**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).

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

uid films. The presence of numerous small bubbles also enhanced the contact area between

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 approxi-

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

–1010 CFU mL−1. The permanent stock was maintained in 20% glycerol at −80°C.

contact efficiency promoted by this apparatus enabled ample penetration of CO<sup>2</sup>

transfers took place both within the interior and exterior sides of the thin liq-

dissolution into water. We hypothesized that the available


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211

transfer efficiency was high. Despite the lower pressures used, the high

and the cell suspension was greatly multiplied in this

into the cell

Hence, CO2

mately 109

gas and water and facilitated CO2

**3. Materials and methods**

**3.1. Microorganism preparation and enumeration**

was re-suspended in 100 mL saline solution.

setup and that the CO2

membranes of *E. coli*.

interfacial contact area between CO2

**Figure 3.** Pictures of an untreated sample and a CO2

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

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

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 211

**Figure 3.** Pictures of an untreated sample and a CO2 -treated sample (the latter contains many small 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 CO<sup>2</sup> into the cell membranes of *E. coli*.
