**3. Materials and methods**

**Figure 1.** Apparatus for forming highly dissolved gas in water.

210 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

**Figure 2.** Representative pictures of liquid film formation with various nozzle diameters at a normal pressure in the pipeline.

## **3.1. Microorganism preparation and enumeration**

Stock cultures of *E. coli* (ATCC 11303) were propagated in Luria-Bertani (LB) broth (Wako Chemical Co., Ltd., Osaka, Japan) containing 30 g L−1 sodium chloride and incubated for 24 h at 37°C by using a reciprocal shaker set to rotate at 150 rpm. The initial enumeration was approximately 109 –1010 CFU mL−1. The permanent stock was maintained in 20% glycerol at −80°C.

The *E. coli* inoculum for each disinfection experiment was prepared by inoculating 100 μL of bacterial glycerol stock into 100 mL of LB broth containing 30 g L−1 sodium chloride. The culture was then incubated for 20 h at 37°C with continuous shaking at 150 rpm. Cells were harvested and washed three times with a 0.9% (w/v) saline solution followed by centrifugation (10 min at 8000 g at room temperature) in a CF15D2 centrifuge (Hitachi, Japan). The pellet was re-suspended in 100 mL saline solution.

*E. coli* were enumerated by using the plate count technique. Briefly, the samples were diluted into a series of 10-fold dilutions by using autoclaved artificial seawater at 3.4% salinity, and 100 μL of either a diluted or an undiluted sample was plated on LB agar (Wako). For samples with a low number of viable cells, 1 mL of the undiluted sample was poured into agar maintained at 45°C. Colonies growing on each plate were counted after incubating the plates overnight at 37°C. Each sample was analyzed in triplicate.

## **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 *E. coli* cultures were added into the artificial/filtered seawater to obtain a bacterial concentration of 5–6 log10 CFU mL−1. The solution was stirred for 30 min to acclimatize the bacteria before starting the experiments. For each batch mode operation, 12 L of samples were prepared, of which 4–5 L were used to restart the system. The pH and temperature of samples were measured with a pH meter (Horiba D-51, Japan).

### **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.

*3.4.2. Experimental procedure for investigating the effect of pressure cycling*

pression [8, 10, 13, 15]. However, such high pressure and CO2

tions (8–550 MPa) and with CO2

**Figure 4.** Setup of the water treatment apparatus.

volume/flow rate.

sures (<1 MPa) and no discharge of CO2

In previous works, the pressure cycling procedure was conducted with high-pressure opera-

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection

an economic standpoint. In order to overcome the above disadvantages, in the present study, we employed a process involving pressure cycling for *E. coli* inactivation but used lower pres-

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

discharges between each cycle of decompression and com-

between each cycle of raised and lowered pressure.

release are undesirable from

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213

### **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 [23]. Each experiment was conducted in triplicate.

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 213

**Figure 4.** Setup of the water treatment apparatus.

*E. coli* were enumerated by using the plate count technique. Briefly, the samples were diluted into a series of 10-fold dilutions by using autoclaved artificial seawater at 3.4% salinity, and 100 μL of either a diluted or an undiluted sample was plated on LB agar (Wako). For samples with a low number of viable cells, 1 mL of the undiluted sample was poured into agar maintained at 45°C. Colonies growing on each plate were counted after incubating the plates over-

212 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

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 *E. coli* cultures were added into the artificial/filtered seawater to obtain a bacterial concentration of 5–6 log10 CFU mL−1. The solution was stirred for 30 min to acclimatize the bacteria before starting the experiments. For each batch mode operation, 12 L of samples were prepared, of which 4–5 L were used to restart the system. The pH and temperature of samples were measured with a pH

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, pres-

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

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 sensitiv-

treatments under different conditions was determined by

pressure (0.2–0.9 MPa) and seawater temperature (11–28°C) for a 25-min

*3.4.1. Experimental procedure for investigating the effects of pressure and temperature*

treatment period [23]. Each experiment was conducted in triplicate.

was also injected into the main chamber. System pressure was adjusted by a gas

night at 37°C. Each sample was analyzed in triplicate.

**3.2. Seawater sample preparation**

meter (Horiba D-51, Japan).

**3.3. Experimental setup**

surized CO2

of the device.

varying the CO2

**3.4. Procedure for disinfection experiments**

ity of bacteria to pressurized CO2

### *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 *E. coli* inactivation but used lower pressures (<1 MPa) and no discharge of CO2 between each cycle of raised and lowered pressure.

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 *E. coli* were immobilized with 2.5% glutaraldehyde in phosphate buffered saline (PBS) for 3 h at 4°C and then rinsed with PBS three times. Next, the samples were soaked in 1.0% osmium tetroxide in cacodylate buffer for 90 min and then washed three times with cacodylate buffer for removal of the fixative. After fixation, the cells were dehydrated by consecutive soaking in increasing concentrations of ethanol solutions (50, 70, 80, 90, 95, and 100%), and this was followed by an ethanol/t-butyl alcohol (v/v = 1:1) treatment for 30 min. The prepared cells were then soaked in t-butyl alcohol two times for 1 h, freeze-dried for 2 h, and sputter coated with gold-palladium. Finally, the cells were examined by using a scanning electron microscope (QuantaTM 3D, FEI Co., USA) at 20 kV [23].

### **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:

$$\mathbf{y}\_{i} = \boldsymbol{\beta}\_{o} + \sum \boldsymbol{\beta}\_{i} \mathbf{x}\_{i} \tag{1}$$

Approximately 5.4–5.7 log reductions of the *E. coli* load were achieved within 10–25 min by

Operating conditions: 0.3–0.9 MPa, 20 ± 1°C, and a working volume ratio (WVR) of 70%. Asterisks (\*) and (\*\*) indicate

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection

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215

only 0.4–0.9 log reductions were achieved after 25 min by the pressurized air treatment; these

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

bacterial inactivation process [12, 20, 21, 25]. However, Dang et al. [24] demonstrated that the low pH alone is not the main cause of the bactericidal activity. Perhaps with the concomitant

treatment (this involved complete inactivation of bacterial cells), whereas

and pressurized air on (a) *E. coli* inactivation and (b) the pH of seawater (SW).

reduced the pH of both filtered seawater and artificial seawater to around 5.0

is probably a major factor driving the

, the low pH prompted the *E. coli* cells to become more

penetration into the cells [24].

the pressurized CO2

**Figure 5.** Effect of pressurized CO<sup>2</sup>

Pressurized CO2

tests involved pressures of 0.3–0.9 MPa (**Figure 5a**).

that the *E. coli* load was completely inactivated after 25 and 10 min, respectively.

that the decrease in pH caused by pressurized CO2

permeable, thereby stimulating the process of CO2

presence of pressure and dissolved CO2

where *y*<sup>i</sup> represents the predicted responses, *x*<sup>i</sup> is a parameter, β<sup>0</sup> is the model intercept, and βi is the linear coefficient.
