**4. Results and discussion**

### **4.1. Bactericidal performance of pressurized CO2 and pressurized air against** *E. coli* **in seawater**

Bactericidal effects of pressurized CO<sup>2</sup> in comparison with pressurized air against *E. coli* in seawater were investigated at three pressure conditions (0.3, 0.7, and 0.9 MPa) and at 20 ± 1°C (**Figure 5**). In general, the disinfection efficiency of the pressurized CO<sup>2</sup> treatment was not different between filtered seawater and artificial seawater. At every operating pressure, the *E. coli* inactivation efficiency of pressurized CO<sup>2</sup> was always higher than that of pressurized air.

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 215

*3.4.3. Experimental procedure for investigating the effect of the working volume ratio*

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

recycle number were calculated as described in Section 3.4.2.

electron microscope (QuantaTM 3D, FEI Co., USA) at 20 kV [23].

represents the predicted responses, *x*<sup>i</sup>

**4.1. Bactericidal performance of pressurized CO2**

Changes in cell morphology after pressurized CO2

**3.5. Scanning electron microscopy**

**3.6. Statistical analysis**

is the linear coefficient.

**4. Results and discussion**

Bactericidal effects of pressurized CO<sup>2</sup>

*coli* inactivation efficiency of pressurized CO<sup>2</sup>

where *y*<sup>i</sup>

**seawater**

βi

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

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

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 val-

*y<sup>i</sup>* = *β*<sup>0</sup> + ∑*β<sup>i</sup> xi* (1)

seawater were investigated at three pressure conditions (0.3, 0.7, and 0.9 MPa) and at 20 ± 1°C

different between filtered seawater and artificial seawater. At every operating pressure, the *E.* 

(**Figure 5**). In general, the disinfection efficiency of the pressurized CO<sup>2</sup>

is a parameter, β<sup>0</sup>

ues of inactivation efficacy were based on the following first-order regression model:

treatment were assessed by using SEM.

is the model intercept, and

treatment was not

 **and pressurized air against** *E. coli* **in** 

was always higher than that of pressurized air.

in comparison with pressurized air against *E. coli* in

**Figure 5.** Effect of pressurized CO<sup>2</sup> and pressurized air on (a) *E. coli* inactivation and (b) the pH of seawater (SW). Operating conditions: 0.3–0.9 MPa, 20 ± 1°C, and a working volume ratio (WVR) of 70%. Asterisks (\*) and (\*\*) indicate that the *E. coli* load was completely inactivated after 25 and 10 min, respectively.

Approximately 5.4–5.7 log reductions of the *E. coli* load were achieved within 10–25 min by the pressurized CO2 treatment (this involved complete inactivation of bacterial cells), whereas only 0.4–0.9 log reductions were achieved after 25 min by the pressurized air treatment; these tests involved pressures of 0.3–0.9 MPa (**Figure 5a**).

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. [24] 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 *E. coli* cells to become more permeable, thereby stimulating the process of CO2 penetration into the cells [24].

### **4.2. Effects of pressure and temperature**

*E. coli* was disinfected in various pressure conditions (0.2–0.9 MPa) at 20°C (**Figure 6**). In general, *E. coli* inactivation significantly increased with increasing pressure, and higher pressures required shorter exposure times to achieve the same log reduction. For example, a treatment application period of 25 min was required to reduce the *E. coli* load by approximately 5.0 log with pressure applications of 0.2–0.4 MPa, whereas pressure applications of 0.5 and 0.6 MPa resulted in a reduction of the treatment period to 20 and 15 min, respectively. The treatment period was further reduced to 10 min with pressure applications of 0.7–0.9 MPa. However, the increased pressure application from 0.7 to 0.9 MPa did not result in significant increase in the rate of bacterial inactivation. These data indicated that the optimal CO2 pressure for inactivating *E. coli* was in the range of 0.7–0.9 MPa, and hence, 0.7 MPa was chosen as the optimal pressure condition for effective bactericidal activity [23].

The disinfection efficiency of pressurized CO<sup>2</sup> substantially increased with increasing temperatures (11–28°C) at 0.7 MPa (**Figure 7**). The *E. coli* load was reduced by more than 5.0 log within 25 min of treatment at 11°C, whereas only 20, 12, and 10 min of pressurized CO2 treatment at 15, 18, and 20–28°C, respectively, were required to reduce the *E. coli* load to a similar extent [23]. Taken together, these findings suggest that *E. coli* inactivation by pressurized CO2 could be efficiently conducted at low-pressure (0.7 MPa) and ambient temperature

conditions. On the other hand, after disinfection and decompression, the pressurized CO2

**Figure 7.** Inactivation of *E. coli* in seawater at various temperatures by using the pressurized CO2

and a working volume ratio (WVR) of 70% [23]. Asterisks (\*) indicate that no colonies were detected.

increased, i.e., no regrowth of bacteria was observed.

of cell membranes [26]. Thus, the increase in CO2

the present study, the solubility of CO2

CO2

the diffusion of CO<sup>2</sup>

treatment [23].

**4.3. Effect of pressure cycling**

cess of CO2

CO2

treated samples were placed at normal conditions to assess the viability of the remaining bacteria. After the 5-d holding period, the number of *E. coli* in the treated samples had not

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

is lipo-hydrophilic in nature, and it can easily penetrate into the phospholipid bilayer

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 pro-

The effect of pressure cycling on *E. coli* inactivation was investigated by using various nozzle diameters (4–8 mm) (a treatment without a nozzle was also tested, where the diameter of

*4.3.1. Effect of pressure cycling at various pump powers and nozzle diameters*

penetration into *E. coli* cells, thereby accelerating the efficiency of the pressurized

into cells and may increase the fluidity of cell membranes [11, 27]. In

pressure and temperature may stimulate

http://dx.doi.org/10.5772/intechopen.68310

into seawater was considerably improved by using


217

treatment at 0.7 MPa

**Figure 6.** Effect of pressure on *E. coli* inactivation during the pressurized CO2 treatment at 20 ± 1.0°C and a working volume ratio (WVR) of 70% [23]. Asterisks (\*) indicate that no colonies were detected.

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 217

**Figure 7.** Inactivation of *E. coli* in seawater at various temperatures by using the pressurized CO2 treatment at 0.7 MPa and a working volume ratio (WVR) of 70% [23]. Asterisks (\*) indicate that no colonies were detected.

conditions. On the other hand, after disinfection and decompression, the pressurized CO2 treated samples were placed at normal conditions to assess the viability of the remaining bacteria. After the 5-d holding period, the number of *E. coli* in the treated samples had not increased, i.e., no regrowth of bacteria was observed.

CO2 is lipo-hydrophilic in nature, and it can easily penetrate into the phospholipid bilayer of cell membranes [26]. Thus, the increase in CO2 pressure and temperature may stimulate the diffusion of CO<sup>2</sup> 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 *E. coli* cells, thereby accelerating the efficiency of the pressurized CO2 treatment [23].

### **4.3. Effect of pressure cycling**

**4.2. Effects of pressure and temperature**

*E. coli* was disinfected in various pressure conditions (0.2–0.9 MPa) at 20°C (**Figure 6**). In general, *E. coli* inactivation significantly increased with increasing pressure, and higher pressures required shorter exposure times to achieve the same log reduction. For example, a treatment application period of 25 min was required to reduce the *E. coli* load by approximately 5.0 log with pressure applications of 0.2–0.4 MPa, whereas pressure applications of 0.5 and 0.6 MPa resulted in a reduction of the treatment period to 20 and 15 min, respectively. The treatment period was further reduced to 10 min with pressure applications of 0.7–0.9 MPa. However, the increased pressure application from 0.7 to 0.9 MPa did not result in significant increase in

tivating *E. coli* was in the range of 0.7–0.9 MPa, and hence, 0.7 MPa was chosen as the optimal

peratures (11–28°C) at 0.7 MPa (**Figure 7**). The *E. coli* load was reduced by more than 5.0 log within 25 min of treatment at 11°C, whereas only 20, 12, and 10 min of pressurized CO2 treatment at 15, 18, and 20–28°C, respectively, were required to reduce the *E. coli* load to a similar extent [23]. Taken together, these findings suggest that *E. coli* inactivation by pressur-

could be efficiently conducted at low-pressure (0.7 MPa) and ambient temperature

pressure for inac-

substantially increased with increasing tem-

treatment at 20 ± 1.0°C and a working

the rate of bacterial inactivation. These data indicated that the optimal CO2

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

pressure condition for effective bactericidal activity [23].

**Figure 6.** Effect of pressure on *E. coli* inactivation during the pressurized CO2

volume ratio (WVR) of 70% [23]. Asterisks (\*) indicate that no colonies were detected.

The disinfection efficiency of pressurized CO<sup>2</sup>

ized CO2

### *4.3.1. Effect of pressure cycling at various pump powers and nozzle diameters*

The effect of pressure cycling on *E. coli* inactivation was investigated by using various nozzle diameters (4–8 mm) (a treatment without a nozzle was also tested, where the diameter of the pipeline inlet was 15 mm) and two pump powers (0.20 and 0.75 kW) to change both the flow rate and ∆P of the input. The disinfection experiments were conducted under 0.7 MPa of pressurized CO2 at 20 ± 1°C with a WVR of 70% for a duration of 25 min (**Figure 8**). In general, larger nozzle diameters led to higher flow rates (**Figure 8c**) and faster fluid recycling in the treatment system (**Figure 8d**). In contrast, increases in the nozzle diameter reduced the pressure difference ΔP (**Figure 8c**). Furthermore, at the same nozzle diameter, stronger pumping powers improved not only the flow rate but also the pressure difference ΔP of the input (**Figure 8c**). At every nozzle diameter, operation of the pump with 0.75 kW of power (**Figure 8b**) yielded greater inactivation efficiencies than those with 0.20 kW of power (**Figure 8a**).

It is hypothesized that pressure cycling enhances the inactivation efficiency by facilitating the

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

can be expected to improve the *E. coli* inactivation. However, our results show that the *E. coli* inactivation efficiency did not increase with higher flow rates or faster recirculation. When 0.20 kW of pumping power was used (**Figure 8a**), the length of treatment periods required for complete inactivation of the *E. coli* load by more than 5.0 log increased with the greater nozzle sizes (i.e., 10 min with the 4-mm nozzle, 15 min with the 5–6-mm nozzles, and 20 min with the 7-mm nozzle, which corresponded to flow rates of 14, 17–19, and 19 L min−1, respectively). Furthermore, the reduction in *E. coli* load was only 3.0 log after 25 min when the device was operated without a nozzle (flow rate = 20 L min−1). A similar finding was found when the pump was operated at 0.75 kW of power (**Figure 8b**); at the higher power, more than a 5.0 log reduction was achieved within 5 min with the 5-mm nozzle (flow rate = 21 L min−1), whereas only a 4.0 log reduction was obtained after 25 min in the treatment lacking a nozzle (flow rate = 26 L min−1). These results indicate that the bactericidal performance of pressurized CO2

ciated with pressure cycling can probably not be attributed to the flow rate alone.

On the other hand, the disinfection efficiency substantially increased with the higher ΔP (**Figure 8**). A 5.4 log reduction in *E. coli* load was achieved within 5 min by the treatment with a ΔP of 0.25 MPa, whereas only a 3.0 log reduction was attained after 25 min by the treatment with a ΔP of 0.05 MPa. When operating the device with the same pump power, as noted above, a larger nozzle diameter resulted in higher water flow rates but weaker ∆P values. Hence, the reduction of ΔP may be considered as a key reason for the phenomenon of low inactivation efficiency at high flow rates. This suggests that the disinfection effect of pressure cycling might be influenced by not only by the frequency of circulation but also by the ΔP.

Noticeably, at the same ΔP value, a faster frequency of circulation substantially augmented the *E. coli* inactivation efficiency (**Figure 8**). For instance, at the same ΔP of 0.12 MPa (generated by a 5-mm nozzle and 0.20 kW pump, and a 7-mm nozzle and 0.75 kW pump), the periods required for complete inactivation of *E. coli* were reduced from 15 to 5 min when the frequency of pressure cycling was raised from 67 cycles/25 min to 92 cycles/25 min, respectively. A similar association between the disinfection efficiency and frequency of pressure cycling was found at ΔP = 0.10 MPa (generated by a 6-mm nozzle and 0.20 kW pump and a 8-mm nozzle and 0.75 kW pump); the associated treatment periods were 15 and 10 min for the recycle numbers corresponding to 71 cycles/25 min and 95 cycles/25 min, respectively. These results affirm the effect of pressure cycling on *E. coli* inactivation during pressurized

**Table 1** summarizes the coefficients of correlation for the inactivation efficiency and param-

*E. coli* inactivation efficiencies were correlated with ΔP values (*r* = 0.63, *p* < 0.0001) and recycle numbers (*r* = 0.66, *p* < 0.0001). The flow rate showed a weak correlation with the inactivation efficiency (*r* = 0.09, *p* = 0.3). Meanwhile, an inverse correlation (*r* = −0.35, *p* = 0.0004) was found between the nozzle diameter and disinfection efficiency. These data indicate that operations with a high flow rate, high ∆P value, large recycle number, and small nozzle diameter will

eters associated with pressure cycling, including the nozzle diameter (*x*<sup>1</sup>

), and recycle number (*x*<sup>4</sup>

into bacterial cell membranes [9, 10]. Thus, an increase in water flow rate

http://dx.doi.org/10.5772/intechopen.68310

asso-

219

), pressure difference

). Based on the Pearson matrix correlation results,

mass transfer of CO2

CO2

ΔP (*x*<sup>2</sup>

treatment.

), flow rate (*x*<sup>3</sup>

yield greater inactivation efficiencies.

**Figure 8.** Effect of pressure cycling on the inactivation of *E. coli* in seawater. Effect of (a) 0.20 kW pump power and (b) 0.75 kW pump power along with various nozzle diameters on the inactivation with pressurized CO2 . Influence of different pump powers and nozzle diameters on the (c) flow rate and pressure difference ΔP, and (d) the circulation number. Operating conditions: 0.7 MPa, 20 ± 1°C, and a working volume ratio (WVR) of 70% within a duration of 25 min. Asterisks (\*) indicate that no colonies were detected.

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 *E. coli* inactivation. However, our results show that the *E. coli* inactivation efficiency did not increase with higher flow rates or faster recirculation. When 0.20 kW of pumping power was used (**Figure 8a**), the length of treatment periods required for complete inactivation of the *E. coli* load by more than 5.0 log increased with the greater nozzle sizes (i.e., 10 min with the 4-mm nozzle, 15 min with the 5–6-mm nozzles, and 20 min with the 7-mm nozzle, which corresponded to flow rates of 14, 17–19, and 19 L min−1, respectively). Furthermore, the reduction in *E. coli* load was only 3.0 log after 25 min when the device was operated without a nozzle (flow rate = 20 L min−1). A similar finding was found when the pump was operated at 0.75 kW of power (**Figure 8b**); at the higher power, more than a 5.0 log reduction was achieved within 5 min with the 5-mm nozzle (flow rate = 21 L min−1), whereas only a 4.0 log reduction was obtained after 25 min in the treatment lacking a nozzle (flow rate = 26 L min−1). These results indicate that the bactericidal performance of pressurized CO2 associated with pressure cycling can probably not be attributed to the flow rate alone.

the pipeline inlet was 15 mm) and two pump powers (0.20 and 0.75 kW) to change both the flow rate and ∆P of the input. The disinfection experiments were conducted under 0.7 MPa

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

general, larger nozzle diameters led to higher flow rates (**Figure 8c**) and faster fluid recycling in the treatment system (**Figure 8d**). In contrast, increases in the nozzle diameter reduced the pressure difference ΔP (**Figure 8c**). Furthermore, at the same nozzle diameter, stronger pumping powers improved not only the flow rate but also the pressure difference ΔP of the input (**Figure 8c**). At every nozzle diameter, operation of the pump with 0.75 kW of power (**Figure 8b**) yielded greater inactivation efficiencies than those with 0.20 kW of power

**Figure 8.** Effect of pressure cycling on the inactivation of *E. coli* in seawater. Effect of (a) 0.20 kW pump power and

different pump powers and nozzle diameters on the (c) flow rate and pressure difference ΔP, and (d) the circulation number. Operating conditions: 0.7 MPa, 20 ± 1°C, and a working volume ratio (WVR) of 70% within a duration of 25 min.

. Influence of

(b) 0.75 kW pump power along with various nozzle diameters on the inactivation with pressurized CO2

Asterisks (\*) indicate that no colonies were detected.

at 20 ± 1°C with a WVR of 70% for a duration of 25 min (**Figure 8**). In

of pressurized CO2

(**Figure 8a**).

On the other hand, the disinfection efficiency substantially increased with the higher ΔP (**Figure 8**). A 5.4 log reduction in *E. coli* load was achieved within 5 min by the treatment with a ΔP of 0.25 MPa, whereas only a 3.0 log reduction was attained after 25 min by the treatment with a ΔP of 0.05 MPa. When operating the device with the same pump power, as noted above, a larger nozzle diameter resulted in higher water flow rates but weaker ∆P values. Hence, the reduction of ΔP may be considered as a key reason for the phenomenon of low inactivation efficiency at high flow rates. This suggests that the disinfection effect of pressure cycling might be influenced by not only by the frequency of circulation but also by the ΔP.

Noticeably, at the same ΔP value, a faster frequency of circulation substantially augmented the *E. coli* inactivation efficiency (**Figure 8**). For instance, at the same ΔP of 0.12 MPa (generated by a 5-mm nozzle and 0.20 kW pump, and a 7-mm nozzle and 0.75 kW pump), the periods required for complete inactivation of *E. coli* were reduced from 15 to 5 min when the frequency of pressure cycling was raised from 67 cycles/25 min to 92 cycles/25 min, respectively. A similar association between the disinfection efficiency and frequency of pressure cycling was found at ΔP = 0.10 MPa (generated by a 6-mm nozzle and 0.20 kW pump and a 8-mm nozzle and 0.75 kW pump); the associated treatment periods were 15 and 10 min for the recycle numbers corresponding to 71 cycles/25 min and 95 cycles/25 min, respectively. These results affirm the effect of pressure cycling on *E. coli* inactivation during pressurized CO2 treatment.

**Table 1** summarizes the coefficients of correlation for the inactivation efficiency and parameters associated with pressure cycling, including the nozzle diameter (*x*<sup>1</sup> ), pressure difference ΔP (*x*<sup>2</sup> ), flow rate (*x*<sup>3</sup> ), and recycle number (*x*<sup>4</sup> ). Based on the Pearson matrix correlation results, *E. coli* inactivation efficiencies were correlated with ΔP values (*r* = 0.63, *p* < 0.0001) and recycle numbers (*r* = 0.66, *p* < 0.0001). The flow rate showed a weak correlation with the inactivation efficiency (*r* = 0.09, *p* = 0.3). Meanwhile, an inverse correlation (*r* = −0.35, *p* = 0.0004) was found between the nozzle diameter and disinfection efficiency. These data indicate that operations with a high flow rate, high ∆P value, large recycle number, and small nozzle diameter will yield greater inactivation efficiencies.


**Table 1.** Correlation coefficients among various operating parameters associated with pressure cycling and the *E. coli* inactivation efficiency.

Regression coefficients, *t*-values, and *p*-values were analyzed for the four factors as shown in **Table 2**. The outcome of the multicollinearity regression model analysis (*R*<sup>2</sup> = 0.77, *p* < 0.001) suggests that the model can explain 77% of the inactivation efficiency of *E. coli*. With bootstrap analysis, the results of multivariate regression analyses were validated. The variables of *x*<sup>1</sup> , *x*<sup>2</sup> , *x*3 , and *x*<sup>4</sup> that were found to be associated with pressure cycling in the original analyses were significantly associated with pressure cycling in approximately 8, 28, 3, and 37%, respectively, of the 1000 iterations of the multivariate analyses. Taken together, these findings suggest that the frequency of recirculation (*x*<sup>4</sup> ) and the ∆P magnitude of the input (*x*<sup>2</sup> ) were key factors that drove the effectiveness pressure cycling.

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,

[23] and (b) a flow rate of 25 L min*<sup>−</sup>*<sup>1</sup>

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

**Figure 9.** Effect of the working volume ratio (WVR) on the inactivation of *E. coli* in seawater by pressurized CO2

circulation number and water level in the main chamber. Asterisks (\*) indicate that no colonies were detected.

WVR (**Figure 9**). Besides, at every WVR, operations with a high flow rate greatly enhanced the disinfection efficiency. When operating the device with a flow rate of 14 L min−1, an approximate 5.7 log reduction of *E. coli* was achieved within 15 min at 80% WVR, whereas only 5 min was required at 50% WVR to reduce the *E. coli* load to a similar extent (**Figure 9a**; [23]). A similar tendency was found in the case of the 25 L min−1 flow rate (**Figure 9b**). The durations required for complete inactivation of *E. coli* were 10 min at 80%, 5 min at 60–70%, and 3 min at 50%.

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 stimu-

(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

transfer across cell membranes and thus improved the bactericidal performance

[11, 28, 29]. In this study, the low inactivation efficiency with a large WVR

Pressure cycling boosts the inactivation efficiency by providing a driving force for CO<sup>2</sup>

significantly increased with decreases in the

transfer

at 0.7

221

. (c) Influence of the WVR on the

http://dx.doi.org/10.5772/intechopen.68310

and from 78 to 125 cycles with the flow rate of 25 L min−1.

*E. coli* inactivation efficacy of pressurized CO<sup>2</sup>

MPa and 20 ± 1°C with (a) a flow rate of 14 L min*<sup>−</sup>*<sup>1</sup>

lated the CO2

of pressurized CO2


*F*-statistic = 78.77 with 4 and 95 degrees of freedom, *p* < 2.2e-16.

**Table 2.** Regression results showing the influence of operating parameters associated with pressure cycling on the inactivation efficiency (at 20 ± 1°C, system pressure = 0.7 MPa, and working volume ratio (WVR) = 70%).

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 221

Regression coefficients, *t*-values, and *p*-values were analyzed for the four factors as shown in

**Table 1.** Correlation coefficients among various operating parameters associated with pressure cycling and the *E. coli*

**Factor Symbol code Unit** *r t***-statistic** *p***-value** Nozzle diameter *x*<sup>1</sup> mm −0.35 −3.64 0.0004\* Pressure difference ∆P *x*<sup>2</sup> Pa 0.63 8.08 1.69e-12\* Flow rate *x*<sup>3</sup> L min−1 0.09 1.05 0.30 Recycle number *x*<sup>4</sup> cycles 0.66 8.73 6.928e-14\*

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

suggests that the model can explain 77% of the inactivation efficiency of *E. coli*. With bootstrap analysis, the results of multivariate regression analyses were validated. The variables of *x*<sup>1</sup>

significantly associated with pressure cycling in approximately 8, 28, 3, and 37%, respectively, of the 1000 iterations of the multivariate analyses. Taken together, these findings suggest that

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.

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

= 0.77; adjusted *R*<sup>2</sup>

**Table 2.** Regression results showing the influence of operating parameters associated with pressure cycling on the

inactivation efficiency (at 20 ± 1°C, system pressure = 0.7 MPa, and working volume ratio (WVR) = 70%).

= 0.76.

**Source Coefficient** *t***-statistic** *p***-value** Intercept −0.63 −0.99 0.33 *x*<sup>1</sup> −0.13 −3.59 0.0005\* *x*<sup>2</sup> 0.01 7.32 7.8e-11\* *x*<sup>3</sup> 0.10 3.40 0.001\* *x*<sup>4</sup> 0.05 11.29 <2e-16\*

that were found to be associated with pressure cycling in the original analyses were

) and the ∆P magnitude of the input (*x*<sup>2</sup>

= 0.77, *p* < 0.001)

) were key factors that

, *x*<sup>2</sup> ,

**Table 2**. The outcome of the multicollinearity regression model analysis (*R*<sup>2</sup>

*x*3 , and *x*<sup>4</sup>

inactivation efficiency.

the frequency of recirculation (*x*<sup>4</sup>

drove the effectiveness pressure cycling.

\**p* < 0.05 (significant at the 95% confidence level); df = 98.

*4.3.2. Effect of pressure cycling at various WVRs*

\*Significant at the 95% confidence level; multiple *R*<sup>2</sup>

*F*-statistic = 78.77 with 4 and 95 degrees of freedom, *p* < 2.2e-16.

**Figure 9.** Effect of the working volume ratio (WVR) on the inactivation of *E. coli* in seawater by pressurized CO2 at 0.7 MPa and 20 ± 1°C with (a) a flow rate of 14 L min*<sup>−</sup>*<sup>1</sup> [23] and (b) a flow rate of 25 L min*<sup>−</sup>*<sup>1</sup> . (c) Influence of the WVR on the circulation number and water level in the main chamber. Asterisks (\*) indicate that no colonies were detected.

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.

*E. coli* inactivation efficacy of pressurized CO<sup>2</sup> significantly increased with decreases in the WVR (**Figure 9**). Besides, at every WVR, operations with a high flow rate greatly enhanced the disinfection efficiency. When operating the device with a flow rate of 14 L min−1, an approximate 5.7 log reduction of *E. coli* was achieved within 15 min at 80% WVR, whereas only 5 min was required at 50% WVR to reduce the *E. coli* load to a similar extent (**Figure 9a**; [23]). A similar tendency was found in the case of the 25 L min−1 flow rate (**Figure 9b**). The durations required for complete inactivation of *E. coli* were 10 min at 80%, 5 min at 60–70%, and 3 min at 50%.

Pressure cycling boosts the inactivation efficiency by providing a driving force for CO<sup>2</sup> 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 CO<sup>2</sup> 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 *E. coli* inactivation efficiency.

Regarding the effect of WVR in pressure cycling treatments, Pearson regression tests showed that *E. coli* inactivation efficiency was strongly correlated with the recycle number (*r* = 0.95*, p* < 0.001). The regression coefficient, *t*-value, and *p*-value were analyzed with regard to the recycle number at various WVRs and flow rates (**Table 3**). According to the regression analysis, the experimental results fit the linear model shown in the following equation:

$$\mathbf{Y} = \mathbf{0}.\mathbf{736} + \mathbf{0}.\mathbf{285} \times \mathbf{x}\_4 \tag{2}$$

**4.4. SEM analyses**

HRT, hydraulic retention time

\*Predicted values calculated based on Eq. (2).

bacterial concentration = 5–6 log10 CFU mL*<sup>−</sup>*<sup>1</sup>

of the pressurized CO2

treated by pressurized CO2

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 *E. coli*

**Table 4.** Validation of model regression for the inactivation efficiency responses to pressure cycling as a function of various working volume ratios (WVRs) and flow rates (at 20 ± 1°C, system pressure = 0.7 MPa, ΔP = 0.12 MPa, and initial

on the cell surface, and some treated cells appeared to be lysed (**Figure 10b**); in contrast, the untreated *E. coli* cells did not have such structures on the surface (**Figure 10a**) [23]. These

23], and that intracellular substance may have leaked out, possibly because of the alterations in cell permeability [20, 23, 30]. The findings also affirm the excellent bactericidal performance

**Figure 10.** Representative scanning electron microscopy (SEM) images of *E. coli* cells that were (a) untreated and (b)

at 0.7 MPa and 20°C for a duration of 25 min [23].

presented several small vesicles

*Y***: Reduction ratio, −log(***Nt*

http://dx.doi.org/10.5772/intechopen.68310

**, cycles Experimental Predicted**

**/***N0* **)** 223


cells (**Figure 10**). The *E. coli* cells treated with pressurized CO2

).

**Flow rate, L min−1 HRT, min Variables Responses**

**WVR, %** *x***<sup>4</sup>**

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

25a 0.20 50 15c 5.2 ± 0.2 5.0\* 25a 0.24 60 21d 5.5 ± 0.0 6.4\* 25a 0.28 70 18d 5.3 ± 0.2 5.8\* 14b 0.36 50 14d 5.7 ± 0.1 4.7\* 14b 0.43 60 19e 5.7 ± 0.0 6.1\* 14b 0.50 70 20f 5.7 ± 0.2 6.5\*

a, bGenerated by a 7-mm nozzle and 0.75 kW pump, and a 5-mm nozzle and 0.20 kW pump, respectively. c, d, e, fExposure times were 3, 5, 8, and 10 min, respectively, when bacteria were completely inactivated.

treatment.

results suggest that the pressurized CO2

Here, *x*<sup>4</sup> is the recycle number (cycles), and *Y* is reduction ratio (−log N/N<sup>0</sup> ) of *E. coli* caused by pressurized CO2 .

As shown in **Table 3**, the *t* values of the regression model were positive and significant (*p* < 0.05), thus indicating that the model result was significant. The outcome of the linear regression model analysis (*R*<sup>2</sup> = 0.91, *p* < 0.001) suggests that 91% of the variation in the *E. coli* inactivation efficiency was explained by the frequency of pressure cycling (ΔP = 0.12 MPa, flow rate = 14–25 L min−1). Predicted values of *E. coli* reduction ratios were calculated based on Eq. (2), and the data are summarized in **Table 4** along with the experimental results. The predicted values were fairly similar to the experimental results, thus suggesting that the model could adequately describe the strong relationship between pressure cycling and bactericidal activity (*p* < 0.05). Taken together, these findings affirm that at the same ΔP, faster pressure cycling can achieve a greater *E. coli* inactivation efficiency.

Dillow et al. [13] 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. [10] 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 .


**Table 3.** Regression results showing the influence of pressure cycling on the inactivation efficiency (at 20 ± 1°C, system pressure = 0.7 MPa, ΔP = 0.12 MPa, flow rate = 14 to 25 L min*<sup>−</sup>*<sup>1</sup> , and initial bacterial concentration = 5–6 log10 CFU mL*<sup>−</sup>*<sup>1</sup> ).

*Escherichia coli* Inactivation Using Pressurized Carbon Dioxide as an Innovative Method for Water Disinfection http://dx.doi.org/10.5772/intechopen.68310 223


HRT, hydraulic retention time

shield interactions [23, 24]. In contrast, the operations with smaller WVRs helped not only to

interactions in the apparatus easily penetrated into the cell membranes, thereby accelerating

Regarding the effect of WVR in pressure cycling treatments, Pearson regression tests showed that *E. coli* inactivation efficiency was strongly correlated with the recycle number (*r* = 0.95*, p* < 0.001). The regression coefficient, *t*-value, and *p*-value were analyzed with regard to the recycle number at various WVRs and flow rates (**Table 3**). According to the regression analy-

*Y* = 0.736 + 0.285 × *x*<sup>4</sup> (2)

As shown in **Table 3**, the *t* values of the regression model were positive and significant (*p* < 0.05), thus indicating that the model result was significant. The outcome of the linear regres-

vation efficiency was explained by the frequency of pressure cycling (ΔP = 0.12 MPa, flow rate = 14–25 L min−1). Predicted values of *E. coli* reduction ratios were calculated based on Eq. (2), and the data are summarized in **Table 4** along with the experimental results. The predicted values were fairly similar to the experimental results, thus suggesting that the model could adequately describe the strong relationship between pressure cycling and bactericidal activity (*p* < 0.05). Taken together, these findings affirm that at the same ΔP, faster pressure cycling can

Dillow et al. [13] reported that an increase of pressure cycling from 3 to 6 cycles using super-

reductions. Silva et al. [10] found that an 8.0 log reduction could be achieved with pressure

interesting from both economic and practical viewpoints. As demonstrated in the present

a low pressure (0.7 MPa) is a promising method to enhance the bactericidal activity of pres-

**Table 3.** Regression results showing the influence of pressure cycling on the inactivation efficiency (at 20 ± 1°C, system

observed with 1 cycle/28 min and 8 MPa. However, high pressure and CO2

**Coefficients Estimate Standard error** *t***-statistic** *p***-value** *R***<sup>2</sup>**

*x*<sup>4</sup> 0.285 0.019 15.30 7.2e-14\* 0.91

Intercept 0.736 0.195 3.77 0.0009\*

(at 20.5 MPa and 34°C) within 0.6 h increased the inactivation from 3 to 9 log

discharge was eliminated during the treatment process, pressure cycling at

at 8 MPa, whereas a 5.0 log reduction was

, and initial bacterial concentration = 5–6 log10 CFU mL*<sup>−</sup>*<sup>1</sup>

= 0.91, *p* < 0.001) suggests that 91% of the variation in the *E. coli* inacti-

sis, the experimental results fit the linear model shown in the following equation:

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

is the recycle number (cycles), and *Y* is reduction ratio (−log N/N<sup>0</sup>

bubble generation but also increased the speed of the

supported by the high pressure and high efficiency of

) of *E. coli* caused

discharge are not

).

promote a greater efficiency for CO<sup>2</sup>

pressure cycling. Consequently, CO2

.

achieve a greater *E. coli* inactivation efficiency.

cycling (5 cycles/140 min) and supercritical CO2

pressure = 0.7 MPa, ΔP = 0.12 MPa, flow rate = 14 to 25 L min*<sup>−</sup>*<sup>1</sup>

the *E. coli* inactivation efficiency.

Here, *x*<sup>4</sup>

critical CO2

study where CO2

\*95% confidence level.

.

surized CO2

by pressurized CO2

sion model analysis (*R*<sup>2</sup>

\*Predicted values calculated based on Eq. (2).

a, bGenerated by a 7-mm nozzle and 0.75 kW pump, and a 5-mm nozzle and 0.20 kW pump, respectively.

c, d, e, fExposure times were 3, 5, 8, and 10 min, respectively, when bacteria were completely inactivated.

**Table 4.** Validation of model regression for the inactivation efficiency responses to pressure cycling as a function of various working volume ratios (WVRs) and flow rates (at 20 ± 1°C, system pressure = 0.7 MPa, ΔP = 0.12 MPa, and initial bacterial concentration = 5–6 log10 CFU mL*<sup>−</sup>*<sup>1</sup> ).

### **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 *E. coli* cells (**Figure 10**). The *E. coli* cells treated with pressurized CO2 presented several small vesicles on the cell surface, and some treated cells appeared to be lysed (**Figure 10b**); in contrast, the untreated *E. coli* cells did not have such structures on the surface (**Figure 10a**) [23]. These results suggest that the pressurized CO2 -treated *E. coli* cells may have been disrupted [19, 20, 23], and that intracellular substance may have leaked out, possibly because of the alterations in cell permeability [20, 23, 30]. The findings also affirm the excellent bactericidal performance of the pressurized CO2 treatment.

**Figure 10.** Representative scanning electron microscopy (SEM) images of *E. coli* cells that were (a) untreated and (b) treated by pressurized CO2 at 0.7 MPa and 20°C for a duration of 25 min [23].

### **5. Summary**

Pressurized CO2 treatments can be used to eliminate *E. coli* from seawater. In this study, the inactivation efficiency was substantially enhanced by pressure cycling, which was conducted at a low pressure (0.7 MPa) and without CO2 release during the treatment period. Bactericidal performance of pressure cycling was concomitantly influenced by two key factors involving the frequency of recirculation and ΔP (*p* < 0.001). At the same ΔP, an increase in the frequency of pressure cycling significantly improved the *E. coli* inactivation efficiency (*p* < 0.001). Additionally, the sensitivity of *E. coli* to pressurized CO2 treatments substantially increased with increased pressures (0.2–0.9 MPa) and temperatures (11–28°C). Under identical treatment conditions (0.7 MPa, 20°C, 25 L min−1, and 50% WVR), more than 5.0 log reductions in the load of *E. coli* were achieved after treatments for 3 min by using pressure cycling (ΔP = 0.12 MPa, 15 cycles). Overall, these findings suggest that pressurized CO<sup>2</sup> technology would be feasible for water disinfection applications such as those used in ballast water treatment.

[2] Fabbricino M, Korshin GV. Formation of disinfection by-products and applicability of differential absorbance spectroscopy to monitor halogenation in chlorinated coastal and deep ocean seawater. Desalination. 2005;**176**69-69. DOI: 10.1016/j.desal.2004.10.026 [3] LeChevallier MW, Au K, editors. Water Treatment and Pathogen Control: Process Efficiency in Achieving Safe Drinking-water. WHO Drinking-water Quality Series. World Health Organization: IWA Publishing; 2004. p. 112. DOI: 10.2166/9781780405858

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

http://dx.doi.org/10.5772/intechopen.68310

225

[4] Von Gunten U. Ozonation of drinking water: Part II. Disinfection and by-product formation in presence of bromide, iodide or chlorine. Water Research. 2003;**37**1487-1487. DOI:

[5] Werschkun B, Sommer Y, Banerji S. Disinfection by-products in ballast water treatment: An evaluation of regulatory data. Water Research. 2012;**46**4901-4901. DOI: 10.1016/j.

[6] Haas GJ, Prescott HE, Dudley E, Dik R, Hintlian C, Keane L. Inactivation of microorganisms by carbon dioxide under pressure. Journal of Food Safety. 1989;**9**265-265. DOI:

[7] Garcia-Gonzalez L, Geeraerd AH, Spilimbergo S, Elst K, Van Ginneken L, Debevere J, Van Impe J, Devlieghere F. High pressure carbon dioxide inactivation of microorganisms in foods: The past, the present and the future. International Journal of Food

[8] Spilimbergo S, Elvassore N, Bertucco A. Microbial inactivation by high-pressure. The Journal of Supercritical Fluids. 2002;**22**63-63. DOI: 10.1016/S0896-8446(01)00106-1

[9] Zhang J, Davis TA, Matthews MA, Drews MJ, LaBerge M, An YH. Sterilization using high-pressure carbon dioxide. The Journal of Supercritical Fluids. 2006;**38**372-372. DOI:

[10] Silva JM, Rigo AA, Dalmolin IA, Debien I, Cansian RL, Oliveira JV, Mazutti MA. Effect of pressure, depressurization rate and pressure cycling on the inactivation of *Escherichia coli* by supercritical carbon dioxide. Food Control. 2013;**29**81-81. DOI: 10.1016/j.

[11] Hong SI, Park WS, Pyun YR. Inactivation of *Lactobacillus* sp. from kimchi by high pressure carbon dioxide. LWT-Food Science and Technology. 1997;**30**685-685. DOI: 10.1006/

[12] Hong SI, Pyun YR. Inactivation kinetics of *Lactobacillus plantarum* by high pressure carbon dioxide. Journal Food Science. 1999;**64**733-733. DOI: 10.1111/j.1365-2621.1999.tb15120.x

[13] Dillow AK, Dehghani F, Hrkach JS, Foster NR, Langer R. Bacterial inactivation by using near and supercritical carbon dioxide. Proceedings of the National Academic of Sciences of the United States of America. 1999;**96**10348-10348. DOI: 10.1073/pnas.96.18.10344 [14] Fraser D. Bursting bacteria by release of gas pressure. Nature. 1951;**167**34-34. DOI:

Microbiology. 2007;**117**28-28. DOI: 10.1016/j.ijfoodmicro.2007.02.018

10.1016/S0043-1354(02)00458-X

10.1111/j.1745-4565.1989.tb00525.x

10.1016/j.supflu.2005.05.005

foodcont.2012.05.068

fstl.1997.0250

10.1038/167033b0

watres.2012.05.034

## **Acknowledgements**

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.

### **Author details**

Tsuyoshi Imai1 \* and Thanh-Loc Thi Dang2,3

\*Address all correspondence to: imai@yamaguchi-u.ac.jp

1 Division of Construction and Environmental Engineering, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Ube, Yamaguchi, Japan

2 Division of Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Ube, Yamaguchi, Japan

3 Department of Environmental Science, College of Sciences, Hue University, Hue, Vietnam

### **References**

[1] Boorman GA, Dellarco V, Dunnick JK, Chapin RE, Hunter S, Hauchman F. Drinking water disinfection byproducts: Review and approach to toxicity evaluation. Environmental Health Perspectives. 1999;**107**(Suppl 1)217-217

[2] Fabbricino M, Korshin GV. Formation of disinfection by-products and applicability of differential absorbance spectroscopy to monitor halogenation in chlorinated coastal and deep ocean seawater. Desalination. 2005;**176**69-69. DOI: 10.1016/j.desal.2004.10.026

**5. Summary**

Pressurized CO2

water treatment.

**Author details**

Tsuyoshi Imai1

**References**

**Acknowledgements**

Economic Research Foundation.

treatments can be used to eliminate *E. coli* from seawater. In this study,

release during the treatment period.

treatments substan-

tech-

the inactivation efficiency was substantially enhanced by pressure cycling, which was con-

Bactericidal performance of pressure cycling was concomitantly influenced by two key factors involving the frequency of recirculation and ΔP (*p* < 0.001). At the same ΔP, an increase in the frequency of pressure cycling significantly improved the *E. coli* inactivation efficiency

tially increased with increased pressures (0.2–0.9 MPa) and temperatures (11–28°C). Under identical treatment conditions (0.7 MPa, 20°C, 25 L min−1, and 50% WVR), more than 5.0 log reductions in the load of *E. coli* were achieved after treatments for 3 min by using pressure cycling (ΔP = 0.12 MPa, 15 cycles). Overall, these findings suggest that pressurized CO<sup>2</sup>

nology would be feasible for water disinfection applications such as those used in ballast

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

1 Division of Construction and Environmental Engineering, Graduate School of Sciences and

2 Division of Environmental Science and Engineering, Graduate School of Science and

3 Department of Environmental Science, College of Sciences, Hue University, Hue, Vietnam

[1] Boorman GA, Dellarco V, Dunnick JK, Chapin RE, Hunter S, Hauchman F. Drinking water disinfection byproducts: Review and approach to toxicity evaluation. Environmental

Technology for Innovation, Yamaguchi University, Ube, Yamaguchi, Japan

ducted at a low pressure (0.7 MPa) and without CO2

\* and Thanh-Loc Thi Dang2,3 \*Address all correspondence to: imai@yamaguchi-u.ac.jp

Engineering, Yamaguchi University, Ube, Yamaguchi, Japan

Health Perspectives. 1999;**107**(Suppl 1)217-217

(*p* < 0.001). Additionally, the sensitivity of *E. coli* to pressurized CO2

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


[15] Ferreira EHdR, Rosenthal A, Calado V, Saraiva J, Mendo S. *Byssochlamys nivea* inactivation in pineapple juice and nectar using high pressure cycles. Journal of Food Engineering. 2009;**95**669-669. DOI:10.1016/j.jfoodeng.2009.06.053

[27] Oulé MK, Tano K, Bernier AM, Arul J. *Escherichia coli* inactivation mechanism by pressur-

[28] Lin HM, Yang Z, Chen LF. Inactivation of *Leuconostoc dextranicum* with carbon dioxide under pressure. The Chemical Engineering Journal. 1993;**52**:B29-B34. DOI: 10.1016/

[29] Garcia-Gonzalez L, Geeraerd AH, Elst K, Van Ginneken L, Van Impe JF, Devlieghere F. Inactivation of naturally occurring microorganisms in liquid whole egg using high pressure carbon dioxide processing as an alternative to heat pasteurization. The Journal of

[30] Kim SR, Rhee MS, Kim BC, Lee H, Kim KH. Modeling of the inactivation of *Salmonella typhimurium* by supercritical carbon dioxide in physiological saline and phosphatebuffered saline. Journal Microbiology Methods. 2007;**70**141-141. DOI: 10.1016/j.

Supercritical Fluids. 2009;**51**82-82. DOI: 10.1016/j.supflu.2009.06.020

. Canadian Journal of Microbiology. 2006;**52**1217-1217. DOI: 10.1139/w06-078

http://dx.doi.org/10.5772/intechopen.68310

227

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

ized CO2

0300-9467(93)80047-R

mimet.2007.04.003


[27] Oulé MK, Tano K, Bernier AM, Arul J. *Escherichia coli* inactivation mechanism by pressurized CO2 . Canadian Journal of Microbiology. 2006;**52**1217-1217. DOI: 10.1139/w06-078

[15] Ferreira EHdR, Rosenthal A, Calado V, Saraiva J, Mendo S. *Byssochlamys nivea* inactivation in pineapple juice and nectar using high pressure cycles. Journal of Food Engineering.

[16] Kobayashi F, Hayata Y, Kohara K, Muto N, Osajima Y. Application of supercritical CO<sup>2</sup> bubbling to inactivate *E. coli* and coliform bacteria in drinking water. Food Science and

[17] Kobayashi F, Yamaza F, Ikeura H, Hayata Y, Muto N, Osajima Y. Inactivation of microorganisms in untreated water by a continuous flow system with supercritical CO<sup>2</sup>

[18] Kobayashi F, Hayata Y, Ikeura H, Tamaki M, Muto N, Osajima Y. Inactivation of

[19] Cheng X, Imai T, Teeka J, Yamaguchi J, Hirose M, Higuchi T, Sekine M. Inactivation of *Escherichia coli* and bacteriophage T4 by high levels of dissolved CO2

Microbiology and Biotechnology. 2011;**90**1500-1500. DOI: 10.1007/s00253-011-3163-0 [20] Vo HT, Imai T, Teeka J, Sekine M, Kanno A, Le TV, Higuchi T, Phummala K, Yamamoto K.

[21] Vo HT, Imai T, Yamamoto H, Le TV, Higuchi T, Kanno A, Yamamoto K, Sekine M. Disinfection using pressurized carbon dioxide microbubbles to inactivate *Escherichia coli*, bacteriophage MS2 and T4. Journal of Water Environment Technology. 2013b;**11**505-505.

[22] Vo HT, Imai T, Ho TT, Dang TTL, Hoang SA. Potential application of high pressure carbon dioxide in treated wastewater and water disinfection: Recent overview and further trends. Journal Environmental Science. 2015;**36**47-47. DOI: 10.1016/j.jes.2015.04.006 [23] Dang TTL, Imai T, Le TV, Vo HT, Higuchi T, Yamamoto K, Kanno A, Sekine M. Disinfection effect of pressurized carbon dioxide on *Escherichia coli* and *Enterococcus* sp. in seawater. Water Science and Technology: Water Supply. 2016a;16(6)1735-1744. DOI: 10.2166/ws.2016.086 [24] Dang TTL, Imai T, Le TV, Nishihara S, Higuchi T, Nguyen KDM, Kanno A, Yamamoto K, Sekine M. Effect of pressure and pressure cycling on disinfection of *Enterococcus* sp. in seawater using pressurized carbon dioxide with different content rates. Journal of Environmental Science and Health, Part A. 2016b;51(11)930-937. DOI: 10.1080/10934529.2016.1191309 [25] Hutkins RW, Nannen NL. pH homeostasis in lactic-acid bacteria. Journal of Dairy

[26] Isenschmid A, Marison IW, von Stockar U. The influence of pressure and temperature of

on the survival of yeast cells. Journal of Biotechnology. 1995;**39**237-237.

Transactions of the ASABE. 2009b;**52**1626-1626. DOI: 10.13031/2013.29113

*coli*. Water Research. 2013a;**47**4293-4293. DOI: 10.1016/j.watres.2013.04.053

bling. Journal of Water and Environment Technology. 2009a;**7**250-250. DOI: 10.2965/

microbubbles at a lower pressure and near room temperature.

, N<sup>2</sup>

O, and N<sup>2</sup>

bub-

. Applied

on *Escherichia* 

2009;**95**669-669. DOI:10.1016/j.jfoodeng.2009.06.053

jwet.2009.241

*Escherichia coli* by CO2

DOI: 10.2965/jwet.2013.497

compressed CO2

DOI: 10.1016/0168-1656(95)00018-L

Technology Research. 2007;**13**22-22. DOI: 10.3136/fstr.13.20

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

Comparison of disinfection effect of pressurized gases of CO<sup>2</sup>

Science. 1993;**76**2365-2365. DOI: 10.3168/jds.S0022-0302(93)77573-6


**Chapter 12**

**Evaluating Meta-Analysis Research of** *Escherichia coli*

This chapter summarizes the progress in *Escherichia coli* research that used the meta-analysis approach. Using systematic searches for *E. coli* literature, we tracked meta-analysis publications and analyzed them based on a number of parameters. These included subject/topic (epidemiology, clinical/intervention/prevention and environmental), geographical region (the Americas, Europe and Australasia) and clinical syndrome (enteric, renal, and sepsis/meningitis). These parameters were plotted in terms of time span to obtain a sense of dynamic change or its absence through the years since the turn of the twentieth century. In terms of region, topic and syndrome, highest meta-analysis productivity was attributed to the Americas, clinical/intervention/prevention and enteric, all of which took place in the last 5 years (2011–2016). Over the combined time span of 16 years, the Americas significantly dominated meta-analysis outputs when compared to Europe and Australasia (P = 0.003). In conclusion, our findings facilitate awareness of the progress in this field wherein the studied parameters were analyzed for patterns over

Published researches on *Escherichia coli* (*E. coli*) have increased in number since the turn of the twentieth century. A search of *E. coli* publications in PubMed reveals an output value of 339,415 (as of July 16, 2016) which when narrowed to *E. coli* in title only, the number is still substantial (96,594). Majority of the *E. coli* publications are primary studies which when addressing the same issue, most often produce contradictory results [1]. Thus when primary studies are reviewed (usually in the narrative style), these contradictions hinder meaningful integration of results. A more systematic way to integrate primary study findings is the use of meta-analysis.

> © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Noel Pabalan, Eloisa Singian, Lani Tabangay and

time and differential rates of publication productivity.

**Keywords:** *Escherichia coli*, meta-analysis

Additional information is available at the end of the chapter

Hamdi Jarjanazi

http://dx.doi.org/10.5772/67337

**Abstract**

**1. Introduction**
