Bacterial Cell Inactivation Using a Single-Frequency Batch-Type Ultrasound Device

Ultrasound technology employs cavitation to generate highpressure soundwaves to disrupt bacterial cells. This study reveals the effectiveness of a single frequency ultrasound device for bacterial cell inactivation. A low-cost ultrasound device having a single frequency, i.e. 22 kHz for lab-scale application, was developed first, and the prototype was mechanically designed and analyzed using the finite-element method to assure the targeted natural frequency could be achieved. The prototype was then tested inactivating bacterial cells, Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), in a simple medium and a food system, and the results were then compared to a commercial system. A treatment time of up to 15 minutes was able to reduce E. coli and B. subtilis cells by 3.3 log and 2.8 log, respectively, and these results were similar to those of the commercial system. The effectiveness of bacterial cell inactivation using the developed single-frequency ultrasound device is then discussed. The findings are useful for designing low-cost ultrasound devices for application in the food industry. © 2021 Tim Pengembang Jurnal UPI Article History: Received 28 Nov 2020 Revised 28 Des 2020 Accepted 13 Jan 2021 Available online 20 Jan 2021 ____________________ Keyword: Ultrasonication, Cavitation, Bubble, Bacterial inactivation Indonesian Journal of Science & Technology Journal homepage: http://ejournal.upi.edu/index.php/ijost/ Indonesian Journal of Science & Technology 6 (1) (2021) 65-80 Poetro Sambegoro et.al. Bacterial Cell Inactivation Using a ... | 66 DOI: https://doi.org/10.17509/ijost.v6i1.31516 pISSN 2528-1410 eISSN 2527-8045


INTRODUCTION
Ultrasound technology has been developed for various applications, among which are microbial cell inactivation and DNA extraction (Chemat et al., 2011). Cavitation occurs when vapor bubbles form in a fluid due to a drop-in pressure below the saturated pressure (Triawan et al., 2019). Using ultrasonic technology, this pressure drop is provided by a fluctuating transducer. After the bubbles are formed, they undergo an expansion and compression phases and collapse. The collapse of the cavitation bubbles can exert a high pressure due to the shock wave and microjet effects. The bursting cavitation bubbles have a highly destructive effect that can be used to damage bacterial cell walls. Water molecules in the vicinity of the bubbles also exert a shear stress effect and cause deformation of the cell membrane. At the same time, the local temperature also rises sharply. These complex phenomena contribute to the overall bacterial inactivation.
Several independent studies on bacterial inactivation using ultrasound have been performed. Ultrasonic waves generate the bubbles and drive them to explode, resulting in high pressure and temperature, and active species in some cases (Wu & Nyborg, 2008). Shear force due to an ultrasonic pressure wave is commonly known to be the primary cell-inactivation mechanism leading to cell disruption and death (Shengpu Gao et al., 2014a;Liao et al., 2018;Piyasena et al., 2003). At higher frequency ranges, this physical effect may be accompanied by a sonochemical reaction in the surrounding medium triggered by the increasing temperature and pressure from the collapsing bubbles (Ashokkumar, 2011).
Heat treatment is the most conventional method applied to bacterial cell inactivation in the food industry (Abdurrahman et al., 2019;Chemat et al., 2011;Piyasena et al., 2003;Raso & Barbosa-Cánovas, 2003). However, the intensity of the heat treatment may have several negative impacts on food quality and nutrition (Chemat et al., 2011). Another common method to inactivate bacteria is by adding chemical preservatives. This method may be effective but some adverse side effects might not be tolerable in the long term. Alternative approaches such as application of natural antimicrobial molecules (Brogden, 2005;Lyu et al., 2019;Xiang et al., 2016) and non-thermal technologies (pulsed electric fields, UV light, high hydrostatic pressure, microwave, ultrasound) have been explored to inactivate cells (Butz & Tauscher, 2002;Leonelli & Mason, 2010;Raso & Barbosa-Cánovas, 2003).
Unlike heat or chemical treatment, mechanical treatments by using membrane filter (Bilad, 2017;Gao et al., 2018;Sharpe et al., 1979) and ultrasound can minimize negative impacts in food quality and prevent harmful health effects (Chemat et al., 2011). While the membrane filter requires high maintenance and does not inactivate the bacteria, the ultrasound treatment has proven to inactivate the bacteria effectively. Ultrasound treatment especially for food preservation can also be assisted with other physical methods such as high pressure (manosonication), heat (thermosonication) and combination of both (manothermosonication) to enhance its effectiveness (Piyasena et al., 2003;Sango et al., 2014;Zupanc et al., 2019). Although, these ultrasounds assisted technology is not within our scope of experiment, but several studies have mentioned their promising results for milk and juices (Bermúdez-Aguirre et al., 2009;Walkling-Ribeiro et al., 2009).
The physical parameters of the ultrasound device are important to achieve maximum inactivation effect. Working frequency and intensity are important parameters that can influence the cavitation effect in the propagating media (Brotchie et al., 2009).
The readily available commercial ultrasound device for cell disruption is normally operated at a single frequency of 20 kHz. However, several studies showed that frequencies higher than 20 kHz cause more effective biocidal effects (Shengpu Gao et al., 2014a;Shengpu Gao et al., 2014b). This motivated us to develop a low-cost ultrasound device prototype with a configuration that allows for different frequency ranges. We designed a batch-type ultrasound device installed with a piezoelectric of 22 kHz.
The batch was designed to have a natural frequency of 22 kHz to ensure that the resonance phenomenon occurs. The prototype was then tested on Escherichia coli and Bacillus subtilis in a simple medium (e.g., phosphate buffer) and a more complex medium (e.g., milk). The results were also compared to those from another experiment using a commercial-probe ultrasound device. Parameters affecting the bacterial cell inactivation were comprehensively discussed.

Ultrasound prototype design and manufacture
The ultrasound prototype presented in this study was of a tubular configuration that had been developed previously (Borthwick et al., 2005). Prior to manufacturing, vibration analysis had to be conducted to assure the prototype could vibrate with the maximum amplitude at the designated frequency, which directly corellated with the prototype efficiency.
The vibration analyses, consisting of modal analysis and harmonic analysis, were performed using the finite element method (FEM). Modal analysis was conducted to get the characteristics of the system, including its natural frequency and corresponding vibration modes (Eslaminejad et al., 2019). These parameters are the foundation for harmonic response analysis, which results in a response at every determined frequency. The peak response corresponds to the natural frequency of the system.
The system was a cylinder with one closed end designed using the ANSYS Design Modeler. The cylinder tank had an outer diameter 45 mm, inner diameter (Di) of 25 mm, height of 22 mm, and hole depth of 18 mm Figure 1. The tank, which was made of steel, was then fitted to a 22 kHz piezoelectric transducer (Steiner & Martins Inc, Doral, FL) and had the mechanical properties presented in Table 1. The steel material was selected because it can convert mechanical vibration generated by the transducer to be pressure waves in liquid inside the tank with low damping (Nurprasetio et al., 2018).
A pressure load to substitute the transducer contact was applied to the outer perimeter of the cylinder surface as the loading condition, which can be seen in Figure 2. The magnitude of the pressure was obtained by dividing the maximum force of the piezoelectric with its surface area; in this case, 0.25 MPa was input into the system. The settings used for harmonic response analysis are shown in Table 2

Bacterial cell preparation
E. coli and B. subtilis cultures were provided by the Microbiology Laboratory at the School of Life Sciences and Technology at ITB. Each culture was grown overnight in Luria Broth media (HiMedia, Mumbai, India) at 37 °C, and it reached 6.5-7 log colonyforming unit per mililiter (CFU/ml). The culture was then harvested and centrifuged down at 10,000 g for 5 minutes. The pellet was washed twice using a phosphate buffer saline solution at a pH of 7.4. The final suspension with the same buffer was used for the ultrasound treatment.

Food sample preparation
Commercial pasteurized cow milk (full cream) was used as a sample. The milk, with a pH of 6.5, was obtained from a local supermarket. Prior to ultrasound treatment, two batches of pasteurized milk were each inoculated with E. coli and B. subtilis and incubated overnight at 37 °C until they reached 6.5-7 log CFU/ml. The inoculated milk was stored at 4°C before the experiment.

Ultrasound experiment
Ultrasound treatment was carried out using the 20 kHz prototype and a 20 kHz probe cell disruptor (Omni Ruptor 4000, GA, USA) for comparison. Five ml of bacterial suspension in pH 7.4 PBS and inoculated milk were exposed to ultrasound for 1, 5, 10, and 15 minutes. The probe cell disruptor was set at 20% and 40% power output (equal to 80W and 160W) with constant sonication. To determine number of surviving cells after treatment, 0.1 ml of each treated sample was spread in Luria Agar (HiMedia, Mumbai, India) and grown at 37 °C for 12 -16 hours until the colony was viable.

Microscopic analysis
To study cell agglomeration of B. subtilis before and after ultrasound treatment, cell suspension was analyzed under an inverted phase contrast microscope (Nikon Eclipse TE 300, NY, USA). The culture suspension was dropped onto a microscope slide (10-20 l) using a glass slide cover. Visualization was performed at 10x, 20x, and 40x magnification.

Genomic analysis
Extraction of genomic DNA of E. coli and B. subtilis after ultrasound treatment was performed according to Rapley. The extracted DNA was then amplified using a mini8 thermal cycler (miniPCR, MA, USA) with a temperature cycling consisting of a

Modal analysis of ultrasound prototype
Natural frequencies of modal analysis obtained from finite-element analysis are presented in Table 3. The first six modes were rigid body movement, which means the tank did not undergo any significant internal deformation. The rest of the modes were the bending modes. Two pairs of modes, 7 th and 8 th , as well as 9 th and 10 th , each had the same natural frequencies, however they had different phases. This was due to the symmetric geometry of the tank.
The 7 th and 11 th modes show bending oscillation in axial and radial movement as presented in Figure 4. These modes follow the loading condition from the piezoelectric (cyclic radial force on the outer surface of the tank).
Modal analysis Figure 5 of a tank with a 25 mm inner diameter (Di) shows the maximum radial displacement (7.8 x 10 -3 mm) that occurs at 37 kHz. Ideally, the prototype should be operated at this frequency to ensure high-power conversion efficiency. However, one of the constraints in the design was the availability of the commercial piezoelectric transducer.
In this work, we limited our work by designing the prototype using a piezoelectric transducer with a working frequency of 22 kHz. Due to this constraint, the radial mode needed to be lowered from 37 kHz to 22 kHz. This could be achieved either by lowering the stiffness or by adding mass. The first option proved to be simpler. The stiffness of the tank was lowered by decreasing the wall thickness between the inner and outer surfaces. This parametric study was performed by increasing the inner diameter (Di) by 1 mm increments until the natural frequency was found to be 22 kHz.
The natural frequency of 22 kHz was obtained when the inner diameter of the tank was 35 mm (23kHz) to 37 mm (21 kHz). For higher frequency accuracy, the resolution of the harmonic response was reduced to 100 Hz, and the harmonic response was conducted between 20 kHz to 25 kHz to shorten processing time. The increment of inner diameter (Di) was reduced to 0.1 mm to improve accuracy. The parametric study is presented in Figure 6. The results show that an inner diameter of 35.7 mm gives a natural frequency of 22 kHz; consequently, 35.7 mm was chosen as the inner diameter of the tank. A linear relationship was observed between the natural frequency and inner diameter for the range of 25 to 38 mm. This indicates that a typical batch can be designed and manufactured for different natural frequencies by simply changing the inner diameter.
Harmonic response analysis was repeated to analyze the tank displacement response between 0 and 40 kHz using the new inner diameter. The resolution was 1 kHz, but near the resonance frequency area, the resolution was lowered to 10 Hz. Moreover, the frequency response of tank stress (normal stress in the radial direction) was also conducted to evaluate the ability of the tank to withstand vibration at the resonance frequency.
The displacement and stress at 22 kHz were 1.7 x 10 -3 mm and 5.7 MPa, respectively. The displacement was 100 times higher than that of the first design. Maximum displacement and stress occurred at 22.04 kHz with 5.5 x 10 -3 mm displacement and 18.5 MPa. Maximum stress was found to be only 18.5 MPa, which was lower than the fatigue strength; thus, the tank could have an infinite lifetime. The FEM analysis ensures that the prototype can be built and used for the experiment.

Manufacturing and prototyping
The cylinder was machined using a standard milling and drilling machine with 100 m accuracy. This accuracy is required to ensure a natural frequency of the cylinder of around 22 kHz. The piezoelectric transducer was force fitted on the outer surface of the cylinder. Since the piezoelectric has an excited frequency of 22 kHz, the cylinder resonates and causes abundant bubble generation. The system was then connected to a function generator and an amplifier to enhance the signal. Figure 7 shows the reduction of viable cell numbers (cell densities) after being treated with a probe cell disruptor. The treatment was carried out at different ultrasonication time periods and power levels. In general, the results indicate a decline in cell numbers, with E. coli being more sensitive to ultrasonication compared to B. subtilis. After a 15-minute sonication at 160 W Figure 7a, the cell density was reduced by 3.6 log for E. coli while B. subtilis was reduced by 3.4 log. At 80 W for 15 minutes, the cell density was reduced by 3.4 log for E. coli and 3.0 log for B. subtilis Figure 7b. The prototype, with a lower power level of 8 W and a 15-minute exposure time, reduced E. coli and B. subtilis cell densities by 3.3 log and 2.8 log, respectively Figure 8. This result was comparable to treatment with a probe cell disruptor at 80 W. The prototype succesfully replicated the tendency of experimental results from commercial probes.

Microbiological and genomic DNA analysis
For treatment of bacterial cells in a more complex matrix such as milk, both commercial probes and our prototype show less inactivation sensitivity. For the commercial probe, cell density was reduced by up to 3.0, whereas for our prototype, the cell density reduction was up to 2.5 log. The different inactivation sensitivities with respect to the medium compositions and viscosities were ultimately influenced by acoustic transfer and distribution of power within the reaction solution (Bermúdez-Aguirre et al., 2009;Fitriyanti & Narsimhan, 2018;Joyce et al., 2011). In the case of milk composition, the lower efficiency at the current treatment intensity might have been due to milk proteins and cell agglomeration, which provided protection against biocidal attack.
It can be seen in Figure 7a and 7b that both power level and exposure time increase cell inactivation. This has also been widely observed in other studies (Bermúdez-Aguirre et al., 2009;Shengpu Gao et al., 2014;Joyce et al., 2011;Joyce et al., 2003;Sango et al., 2014;Tiwari et al., 2009). A study conducted by (Joyce et al., 2011;Fülscher & Roos, 1994) observed that ultrasound treatment with an intensity of 0.24 W/cm 3 (20 kHz probe sonicator) is more effective in reducing B. subtilis cells than treatment with an intensity of 0.18 W/cm 3 (38 kHz batch sonicator). Frequency and power level are two important factors in determining the effect of ultrasound because they influence the size and number of cavitation bubbles (Joyce et al., 2011). It has been found that at higher frequencies (>500 kHz) and lower power levels, cavitation bubbles are smaller and collapse with less energy due to a shorter acoustic cycle, which means a shorter time for bubble formation. As a consequence of this phenomenon, a bacterial suspension treated with a higher frequency and a lower power level experiences a significantly lower mechanical effect (Joyce et al., 2011).
The difference in sensitivity between the two tested bacterial cells, E. coli and B. subtilis, may be due to differences in their cell wall characteristics. As mentioned in previous studies, gram-negative bacteria with thinner cell walls such as E. coli, E. aerogenes, and K. pneumoniae are more sensitive to ultrasonication than grampositive bacteria with thicker cell walls such as B. subtilis and A. pullulans (Fitriyanti & Narsimhan, 2018;Shengpu Gao et al., 2014;Joyce et al., 2011). It has also been observed that cocci, or spherical bacteria, are more resistant than bacilli, or rod shaped bacteria (Joyce et al., 2011). This corresponds to our experimental results, where E. coli cell reduction was higher than that of B. subtilis. While Figure 7 shows the predicted trend in terms of treatment time, the inactivation rate seems to vary with differing time periods. This may be due to the agglomeration of the bacterial cells. Fülscher reported that ultrasound treatment (20 and 38 kHz) for 1 to 10 minutes did not have a dramatic effect on the cell viability, except for a small drop after 15 minutes of treatment. This insignificant result suggested a declumping of bacterial agglomerates with little deactivation. We also observed similar agglomeration as shown in Figure 9. Agglomeration of cells or biofilm formation were indicated in untreated B. subtilis suspension. After 15 minutes of treatment, a declumping of bacterial cells occurred, resulting in distinct individual cells. Outside the conventional power ultrasound frequency range (100 kHz < frequency < 2 MHz), acoustic cavitation may induce mechanical effects and sonochemical reactions (production of free radicals) that can damage DNA, enzymes, liposomes, and membranes (Butz & Tauscher, 2002;Shengpu Gao, Hemar, et al., 2014;Wu & Nyborg, 2008). As observed by Gao et al. (Shengpu Gao et al., 2014) ultrasound at 850 kHz was highly efficient in inactivating bacteria such as (B. subtilis, Enterobacter aerogenes, Staphylococcus epidermidis) and yeast (Aureobasidium pullulans) with inactivation rates of up to 99%. The inactivation is due to mechanical effects and generation of hydroxy radicals that cause damage to DNA and proteins. Based on these studies, we performed a genomic DNA analysis to examine the effect of prototype power intensity on bacterial cell DNA. Figure  9 presents genomic DNA bands of E. coli (B and C) and B. subtilis (D and E) after treatment with our prototype.
The fluorescent density of DNA bands decreased with longer ultrasound treatment time. Based on these results, the cells disintegrated, causing DNA molecules to be detected in the surrounding medium using gel electrophoresis. The results were consistent with another report which also observed a decrease in DNA bands for cells treated with longer sonication (Liao et al., 2018).

CONCLUSION
The readily available ultrasound probe used in the lab for cell disruption works mainly at 20 kHz. This limitation creates an opportunity for exploring a low-cost prototype. Due to the constraints imposed by the availability of piezoelectric transducers, the prototype geometry was optimized by changing the tank's inner diameter. The peak resonance with a frequency of 22 kHz was obtained for a diameter of 35.7 mm. The relationship between the natural frequency and the inner diameter of the tank was revealed. This current prototype design will later allow further modifications such as the use of transducers of different frequencies and a continuous flow-through system. The current ultrasound prototype was able to inactivate bacterial cells, E. coli and B. subtilis in a simple medium (PBS, pH 7.4) and in a food system (milk, pH 6.5). The results were comparable to treatment with a commercial probe cell disruptor. From the experiment, it was confirmed that B. subtilis has more resistance to cavitation bubbles generated by an ultrasonicator than E. coli. The experiment also confirmed that the cell density reduction in milk is lower. Further biochemical analysis and ultrasound power intensity variations need to be performed to understand this phenomenon better.

ACKNOWLEDGEMENT
This research was supported by the Institute of Research and Community Service, Institut Teknologi Bandung, under the 2020 Multidisciplinary Research Program.

AUTHORS' NOTE
The authors declare that there is no conflict of interest regarding the publication of this article. Authors confirmed that the paper was free of plagiarism.
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