Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 8, No. 1, January 2023 Research Paper The Effects of Reactive Oxygen and Nitrogen Species (RONS) Produced by Surface Dielectric Barrier Discharge (SDBD) Non-Thermal Plasma with Treatment Time and Distance Variations to Kill Escherichia coli Renaldo Apriandi Kasa1*, Unggul Pundjung Juswono2, Dionysius J. D. H. Santjojo2 1Master of Physics Study Program, Universitas Brawijaya, Malang, 65145, Indonesia2Departement of Physic, Universitas Brawijaya, Malang, 65145, Indonesia *Corresponding author: randi−kasa@student.ub.ac.id AbstractResearch on the inactivation of Escherichiacoli causing diarrheal disease using non-thermal plasma SDBD has been carried out. SDBDis a new technique for non-thermal plasma generation with several advantages: low power generation, comprehensive treatmentarea coverage, and reducing the potential effects of burning and drying tissue. This study aimed to analyze the effect of treatmenttime variations, namely 0 as control, 60, 75, 90, 105, and 120 seconds and treatment distance variations of 3, 6, 9, 12, and 15 mmof non-thermal plasma treatment of SDBD on E. coli. The results of the non-thermal plasma SDBD treatment with variations intime and distance showed that the longer the treatment time, the more bacterial cells died. Colony counts decreased to 4.33 x 107CFU/mL compared to the control, 409 x 107 CFU/mL, with a treatment time variation of 120 seconds, yielding the best treatmentresults. At the same time, the results of the treatment for variations in the non-thermal plasma distance of SDBD showed that thesmaller the treatment distance, the greater the bacterial death rate, with the best treatment results at a 3 mm treatment interval,with colony counts of 8 x 107 CFU/mL, compared to 409 x 107 CFU/mL in control. Based on these results, SDBD non-thermalplasma treatment can be used to inactivate or kill bacteria with effectiveness in killing bacteria depending on the length of treatmenttime and the distance of treatment. KeywordsNon-thermal Plasma, SDBD, E. Coli, RONS, Treatment Time, Treatment Distance Received: 18 September 2022, Accepted: 13 Desember 2022 https://doi.org/10.26554/sti.2023.8.1.45-51 1. INTRODUCTION Infectious diseases are one of signicant health problems in almost all developing countries, including Indonesia. One of the most common infectious diseases is diarrhea. According to Ragil and Dyah (2017), diarrhea is one of the leading causes of illness and death in almost all geographic areas, and all age groups can be aected. In Indonesia, diarrheal disease is a potential endemic disease of Extraordinary Events (KLB), and it is frequently fatal. According to Kemenkes RI (2018), the number of suerers of diarrheal disease based on the results of the diagnosis by health workers was 6.8%, while based on the symptoms experienced, 8%. The number of patients with diarrheal diseases classied according to age found that the age group of 1 to 4 years had the highest number of suerers, namely 11.5%. In addition, the age group of 75 years and over has a relatively high number of suerers, namely 7.2% (Prab- hakara, 2010). Microorganisms that generally cause disease are called pathogens. Pathogens include bacteria, protozoa, viruses, prions, fungi, and worms. These pathogens can cause various symptoms and diseases, including diarrhea (Levy et al., 2018). Pathogens that cause diarrheal disease can come from viruses, for example, Rotavirus (40-60%), Escherichia coli (20- 30%), Shigella sp. (1-2%), and the parasite Entamoeba histolytica (Ragil and Dyah, 2017). Several conventional sterilization techniques have been de- veloped to inactivate disease-causing microorganisms, such as sterilization using dry heat (oven), moist heat (autoclaving), and chemicals such as glutaraldehyde and the use of gamma irradiation (Moisan et al., 2001; Morent and De Geyter, 2011; Park et al., 2003). This conventional technique that has been used has several disadvantages, such as high processing temper- atures, long sterilization times, and the use of toxic chemicals, resulting in changes to the material during sterilization, and this technique is quite expensive to use (Moisan et al., 2001; Morent and De Geyter, 2011). Based on the limitations of con- ventional methods, it is necessary to have a new and alternative https://crossmark.crossref.org/dialog/?doi=10.26554/sti.2023.8.1.45-51&domain=pdf https://doi.org/10.26554/sti.2023.8.1.45-51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 sterilization method, namely pretreatment with non-thermal plasma (plasma sterilization) (Hati et al., 2012). Plasma is the fourth matter after solids, liquids, and gases. Plasma is dened as an ionized gas of free particles (Putra et al., 2021; Scholtz et al., 2021; Šimončicová et al., 2019). Based on the temperature, plasma is divided into thermal plasma and non-thermal plasma (Morent and De Geyter, 2011). Cold or non-thermal plasma is produced by applying an electric or electromagnetic eld to a gas. Plasma contains excited states of molecules and atoms, cations and anions, free radicals, elec- trons, UV radiation, ozone, superoxide, hydroxyl radicals, sin- gleoxygen, atomicoxygen, nitrogenoxides, ornitrogen dioxide (Puligundla and Mok, 2017; Zheng et al., 2016). These species exhibit antimicrobial activity against various microorganisms, including bacteria, yeasts, and even bacterial and fungal spores (López et al., 2019). Surface dielectric barrier discharge is a new technique for non-thermal plasma generation with several advantages: low powergeneration, comprehensive treatment area coverage, and reducing the potential eects of burning and drying tissue. Sev- eral non-thermal plasma sterilization techniques have been carried out, but further research is needed to optimize non- thermal plasma techniques. Therefore, this study aimed to an- alyze the eect of treatment time and distance of non-thermal SDBD plasma treatment on E. coli. 2. EXPERIMENTAL SECTION 2.1 Materials The non-thermal plasma system used in this study is a sur- face dielectric barrier discharge. The non-thermal plasma of the surface dielectric barrier discharge is generated using a 20 V DC voltage source which is then transformed into a high voltage source to create the plasma. The scheme used in this study is shown in Figure 1. The plasma non-thermal discharge surface dielectric barrier uses two copper electrodes. A dielec- tric separates two copper electrodes. The two electrodes are connected to a voltage source, with one electrode connected to the ground and the other connected to a high-voltage source. The sample used in this study was E. coli obtained from the microbiology laboratory, Faculty of Medicine, Brawijaya Uni- versity. In addition, some materials, namelynutrient agar (NA), are used as media for bacterial growth and sterile physiological NaCl for dilution. 2.2 Methods 2.2.1 E. coli Sample Preparation and SDBD Non-thermal Plasma Treatment E. coli isolates that had been incubated for 24 hours were di- luted with serial dilutions up to 10−6 with one loop of bacterial isolates homogenized with 9 mL of sterile physiological NaCl (10−1 dilution).1 mL of the 10−1 dilution of bacterial suspen- sion was homogenized with 9 mL of sterile physiological NaCl (10−2 dilution). This step was carried out until a dilution of 10−6. The results of the dilution were then treated using non- thermal plasma with the surface dielectric barrier discharge Figure 1. Schematic of SDBD Non-thermal Plasma with variations in treatment time (0 as control, 60, 75, 90, 105, 120 seconds) and treatment distance (3 mm, 6 mm, 9 mm, 12 mm, 15 mm). The results of the treatment were then spidered using a triangular rod. After that, it was incubated at 37°C for 24 hours by placing the petri dish in an inverted position. The next step was to count the number of colonies in each treatment. Each experiment was repeated three times. 2.2.2 Optical Emission Spectroscopy Optical emission spectroscopy (OES) was used to characterize reactive plasma species and to analyze plasma composition, which can explain the relationship or mechanism between re- active spaces formed in plasma and the ability to inactivate bacteria (Wiegand et al., 2014). The optical emission spec- trum was measured using Aurora 4000 at a wavelength of 200 to 900 nm with an integration time of 5000 ms and 3 repe- titions of the spectrum capture and then averaged to obtain the optical emission spectrum from the plasma. The emission spectrumobtainedwas thenanalyzedqualitativelytodetermine the chemical species at each wavelength peak. The results of identifying the wavelength peaks were then analyzed using the National Institute of Standards and Technology atomic spec- trum database and previous journal publications to identify chemically active species (Sarangapani et al., 2016). 2.2.3 Data Analysis The data obtained were based on the eect of treatment time (0, 60, 75, 90, 105, 120 seconds) and treatment distance (3, 6, 9, 12, 15 mm) on the number of bacteria, tabulated and analyzed using ANOVA using SPSS software. 26. If p-value < 0.05 H0 was accepted, then the length of treatment time and treatment distance aect the number of bacteria. 3. RESULT AND DISCUSSION 3.1 Optical Emission Spectroscopy of SDBD Non-thermal Plasma An optical emission spectrum (OES) can measure and analyze reactive species produced by non-thermal plasma. The spec- trum results obtained can be analyzed for reactive species by looking at the spectrum peaks. In the following section, Fig- © 2023 The Authors. Page 46 of 51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 Figure 2. OES Spectrum of SDBD Non-thermal Plasma with Treatment Time Variation and Treatment Distance Variation ure 2 depicts the spectrum obtained from a surface discharge dielectric barrier non-thermal plasma. Spectrum results were obtained using OES for each vari- ation of treatment time and distance. The time variation is taken every 60 seconds, 90 seconds, and 120 seconds, while the distance variation is taken every 3 mm. 6mm, and 9mm. The spectrum results with time variations show the increasing intensity with longer treatment time. In contrast, for variations in the distance, the intensity will be smaller if the distance from the plasma source is further away. The resulting spectrum was measured using OES at a wave- length of 200 to 900 nm from the plasma source. The intensity emitted from the plasma source is recorded at each wavelength. In the formation of plasma, various chemical species are in an excited state. The chemical species produced in the gas phase were observed using OES during plasma release (Sarangapani et al., 2016). In the UV region, the emission spectrum shows that N2 and N2+ excitation species’ emission shows dierent peaks. A small peaks of OH appear at wavelengths 296,1 nm (Adhikari et al., 2021; Dhungana et al., 2020; Hosseini et al., 2018; Naz et al., 2021; Sarangapani et al., 2016). The low intensity of singlet oxygen is at a wavelength of 777.5 nm (Sarangapani et al., 2016). At the same time, the N2 Second Positive System (SPS) has a prominent peak at a wavelength of 313-390 nm, N2 rst negative system (FNS) at a wavelength of 390-450 nm (Akter et al., 2020; Misra et al., 2015). From the reactive species produced in the gas phase plasma, long-lived or short-lived reactive species such as hydrogen peroxide (H2O2) and ozone (O3) are formed as long-lived reactive species, and short-lived reactive species such as hydroxyl radicals (•OH), singlet oxygen (1O2), superoxide anion (O2−), atomic oxygen (O), nitrite oxide (NO), and peroxynitrite (ONOO−), all of these reactive species exhibit antimicrobial activity (Xu et al., 2018). 3.2 Eect of Treatment Time and Distance of SBDB Non- thermal Plasma Teatment on the Number of Colonies Non-thermal plasma can decontaminate bacteria. The mecha- nism and level of decontamination capability vary depending on the length of treatment time, the distance of treatment, the amount of voltage used, and the source of the gas used. All play a role in how eective non-thermal plasma is at decontam- inating bacteria (Amalda et al., 2020). In research on bacterial inactivation using SDBD non-thermal plasma treatment, vari- ations in treatment time and treatment distances were used. For the treatment time, variations in treatment time were used, namely 60 s, 75 s, 90 s, 105 s, and 120 s. Various treatment distances were also used, namely 3 mm, 6 mm, 9 mm, 12 mm, and 15 mm. From the results of the treatment obtained, it can be seen in Table 1 and Table 2 that non-thermal plasma inu- ences bacterial inactivation for variations in treatment time and variations in treatment distance. The longer the treatment time, the more bacteria are inactivated, or the number of inactivated bacteria is directly proportional to the length of treatment time. The eect of treatment time on colony number can be seen in Figure 3. Meanwhile, treatment results with distance variations and SBDB non-thermal plasma still aect the inactivation of bacteria. The smaller the distance of treatment used for treat- ment, the more bacteria will die, or the ability of the bacteria to live will be smaller. The eect of treatment distance on the number of colonies can be seen in Figure 5. The results of the treatment showed that the treatment time of 120 seconds had a better ability to inactivate or kill bacteria than other treatments. The number of bacteria that grewafterbeing treated for120 seconds was an average of 4.3 x 107 CFU/mL. This number was much lower than the number of bacteria that grew without non-thermal plasma treatment, whichwas409x107 CFU/mL.While fortheshorter treatment time, the number of colonies that grew more and more was 105 s, 90 s, 75s, and 60 s, with the average number of colonies growing was 11 x 107 CFU/mL, 13 x 107 CFU/mL, 46.33 x 107 CFU/mL, 56 x 107 CFU/mL. Pictures of the number of colonies before (control) and after treatment with treatment time variations can be seen in Figure 4. Meanwhile, from the results of the variation in treatment distance, 3 mm has a more extraordinary ability to inactivate bacteria than other treatment distances. The number of bac- teria that grew after being treated at 3 mm averaged 8 x 107 CFU/mL. This number is much decreased compared to the number of bacteria that grow when the treatment is carried out at a longer distance. At the furthest distance carried out in this study, which was 15 mm, the number of colonies growing on average was 253 x 107 CFU/mL. At the same time, the number of bacterial colonies in the control treatment was 409 x 107 CFU/mL. Images of colony count before (control) and after treatment with treatment distance variations can be seen in Figure 6. © 2023 The Authors. Page 47 of 51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 Table 1. Number of Colonies Before (Control) and After the Treatment with Treatment Time Variations (P < 0.05) Time (Second) Distance (mm) Number of Colonies (x 107 CFU/mL) Treatment I Treatment II Treatment III Control 410 418 399 60 3 54 46 68 75 3 60 43 36 90 3 13 15 11 105 3 7 3 23 120 3 4 3 6 Figure 3. Graph of the Number of Colonies Before (Control) and After Treatment with Treatment Time Variations (60 s, 75 s, 90 s, 105 s, 120 s) (R2 = 0.98188) The treatment of time variation and distance variation shows that there is an eect of non-thermal plasma treatment of SDBD. This eect is due to the reactive species formed during plasma generation. The RONS formed to have an im- portant role in the inactivation of bacteria. RONS can disrupt the bonding of microbial cell structures with lipid peroxidation, damaging the RONS membrane as reactive free radicals (NO, •OH, and superoxide) or strong oxidizing agents (H2O2 and O3) can penetrate microorganisms. Furtherchemical reactions can occur in the cytoplasm that can oxidize cellular proteins or microbial DNA (Klämp et al., 2012). One of the molecules that are the main agent of bacterial inactivation is NO (Nitrogen Oxide). NO can destroy cells by dimerizing thymine bases on DNA strands, disrupting DNA replication (Amalda et al., 2020; Tian et al., 2010). Besides NO, the reactive species formed during plasma formation is H2O2 which has the potential to cause oxidative damage. Be- sidesbeingable tocauseoxidativedamage,H2O2 alsofunctions as a more potent hydroxyl radical (•OH) precursor. The •OH radical is a reactive oxygen species with excellent reactive abil- Figure 4. Picture of the Number of Colonies Before (Control) and After Treatment with Treatment Time Variations © 2023 The Authors. Page 48 of 51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 Table 2. Number of Colonies Before (Control) and After the Treatment with Treatment Distance Variations (P < 0.05) Time (Second) Distance (mm) Number of Colonies (x 107 CFU/mL) Treatment I Treatment II Treatment III Control 410 418 399 120 3 10 6 8 120 6 12 13 14 120 9 49 70 85 120 12 137 109 224 120 15 284 242 233 Figure 5. Graph of the Number of Colonies Before (Control) and After Treatment with Treatment Distance Variations (3 mm, 6 mm, 9 mm, 12 mm, 15 mm) (R2 = 0.99727) ity and can produce oxidative damage to cell components (Pai etal.,2018). The•OHradical is a strongoxidant thatcancause a decrease in ATP, thereby causing low energy in cells. The •OH radical can also break the phosphodiester bond of DNA molecules, which causes DNA fragmentation, and can become lipids in cell membranes, resulting in cells being unable to repli- cate or causing cell death (Feng and Wang, 2020). In addition, another strong oxidizing agent plays a role in the inactivation of bacteria, namely ozone (O3). Ozone has a considerable ox- idation potential, which can damage cell walls and bacterial cytoplasmic membranes, increasing membrane permeability. As a result, there is a decrease in surface tension which results in cell leakage. Furthermore, ozone and other reactive species easily enter the cell and damage bacterial nucleic acids, damag- ing the pyramidal rings and breaking the bonds between the pyramidal rings and the sugar groups in nucleic acids. This nu- cleic acid damage will result in cell death (Kristanti and Dessy, 2012). The concentration of reactive species formed during non- thermal plasma treatment signicantly aects the sterilization Figure 6. Picture of the Number of Colonies Before (Control) and After Treatment with Treatment Distance Variations © 2023 The Authors. Page 49 of 51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 ecacy. The longer the treatment time, the more reactive the plasma species contains and the more eective it is in killing bacteria. Similarly, the variation in treatment distance showed a relationship between the ability of bacteria to survive and the number of reactive species produced during plasma treat- ment. The results of the OES spectrum in Figure 3. show that the farther the treatment distance, the smaller the intensity of the spectrum, which indicates that the fewer reactive species formed, the lower the ability to kill bacteria. 4. CONCLUSION The results of research that have been carried out on the eect of RONS produced by SDBD non-thermal plasma to kill Es- cherichia coli show that the time and distance of treatment aect the numberof colonies that live before and after treatment. For variations in treatment time, the longer the treatment time, the more bacteria are inactivated, or the more bacteria die where the numberof colonies that growwhen treated with a treatment time of 120 s, which is 4.3 x 107 CFU/mL, is much lower than the control, which is 409 x 107 CFU/mL. As for variations in treatment distance, the farther the treatment distance, the more the number of colonies that live, where the number of colonies that grow when treated with 15 mm, namely 253 x 107 CFU/mL, signicantly decreased when treated with 3 mm, namely 8 x 107 CFU/mL. Based on these results, further re- search can be investigated more deeply by looking at the eect of non-thermal plasma treatment on DNA, lipids, proteins, and bacterial cell morphology to determine the mechanism of non-thermal plasma treatment that kills bacteria. Additionally, several variations of parameters, such as gas source and volt- age, can be carried out in order to obtain the most eective non-thermal plasma composition. 5. ACKNOWLEDGMENT The author would like to thank Lembaga Pengelola Dana Pen- didikan (LPDP), which has funded this research, and all parties who have helped and been involved in completing this research. REFERENCES Adhikari, B. C., P. Lamichhane, J. S. Lim, L. N. Nguyen, and E. H. Choi (2021). Generation of Reactive Species by Naturally Sucked Air in the Ar Plasma Jet. Results in Physics, 30; 104863 Akter, M., D.K.Yadav, S.H.Ki, E.H.Choi, andI.Han(2020). Inactivation of Infectious Bacteria Using Nonthermal Bio- compatible Plasma Cabinet Sterilizer. International Journal of Molecular Sciences, 21(21); 8321 Amalda, E. C., F. Alhamidah, Y. Oktanella, and M. Khuzain (2020). Kajian Artikel: Potensi Plasma Non Termal Se- bagai Kandidat Terapi Mastitis Subklinis. VITEK: Bidang Kedokteran Hewan, 10; 1–9 (in Indonesia) Dhungana, S., R. P. Guragain, H. Baniya, G. Panta, G. K. Chhetri, and D. Subedi (2020). Electrical and Optical Char- acterization of Gliding Arc Discharge (GAD) Operated at Line Frequency (50 Hz) Power Supply. Journal of Nepal Physical Society, 6(2); 26–33 Feng, T. and J. Wang (2020). Oxidative Stress Tolerance and Antioxidant Capacity of Lactic Acid Bacteria As Probiotic: A Systematic Review. Gut Microbes, 12(1); 1801944 Hati, S., S. Mandal, S. Vij, P. Minz, S. Basu, Y. Khetra, D. Ya- dav, and M. Dahiya (2012). Nonthermal PlasmaTechnology and Its Potential Applications Against Foodborne Microor- ganisms. Journal of Food Processing and Preservation, 36(6); 518–524 Hosseini, S. I., S. Mohsenimehr, J. Hadian, M. Ghorbanpour, and B. Shokri (2018). Physico-chemical Induced Modi- cation of Seed Germination and Early Development in Artichoke (Cynara scolymus L.) Using Low Energy Plasma Technology. Physics of Plasmas, 25(1); 013525 KemenkesRI (2018). HasilRisetKesehatanDasarTahun2018. Kementrian Kesehatan RI, 53(9); 1689–1699 (in Indonesia) Klämp, T. G., G. Isbary, T. Shimizu, Y. F. Li, J. L. Zim- mermann, W. Stolz, J. Schlegel, G. E. Morll, and H. U. Schmidt (2012). Cold Atmospheric Air Plasma Steriliza- tion Against Spores and Other Microorganisms of Clinical Interest. Applied and Environmental Microbiology, 78(15); 5077–5082 Kristanti, Y. and D. Dessy (2012). Pengaruh Ozonated Wa- ter Sebagai Antiseptik Dalam Menghambat Pertumbuhan Staphilococcus Aureus (in Vitro). Majalah Kedokteran Gigi Indonesia, 19(1); 25–28 (in Indonesia) Levy, K., S. M. Smith, and E. J. Carlton (2018). Climate Change Impacts on Waterborne Diseases: Moving Toward Designing Interventions. Current Environmental Health Re- ports, 5(2); 272–282 López, M., T. Calvo, M. Prieto, R. Múgica Vidal, I. Muro Fraguas, F. Alba-Elías, and A. Alvarez-Ordóñez (2019). A Review on Non-thermal Atmospheric Plasma For Food Preservation: Mode of Action, Determinants of Eectiveness, and Applications. Frontiers in Microbiology, 10; 622 Misra, N., K. Keener, P. Bourke, and P. Cullen (2015). Gener- ation of In-package Cold Plasma and Ecacy Assessment Using Methylene Blue. Plasma Chemistry and Plasma Process- ing, 35(6); 1043–1056 Moisan, M., J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, and Y. L’H (2001). Low Temperature Sterilization Using Gas Plasmas: A Review of the Experiments and An Analy- sis of the Inactivation Mechanisms. International Journal of Pharmaceutics, 226(1-2); 1–21 Morent, R. and N. De Geyter (2011). Inactivation of Bacteria by Non-thermal Plasmas. Biomedical Engineering-Frontiers and Challenges; 25–50 Naz, M., S. Shukrullah, S. Rehman, Y. Khan, A. Al Arainy, and R. Meer (2021). Optical Characterization of Non-thermal Plasma Jet Energy Carriers for Eective Catalytic Processing of Industrial Wastewaters. Scientic Reports, 11(1); 1–13 Pai, K., C. Timmons, K. D. Roehm, A. Ngo, S. S. Narayanan, A. Ramachandran, J. D. Jacob, L. M. Ma, and S. V. Madi- © 2023 The Authors. Page 50 of 51 Kasa et. al. Science and Technology Indonesia, 8 (2023) 45-51 hally (2018). Investigation of The Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge. Scientic Reports, 8(1); 1–13 Park, B. J., D. Lee, J. C. Park, I. S. Lee, K. Y. Lee, S. Hyun, M. S. Chun, and K. H. Chung (2003). Sterilization Using A Microwave-induced Argon Plasma System at Atmospheric Pressure. Physics of Plasmas, 10(11); 4539–4544 Prabhakara, G. (2010). Health Statistics (Health Information System). Short Textbook of Preventive and Social Medicine; 28–28 Puligundla, P. and C. Mok (2017). Potential Applications of Nonthermal Plasmas Against Biolm-associated Micro- organisms In Vitro. Journal of Applied Microbiology, 122(5); 1134–1148 Putra, V. G. V., I. Irwan, I. Purnama, J. N. Mohamad, and Y. Yusuf (2021). A Study of Anti-Radiation Weaving Fabric with Plasma Corona Treatment. Indonesian Journal of Applied Physics, 11(1); 59–67 Ragil, D. and Y. Dyah (2017). Hubungan Antara Pengetahuan dan Kebiasaan Mencuci Tangan Pengasuh Dengan Kejadian Diare Pada Balita. Jurnal of Health Education, 2(1); 39–46 (in Indonesia) Sarangapani, C., N. Misra, V. Milosavljevic, P. Bourke, F. O’Regan, and P. Cullen (2016). Pesticide Degradation in Water Using Atmospheric Air Cold Plasma. Journal of Water Process Engineering, 9; 225–232 Scholtz, V., E. Vaňková, P. Kašparová, R. Premanath, I. Karunasagar, and J. Julák (2021). Non-thermal Plasma Treatment of ESKAPE Pathogens: a Review. Frontiers in Microbiology, 12; 2919 Šimončicová, J., S. Kryštofová, V. Medvecká, K. Ďurišová, and B. Kaliňáková (2019). Technical Applications of Plasma Treatments: Current State and Perspectives. Applied Micro- biology and Biotechnology, 103(13); 5117–5129 Tian, Y., P. Sun, H. Wu, N. Bai, R. Wang, W. Zhu, J. Zhang, and F. Liu (2010). Inactivation of Staphylococcus aureus and Enterococcus faecalis by A Direct-current, Cold Atmospheric- pressure Air Plasma Microjet. Journal of Biomedical Research, 24(4); 264–269 Wiegand, C., O. Beier, K. Horn, A. Pfuch, T. Tölke, U. C. Hipler, and A. Schimanski (2014). Antimicrobial Impact of Cold Atmospheric Pressure Plasma on Medical Critical Yeasts and Bacteria Cultures. Skin Pharmacology and Physiol- ogy, 27(1); 25–35 Xu, Z., C. Cheng, J. Shen, Y. Lan, S. Hu, W. Han, and P. K. Chu (2018). In Vitro Antimicrobial Eects and Mechanisms ofDirectCurrentAir-liquidDischargePlasmaonPlanktonic Staphylococcus aureus and Escherichia coli in Liquids. Bioelec- trochemistry, 121; 125–134 Zheng, C., Y. Kou, Z. Liu, C. Li, Y. Huang, and K. Yan (2016). Rapid Disinfection Performance of A Touchable Pulsed Sdbd Nonthermal Plasma. IEEE Transactions on Plasma Science, 44(11); 2667–2672 © 2023 The Authors. Page 51 of 51 INTRODUCTION EXPERIMENTAL SECTION Materials Methods E. coli Sample Preparation and SDBD Non-thermal Plasma Treatment Optical Emission Spectroscopy Data Analysis RESULT AND DISCUSSION Optical Emission Spectroscopy of SDBD Non-thermal Plasma Effect of Treatment Time and Distance of SBDB Non-thermal Plasma Teatment on the Number of Colonies CONCLUSION ACKNOWLEDGMENT