Inactivation of Coliforms in Sludge Through Cavitation Phenomena by Ultrasonic Waves

Inactivation of coliforms in sludge through cavitation phenomena by ultrasonic


Introduction
Disinfection is a crucial part of a wastewater treatment plant.As the health standards of people's life increases, its role becomes more significant. 1,2There are different types of pathogens in the sludge such as viruses, bacteria, parasites, and fungi.There are also different indicator pathogens such as fecal coliforms, clostridium perfringens, helminth ova, salmonella, enteric viruses, etc. 3 Coliforms are the major indicators of pathogens in sludge or wastewater.The primary goal of this process is to remove pathogenic microorganisms to alleviate their health hazard. 4,5ifferent techniques are applied for disinfection purpose.][8] Despite the acceptable efficiency of these techniques, their limitation and side products vitalize emerging, safer and more promising technologies.0][11] Additionally, application of UV technique is limited due to the requirement of highly light scattering or absorbing medium and self-repair of disinfected microorganisms. 11trasound waves are one of the alternative methods for conventional disinfection techniques, which not only do not produce undesirable by-products but also have good efficiency in inactivation of microbes and viruses from water and wastewater. 12,13The disinfection of ultrasound is based on the presence of cavitation occurrences. 14everal physical, chemical and mechanical processes are taking place during cavitation phenomena.During the cavitation process, micro and Nanobubbles are produced that is followed by expansion and implosion.Contraction and refraction cycle of ultrasound wave causes the reduction and expansion of the average diameter between molecules in a liquid.If the refraction force of ultrasonic waves is intense enough to break the attraction force of molecules, cavitation bubbles are produced. 15The generated bubbles also experience contraction and refraction cycle.If the expansion is sufficient to reach an unstable phase, the implosion will occur which unleash high energy and causes sonochemical reactions. 16,17By implosion of gas bubbles, physical and mechanical impact of cavitation process are imposed by increase in pressure and temperature (locally up to 5000°C and 2000 atmosphere) as well as shear stress and turbulence that can break cell membranes and release intercellular molecules in the external environment. 18,19avitation phenomena has also chemical effect which is produced by the OH -and H + radicals that lacerate cell wall by oxidation and pathogenic microorganism. 19,20he final product of oxidation is H 2 O 2 and H 2 O that not only are hazardous, but also H 2 O 2 is an oxidizing agent which enhances the oxidation process.One of the most critical indicators of existing pathogenic agents in water and wastewater is total coliform, elimination of which is an essential target of disinfection.Several studies have examined the application of ultrasonic waves. 19,21Vázquez-López et al studied the removal of total coliform in wastewater by ultrasound, and reached to almost complete removal of total coliform in frequency of 26 kHz and time of 30 minutes. 21In another research, 99.99% of coliform removal rate was achieved in 0.2250 KW.h/m 3 of specific energy consumption. 13he objective of present study was to determine the impact of ultrasound on disinfection of sludge and find the optimal range of operating parameters by ultrasound to reduce the total coliform bacteria in the sludge by response surface methodology.

Materials and Methods
To estimate the efficiency of ultrasound on sludge disinfection, the samples of sludge were taken after digesters from sewage treatment plant of the south of Tehran.For this purpose, COD was measured by Hatch DR 5000 spectrophotometer based on standard procedures test number of 5220.Total coliform and E. coli experiments were analyzed by MPN standard method and test number of 9221.Also, pH and temperature were measured by Metrom 691 device according to test numbers of 2310 and 2550.All tests were carried out by the standard methods. 22able 1 shows the sludge samples characters.
A homogenizer ultrasound with a 5-mm sonotrode was used.The output of electric power was reached up to 750 W, and the frequency of operating was set to 20 kHz.
Data analysis was done by Design Expert 10.2.1.For experiment's design, Box-Behnken design based on the RSM was used to evaluate the impact of variables on coliform inactivation.
Box-Behnken design is more proficient and powerful than other designs such as the three-level full factorial, central composite design (CCD) and Doehlert design.][25][26] The tests were evaluated at times of 30, 15, 10, 5 and 1 minute.Also, the density of ultrasound levels was 2.5, 1.3, 1, 0.75 and 0.375 W/mL.

Results and Discussion
This research was performed to determine ultrasonic wave's effect on disinfection of sludge and find the optimal operating parameters range using ultrasound waves to inactivate total coliform of sludge.Table 2 shows the response values of total coliform inactivation.The maximum coliform inactivation was 99.44% in ultrasonic density of 2.5 W/mL (P=0.0001) and time of 30 minutes.
Table 3 shows the results of variance analysis and regression.A and B are time (minutes) and density of ultrasonic (W/mL), respectively.
Equation 1 shows the expected second order polynomial equation.High coefficient of correlation (R 2 = 98.95) indicated that the model was reproducible.(1) Based on Table 2 and the statistical analysis, there was no significant differences between time of sonification and ultrasound density (p>0.05).This may be due to heterogeneity of physico-chemical composition of the samples between the repetitions.The studies conducted by Gholami et al, 27 Lazarotto et al 28 and Mahvi et al 29 found similar results.
Figure 1 shows the changes in total coliform population during time by different ultrasound densities.During the test period and formation of micro and nano-bubbles, more than 2 log reduction in total coliform at 30 minutes was observed.Some mechanisms that affect the total coliform inactivation are as follow: A) The temperature of samples will rise by increase in time of sonication which accelerates the ions diffusion process in the cell membranes.
B) Decomposition of pollutants using ultrasonic waves occur under the oxidization process by the formation of OH˙ radicals as a result of ultrasonic dynamic mixing, thermal destruction, and shearing process. 16,27,30s seen in Figure 1, with increase in time of sonication up to 30 minutes, the removal of total coliform reaches their maximum level.With increase in time of sonication, this reduction continued, though the optimal time was 30 minutes due to the cost-effectiveness of the process.
A research on the total coliform inactivation established that by increase in time of ultrasonic waves up to 45 minutes, elimination of the total coliform bacteria would be over 99%. 31Al-Juboori's study findings confirm the results of our study by showing the relationship between increase in time and the rise in total coliform inactivation. 10n a similar study conducted by Ali et al, ultrasonic wave was used for inactivation of coliforms of water.In the study, experiments showed that higher contact time up to 60 minutes and higher frequency of 60 Hz have the most effectiveness in removal of coliforms. 32igure 1 indicates a significant reduction of total coliform population by the increase in ultrasonic density from 0.375 to 2.5 W/mL.The optimum removal of total coliform was 99.44 percent which is equal to total coliform inactivation.This was achieved at ultrasonic density of 2.5 W/mL.
The pulse cycle in this research was 9 seconds, and according to the results of previous studies, more time is need to the active surface of bacteria ultrasonic cavitation of micro and nano-bubbles that lead to more bacteria inactivation. 16,31ltrasound waves on coliforms punctures their cell walls and produces OH˙ radicals, and extrusion of the intracellular matrix ultimately kills the coliforms.In general, different factors such as volume of sample, type of microorganism, wave amplitude, time and ultrasonic power have the most important role on removal of coliforms by sonification. 33 research by Ayyildiz et alon inactivation of total coliform using ultrasound waves observed that the removal rate was increased over 90 percent in 10 minutes when the ultrasonic frequency reached to 20 kHz and ultrasonic density was between 75 to 300 W/L.34 Zhou et al 35 investigated the impact of increasing ultrasonic density on inactivation of total coliform.The results revealed that by increasing ultrasonic density from 2 to 12 W/mL, the rate of inactivation enhanced considerably

Conclusion
In this study, we investigated the pilot scale sonolytic disinfection and inactivation of total coliform in wastewater sludge.The results revealed that inactivation of almost all the total coliform could be reached by applying high power and low-frequency ultrasound waves at relatively short irradiation times through formation of micro and nano cavitation bubbles.Moreover, the critical parameters of ultrasonic disinfection of sludge were ultrasound density and irradiation time.
According to the results, the maximum of total coliform inactivation was 99.44% in operating conditions of ultrasonic density, irradiation time and ultrasonic frequency of 2.5 W/mL, 30 minutes and 20 kHz respectively.The results also proved the apparent effectiveness of ultrasound waves on inactivation of the total coliform bacteria in wastewater sludge.

Figure 1 .
Figure 1.The effect of time on total coliform population (A) and inactivation (B).

Table 1 .
Sludge Samples Characteristics

Table 2 .
Response Values of Total Coliform Inactivation

Table 3 .
Variance Results of RSM Analysis for Inactivation of Total Coliform