Physical and chemical properties of plasma deviceThe physical and chemical properties of the plasma source for microbial activity were examined by measuring the characteristics of the high voltage applied to the electrode and the concentration of ozone, a key reactive species. Figure 2 shows the electrical characteristics of the plasma device. At a pulse frequency of 0.5 kHz, the voltage width varied between 100 and 250 μs due to changes in pulse width (Fig. 2a). The voltage could reach up to 3.8 kV, with the duration of the saturated voltage increasing as the pulse width increased. Numerous discharge current peaks appeared during voltage rises and falls, while no discharge occurred when the voltage was constant. The dissipated power (Fig. 2c) remained approximately 5 mW, showing no significant changes with pulse width variation. However, with a fixed pulse width of 100 μs (Fig. 2d), the dissipated power increased with higher pulse frequencies, reaching up to 12.9 mW at 1 kHz.Figure 2(a) Voltage waveforms at different pulse widths with a pulse frequency of 0.5 kHz. (b) Current waveforms at different pulse widths with a pulse frequency of 0.5 kHz. (c) Power dissipation as a function of pulse width. (d) Power dissipation as a function of frequency.To assess the concentration of reactive species generated in the plasma, real-time measurements were conducted for ozone, nitric oxide, and nitrogen dioxide concentrations. As shown in Fig. 3, ozone was the dominant reactive oxygen species generated from plasma discharge. Ozone is expected to be produced through pathways such as \({\varvec{O}}+{{\varvec{O}}}_{2}+{\varvec{M}}\to {{\varvec{O}}}_{3}+{\varvec{M}}\, ({\varvec{where}}\,{\varvec{M}}\,{\varvec{is}}\, {\varvec{air}}\, {\varvec{molecules}})\). During discharge, O3 is generated by a combination of O2 contained in the CDA, which is used as the discharge gas, and O generated by the plasma. The generated O3 is partially photo-decomposed by ultraviolet radiation in the range of 240–310 nm generated in the plasma, such as \({{\varvec{O}}}_{3}+{\varvec{h}}{\varvec{\nu}}\to {{\varvec{O}}}_{2}+{\varvec{O}}\). Additionally, a specific ozone concentration is maintained over time due to the ozone loss caused by the plasma treatment system’s pump. The maximum ozone concentration consistently reached 200–202 ppm, regardless of variations in the plasma pulse width (Fig. 3a).Figure 3(a) Ozone concentration over time with varying pulse widths. (b) Average (Avg) and maximum (Max) ozone concentrations with different pulse widths. (c) Ozone production over time with frequency changes. (d) Average (Avg) and maximum (Max) ozone concentrations with different frequencies.An increase in pulse frequency led to higher ozone production. At 20 Hz, 11.5 ppm of ozone was generated, and at 1 kHz, the maximum reached 248.0 ppm. However, this increase was not linear and was likely related to the plasma source temperature (see Supplementary Fig. 1). Although not detailed in the experimental results, nitric oxide and nitrogen dioxide concentrations measured with an NOX gas analyzer (EcoPhysics CLD 60) were below the detection limit, indicating minimal occurrence due to the plasma treatment. The plasma frequency range of 20 Hz to 1 kHz did not cause thermal damage to the bio-samples.The long off-pulse period primarily resulted in diffuse surface discharge without filament discharge. These conditions involve relatively low electron energy and low rotational and vibrational temperatures, which do not favor the production of nitrogen oxides (NOX). Consequently, these mild plasma operating conditions are advantageous for ozone generation as they minimize ozone quenching. Thus, the plasma is operated to maximize ozone production while reducing the formation of undesirable NOX compounds 33,34.The role of evaporated moisture from the microbial suspension is crucial in the formation of hydroxyl radicals (•OH), which are strong oxidizing agents. During plasma treatment, the presence of water vapor can lead to the generation of •OH through reactions such as \({{\varvec{H}}}_{2}{\varvec{O}}+{{\varvec{e}}}^{-}\to {\varvec{H}}+\cdot {\varvec{O}}{\varvec{H}}+{{\varvec{e}}}^{-}\) and \({{\varvec{O}}+{\varvec{H}}}_{2}{\varvec{O}}\to 2\cdot {\varvec{O}}{\varvec{H}}\). Additionally, gas-phase ozone can contribute to the formation of •OH radicals through the following pathways: \({{\varvec{O}}}_{3}+{{\varvec{H}}}_{2}{\varvec{O}}\to 2\cdot {\varvec{O}}{\varvec{H}}+{{\varvec{O}}}_{2}\)35,36.The generated •OH radicals can enhance microbial activation by inducing stress responses that promote microbial activity. However, excessive •OH can lead to microbial inactivation through oxidative damage. Therefore, controlling ambient humidity and monitoring evaporated moisture during plasma discharge is important to optimize microbial activation. Therefore, hydroxyl radicals oxidized due to moisture in the microbial suspension were identified by chemical methods (Supplementary Fig. 2).Plasma stability and reproducibilityAn optical diagnosis was performed to confirm the stability of the plasma. The optical emission spectrum (OES) of the plasma was measured and is shown in Supplementary Fig. 3a. The spectrum of the nitrogen band, including the N2 second positive system (SPS) in the 300–380 nm range, N2 first negative system (FNS) in the 380–500 nm range, and N2 first positive system (FPS) in the 500–800 nm range, was predominantly recorded. These emissions were attributed to the nitrogen molecules in the CDA used as the discharge gas. To monitor emission stability, the intensities of the prominent N2 SPS peaks at 296.2, 315.8, and 337.2 nm were tracked over 15 min. The results confirmed that after ignition, the plasma discharge remained stable with no significant fluctuations throughout the treatment period.Figure 4 shows a discharge image of the plasma, demonstrating a stable diffuse surface discharge without filamentary behavior under various operating conditions. Changes in pulse width had minimal effect on discharge intensity; however, increasing the pulse frequency did affect discharge intensity. Since discharge did not occur during the pulse-off state, discharge intensity noticeably decreased at 50 Hz due to the prolonged pulse width interval. By comparing pixel intensities from the discharge images, changes in intensity due to axis variations can be confirmed (Supplementary Fig. 4). Under 100 μs, 0.5 kHz conditions, almost uniform pixel intensity was observed along the x-axis of the linear electrode, indicating a consistent discharge. For the y-axis, the intensity increased in the 41 lines where the discharge occurred. Although the intensity was higher in the central part of the plasma source, the error was within 10% and not significant. Lowering the pulse frequency decreased overall intensity and discharge uniformity, depending on the source location.Figure 4Visualization of plasma discharge. Image of the plasma discharge area with the actuation parameters used in this study, demonstrating a stable diffuse surface discharge without filamentary behavior.Temperature measurements during plasma operation were used to determine potential temperature changes affecting bio-samples (Supplementary Fig. 1). An increase in pulse width at a pulse frequency of 0.5 kHz did not significantly affect the temperature of the plasma source. After the initial discharge, the temperature increased by 27 °C, from 23 °C to 50 °C, and then remained stable for 15 min. At a position 45 mm away from the source, where the sample was treated, the temperature change was less pronounced, showing a variation of less than 3 °C. However, changes in pulse frequency did affect the temperature changes (Supplementary Fig. 1c). At a low pulse frequency of 20 Hz, the plasma source temperature remained nearly unchanged within the error range before and after discharge. Temperature changes increased linearly with frequency, reaching 81.0 °C at 1 kHz. However, even at this frequency, the temperature changes at the sample location remained below 5 °C, avoiding sample damage.Plasma electrodes fabricated using LTCC technology notably reduced susceptibility to ions, radical species, and ultraviolet radiation commonly found in DBD plasma. These challenges are frequently encountered with polymer-based dielectrics37,38. Ceramic materials boast a high dielectric constant of 9.7, surpassing that of polymer materials, thereby ensuring the maintenance of a uniform non-thermal discharge across the entire electrode area. Moreover, the aluminum oxide (Al2O3) dielectric layer protects the structure and significantly reduces electrode oxidation and damage (Fig. 5a). The high thermal conductivity of these materials also facilitated an almost uniform temperature change across the entire plasma source during discharge (Fig. 5b). The temperature uniformity of the plasma electrode contributed to the stable generation of reactive species during long-term plasma treatment (Fig. 5c). The stable discharge of LTCC-based electrodes can play an important role as one of the characteristics of plasma devices that can activate bacteria.Figure 5(a) Comparison of electrode oxidation and damage before and after plasma discharge. (b) Assessment of temperature gradients across the plasma source during operation. (c) Monitoring of ozone concentration and temperature variations over extended treatment periods (100 h).Improvement of cell proliferation and viability of P. putida by plasma treatmentBecause pulse frequency is an important variable in plasma characteristics, the degree of microbial activation according to frequency change was compared. The results show that as the pulse frequency increases, the degree of activation also increases, with the microbial population being 2.97 times greater at 0.5 kHz compared to the control group (Fig. 6). However, when plasma treatment was applied at frequencies above 0.5 kHz, the P. putida strain was inactivated. These results suggest that the optimal condition for activating microorganisms is a frequency of 0.5 kHz and that excessive reactive oxygen species concentrations may damage microorganisms at frequencies above that. Therefore, the plasma operating conditions for activating plastic-degrading microorganisms were fixed at 0.5 kHz and 100 μs.Figure 6Degree of activation of P. putida bacteria according to changes in plasma pulse frequency.During real-time monitoring of microbial cell concentration over the incubation period, it was observed that the microbial cells exposed to air plasma for 3 min exhibited a more rapid increase in concentration over time compared to untreated microorganisms (Fig. 7a). After 5 h, the plasma-treated microbes entered a dramatically increased log phase compared to the untreated microbes. This phase of exponential cell growth was sustained until approximately 17 h, after which the cell concentration gradually began to decrease; however, it remained higher than that of untreated microorganisms. The elevated cell population of plasma-treated microorganisms during this active cell division period suggests that plasma treatment may enhance microbial cell viability.Figure 7P. putida cell viability post plasma treatment. (a) OD600 growth curves for 40 h post-treatment comparing plasma-treated and untreated groups. (b) Number of live and dead cells. (c) Viability ratio of live to dead cells after plasma treatment. (d) CLSM images of P. putida stained using the LIVE/DEAD BacLight microbial viability kit. Green indicates viable bacteria, and red indicates dead bacteria. Microbial cells used as a negative control were killed by 70% isopropyl alcohol. Comparisons are shown between untreated control P. putida and P. putida treated with plasma for 3 min. Scale bar = 50 µm. Microbial cell observation was repeated three times and performed under 40X magnification using fluorescence microscopy.To assess the viability of microbial cells, the fluorescent probes SYTO 9 and propidium iodide were used to selectively stain live and dead cells, respectively. CLSM images (Fig. 7d) show the populations of live and dead cells for each treatment. Figure 7b shows that when plasma-treated P. putida exhibited cell damage, the number of viable cells was approximately two-fold higher than that in the untreated group. Comparing the ratio of dead cells to total living cells confirmed that the difference was more than eight times greater in plasma-treated P. putida (Fig. 7c). The survival rate of cells treated with plasma for 3 min was significantly higher than that of the untreated group, confirming that plasma treatment enhanced microbial proliferation and viability.When microorganisms are directly exposed to plasma, they undergo both physical and chemical effects. Physical effects include exposure to ultraviolet rays and electromagnetic fields, while chemical effects involve reactive oxygen species (ROS) and reactive nitrogen species (RNS). Building upon these findings, our plan is to identify the key parameters of plasma that activate plastic-degrading microorganisms by investigating the interaction between plasma and microorganisms.
