DBPs have boosted efforts to develop chlorination alternatives. Ozonation is effective at inhibiting a variety of pathogens; however, its disadvantages include the high cost and the potential formation of DBPs such as bromate in seawater [4, 5]. Other water treatment methods such as ultraviolet (UV) radiation, ultrasound, cavitation, or heat application can be used for the inactivation of organisms. Although these methods do not produce DBPs or other problematic chemical residues, they require substantial energy consumption and have high operational costs . Besides, the efficiency of UV disinfection is greatly dependent on water quality because the activity of UV light is substantially decreased by turbidity or organic matter present in water .
Sterilization by using pressurized CO2 has been an active research field for decades [6, 7]. CO2 has been used extensively to sterilize dried food and liquid products via a nonthermal sterilization method  because of its effectiveness in inactivating microbes, nontoxicity, and low cost . Prior research on high-pressure CO2 treatments has investigated the effects of several factors such as pressure, temperature, type of microorganisms, agitation speed, decompression rate, and pressure cycling on the inactivation capacity of this method [6, 8, 10–15]. Most studies have reported that high-pressure operating conditions (4–50 MPa) are required to inactivate significant numbers of pathogens [7, 9]. Subsequently, certain concerns involving high-pressure operations (i.e., the need for heavy-duty pressure equipment, high initial investment costs, energy consumption concerns, and pressure control and management issues) have hampered the implementation of high pressurized CO2 preservation technology at a large scale within the food industry.
In recent years, pressurized CO2 has shown great potential as a sustainable disinfection technology in water and wastewater treatment applications [16–22] largely because this method does not generate DBPs [9, 22]. Kobayashi et al. [16, 17] employed CO2 microbubbles in the treatment of drinking water and succeeded in inhibiting
Previous research has shown that pressure cycling is a potential means to improve bacterial inactivation during pressurized CO2 treatments [8–10, 13, 15]; nevertheless, the inactivation mechanism is still unknown for this process. Pressure cycling is defined as a repetitive procedure that involves the decompression and compression of CO2 [9, 10]. Evidence so far suggests that the decompression process may lead to mechanically induced explosive cell ruptures , while the compression process may intensify the mass transfer of CO2 across cell membranes [11–13]. In previous works, the pressure cycling procedure has been conducted with high-pressure operations (8–550 MPa) and with CO2 discharges between each cycle of decompression and compression [8, 10, 13, 15]. Despite the good bactericidal performance of pressurized CO2 technology enhanced by pressure cycling [11–13, 15], the high pressure and CO2 release requirements are drawbacks owing to the costly and complex operating procedures. Presently, it is not clear whether pressure cycling with low-pressure CO2 treatments (<1.0 MPa) will enhance the bactericidal activity. Therefore, in this study, we examined the effect of pressure cycling on the bactericidal performance of CO2 at low pressures and with no release of CO2 between each cycle of raised/lowered pressure.
This study investigated the use of pressurized CO2 at less than 1.0 MPa for seawater disinfection applications such as ballast water treatment. Comparisons of
2. Novel idea: apparatus for forming highly dissolved gas in water
For pressurized CO2 methods in the field of food preservation, the interaction efficiency between CO2 and pathogens in the foodstuffs is probably limited at low pressures and ambient temperatures, and consequently, high-pressure (4–50 MPa) or ultra-high-pressure (200–700 MPa) conditions are vital for sufficient inactivation. However, to be more attractive in terms of its economic feasibility, pressurized CO2 technology needs to be implemented at lower pressures. In this study, we employed the use of a liquid-film-forming apparatus, which enabled improvements in the interaction efficiency but with lower pressures (<1 MPa) for the water disinfection purposes.
The experimental apparatus for disinfection was a stainless steel chamber with an internal volume of 10 L and pressure tolerance up to 1.0 MPa. The device was designed with a solid stream nozzle and shield to enable vigorous agitation of the influent in such a way that produced liquid films along with fine bubbles (Figures 1–3). The device was supplemented with CO2 pressure prior to the treatments. Sample water was then pumped into the device at high speed through a small nozzle and directed onto the shield. The highly pressurized fluid stream thus collided with the bubble-generating shield. Subsequently, numerous gas bubbles, which were generated from inside the shield, were entrained by the ascending bubbles and overcame the shield; these bubbles then floated into the main chamber (outside the shield). Hence, CO2 transfers took place both within the interior and exterior sides of the thin liquid films. The presence of numerous small bubbles also enhanced the contact area between gas and water and facilitated CO2 dissolution into water. We hypothesized that the available interfacial contact area between CO2 and the cell suspension was greatly multiplied in this setup and that the CO2 transfer efficiency was high. Despite the lower pressures used, the high contact efficiency promoted by this apparatus enabled ample penetration of CO2 into the cell membranes of
3. Materials and methods
3.1. Microorganism preparation and enumeration
Stock cultures of
3.2. Seawater sample preparation
The artificial seawater was prepared by adding artificial sea salt (GEX Inc., Osaka, Japan) to distilled water to obtain a final salinity of 3.4%, as measured with a salinity meter (YK-31SA, Lutron Electronic Enterprice Co., Ltd., Taiwan). As for the preparation of filtered natural seawater, natural seawater (pH = 8.3, salinity 3.3%) was first filtered through a glass fiber filter (GA-100, Advantec, Toyo); then, the seawater was filtered through a membrane filter with a pore size of 0.45 μm (Millipore, Ireland). For all experiments, prepared
3.3. Experimental setup
Disinfection experiments were conducted in batch mode (Figure 4). Sample water, as the influent, was pumped in one shot into the device. Following the first influx of water, pressurized CO2 was also injected into the main chamber. System pressure was adjusted by a gas pressure regulator and gas exhaust valve. The fluid was then circulated by pumping inside the system for 25 min. A pump was used to apply a higher pressure than that inside the main chamber to accelerate gas solubilization in water. During the treatment period, the outer wall of the device was kept in contact with cool water by using a water jacket to maintain the initial temperature of the sample at ±1.0°C. The treated water was then collected from a bottom valve of the device.
3.4. Procedure for disinfection experiments
3.4.1. Experimental procedure for investigating the effects of pressure and temperature
To investigate the effects of pressure and temperature, 7 L of sample were pumped into the main chamber by using a 0.2 kW pump (Iwaya-WPT-202), and the fluid was circulated inside the system at a flow rate of 14 L min−1 (hydraulic retention time, HRT = 0.5 min). The pump was used to apply 0.12 MPa higher pressure than that inside the main chamber. The sensitivity of bacteria to pressurized CO2 treatments under different conditions was determined by varying the CO2 pressure (0.2–0.9 MPa) and seawater temperature (11–28°C) for a 25-min treatment period . Each experiment was conducted in triplicate.
3.4.2. Experimental procedure for investigating the effect of pressure cycling
In previous works, the pressure cycling procedure was conducted with high-pressure operations (8–550 MPa) and with CO2 discharges between each cycle of decompression and compression [8, 10, 13, 15]. However, such high pressure and CO2 release are undesirable from an economic standpoint. In order to overcome the above disadvantages, in the present study, we employed a process involving pressure cycling for
To investigate the effect of pressure cycling, two pumps (0.20 kW, Iwaya-WPT-202, Japan; 0.75 kW, 32 mm × 32 mm SUP-324 M, Toshiba, Japan) and nozzles with various sizes (15 mm height × 4–8 mm diameter) were used to change the flow rate and pressure power of the input (a treatment without a nozzle was also used, whereby the diameter of the pipeline inlet was 15 mm). Pumping pressure and system pressure were measured by pressure gages. The pressure difference ΔP = pumping pressure (MPa) − pressure inside the main chamber (MPa). The water flow rate was measured by a flow meter (GPI, Nippon Flow Cell Co., Ltd., Japan). The recycle number was calculated in relation to the treatment time and HRT, wherein HRT = sample volume/flow rate.
3.4.3. Experimental procedure for investigating the effect of the working volume ratio
The WVR is defined as the ratio between the sample volume and apparatus volume. To examine the effect of WVR, different sample volumes (5, 6, 7, and 8 L) were used to vary the sample volume ratios (50, 60, 70, and 80%). The experiment was conducted with the following two flow rate levels: 14 and 25 L min−1. The water level was measured by using a gauge to evaluate the effect of WVR on the bubble-generating shield inside the main chamber. The HRT and recycle number were calculated as described in Section 3.4.2.
3.5. Scanning electron microscopy
Changes in cell morphology after pressurized CO2 treatment were assessed by using SEM. The pellets of
3.6. Statistical analysis
The statistical analysis was done by using the statistical computer program R (version 3.2.2, available at http://cran.R-project.org). Multicollinearity regression was performed to evaluate statistically significant variables of the system with a significance level of 0.05. Predicted values of inactivation efficacy were based on the following first-order regression model:
4. Results and discussion
4.1. Bactericidal performance of pressurized CO2 and pressurized air against
E. coliin seawater
Bactericidal effects of pressurized CO2 in comparison with pressurized air against
Pressurized CO2 reduced the pH of both filtered seawater and artificial seawater to around 5.0 after the first few minutes of exposure time, whereas the pH of pressurized air-treated seawater remained around 8.3 during the treatment period (Figure 5b). It has been hypothesized that the decrease in pH caused by pressurized CO2 is probably a major factor driving the bacterial inactivation process [12, 20, 21, 25]. However, Dang et al.  demonstrated that the low pH alone is not the main cause of the bactericidal activity. Perhaps with the concomitant presence of pressure and dissolved CO2, the low pH prompted the
4.2. Effects of pressure and temperature
The disinfection efficiency of pressurized CO2 substantially increased with increasing temperatures (11–28°C) at 0.7 MPa (Figure 7). The
CO2 is lipo-hydrophilic in nature, and it can easily penetrate into the phospholipid bilayer of cell membranes . Thus, the increase in CO2 pressure and temperature may stimulate the diffusion of CO2 into cells and may increase the fluidity of cell membranes [11, 27]. In the present study, the solubility of CO2 into seawater was considerably improved by using the liquid-film-forming apparatus. Hence, we speculate that simultaneous effects of pressure, temperature, and high efficiency of contact with this apparatus may have stimulated the process of CO2 penetration into
4.3. Effect of pressure cycling
4.3.1. Effect of pressure cycling at various pump powers and nozzle diameters
The effect of pressure cycling on
It is hypothesized that pressure cycling enhances the inactivation efficiency by facilitating the mass transfer of CO2 into bacterial cell membranes [9, 10]. Thus, an increase in water flow rate can be expected to improve the
On the other hand, the disinfection efficiency substantially increased with the higher ΔP (Figure 8). A 5.4 log reduction in
Noticeably, at the same ΔP value, a faster frequency of circulation substantially augmented the
Table 1 summarizes the coefficients of correlation for the inactivation efficiency and parameters associated with pressure cycling, including the nozzle diameter (
Although the use of small nozzle diameters was associated with effective inactivation, operating conditions at high ΔP values and low flow rates may be more complex and of lesser economical interest. The highest inactivation efficiency was observed when 5–7 mm nozzle diameters and the 0.75 kW pump were used (Figure 8b). Since a large processing capacity is of great commercial interest, the 7 mm nozzle and 0.75 kW pump were used for subsequent experiments.
4.3.2. Effect of pressure cycling at various WVRs
The effect of WVR was investigated at four ratios (50, 60, 70, and 80%) by applying a pressure of 0.7 MPa at a temperature of 20 ± 1°C and two flow rates (14 and 25 L min−1) for 25 min (Figure 9). As shown in Figure 9c, decreasing WVR from 80 to 50% resulted in a decrease in the water level (22–11 cm) and a faster frequency of pressure cycling. In regard to pressure cycling, the circulation number increased from 44 to 72 cycles with the flow rate of 14 L min−1, and from 78 to 125 cycles with the flow rate of 25 L min−1.
Pressure cycling boosts the inactivation efficiency by providing a driving force for CO2 transfer efficiency [9–13]. Recall that at the same flow rate and ΔP, a decrease in WVR increased the frequency of pressure cycling. Hence, it is hypothesized that a smaller WVR may have stimulated the CO2 transfer across cell membranes and thus improved the bactericidal performance of pressurized CO2 [11, 28, 29]. In this study, the low inactivation efficiency with a large WVR (i.e., 80%) may be related to the high water level (20–22 cm; Figure 9c), which led to submergence of the shield inside the device; this may have in turn decreased bubble formation via shield interactions [23, 24]. In contrast, the operations with smaller WVRs helped not only to promote a greater efficiency for CO2 bubble generation but also increased the speed of the pressure cycling. Consequently, CO2 supported by the high pressure and high efficiency of interactions in the apparatus easily penetrated into the cell membranes, thereby accelerating the
Regarding the effect of WVR in pressure cycling treatments, Pearson regression tests showed that
As shown in Table 3, the
Dillow et al.  reported that an increase of pressure cycling from 3 to 6 cycles using supercritical CO2 (at 20.5 MPa and 34°C) within 0.6 h increased the inactivation from 3 to 9 log reductions. Silva et al.  found that an 8.0 log reduction could be achieved with pressure cycling (5 cycles/140 min) and supercritical CO2 at 8 MPa, whereas a 5.0 log reduction was observed with 1 cycle/28 min and 8 MPa. However, high pressure and CO2 discharge are not interesting from both economic and practical viewpoints. As demonstrated in the present study where CO2 discharge was eliminated during the treatment process, pressure cycling at a low pressure (0.7 MPa) is a promising method to enhance the bactericidal activity of pressurized CO2.
4.4. SEM analyses
Comparative SEM images of untreated samples and samples treated with pressurized CO2 (0.7 MPa and 20°C for a duration of 25 min) revealed changes in the morphology of
Pressurized CO2 treatments can be used to eliminate
This study was supported by the Ministry of Education and Training of Vietnam under the Ph.D. Program No. 911, Yamaguchi University (Japan), and the Takahashi Industrial and Economic Research Foundation.