Large-Scale Plasma-Activated Water Reactor: The Differential Impact on the Growth of Tomato and Bell Pepper Plants in Nutrient-Rich and Nitrogen-Free Substrates

Abstract

In this study, plasma-activated water (PAW) was generated using a large-volume (5 L) plasma reactor with a quasi-stationary, water-cathode glow-type discharge in atmospheric pressure air. Tap water was activated up to 75 min. PAW exhibited high concentrations of long-lived reactive nitrogen species (RNSs), reaching 8 mM, which is between 4 and 26 times higher than those reported in previous studies. The reactor reached an RNS synthesis efficiency of 61 nmol/J and an RNS production rate of 526 μmol/min, both among the highest reported. PAW was evaluated on tomato and bell pepper. Seedling emergence was determined in a nutrient-free substrate. To assess plant growth, seedlings were transplanted into pots filled with either nitrogen-free or nutrient-rich substrate. PAW-irrigation significantly promoted seedling emergence and leaf expansion, especially in tomato plants. The plant growth-stimulating effects of PAW were more pronounced in nitrogen-free substrate: fresh weight of tomato and bell pepper increased up to 13.1-fold and 2.6-fold, respectively. In contrast, the effect on the nutrient-rich substrate was negligible. Tomato plants grown in the nitrogen-free substrate and irrigated with 75-min PAW reached a dry weight comparable to those grown in nutrient-rich substrate. PAW irrigation did not induce oxidative stress, as confirmed by malondialdehyde (MDA) levels and antioxidant enzyme activity.

1. Introduction

Nowadays, one of the most extensively studied areas in non-thermal electrical discharges is the field known as “Plasmas in Agriculture” [1,2,3]. In recent years, about 2000 scientific articles have been published annually in this area [4]. Plasma applications in agriculture encompass a wide range of activities, spanning from pre-harvest to post-harvest processes.

Treatments in agriculture with non-thermal plasma can be classified into two main categories: “direct” and “indirect” treatments. In direct treatments, substrates such as seeds or fruits are directly exposed to UV radiation, intense electric fields, and reactive plasma species (including electrons, ions, and excited neutral particles). In contrast, in indirect treatments, the substrates are not located in the plasma region, but this treatment is based on the effect of the long-lived species generated by the plasma. This type of treatment involves two cases. The first is the exposure of substrates to long-lived reactive species generated by the plasma in the gas phase. These species are the products of what is called afterglow, i.e., post-discharge. The treatment of seeds and plants with ozone can be included in this category. The other case of indirect treatment involves the use of liquids that have been previously exposed to plasma. This emerging technique uses plasma-activated liquids as a nutrient source to improve seed germination and growth of plants [1,2,3,5].

Water is the most commonly used medium in indirect treatments, called plasma-treated or, more commonly, plasma-activated water (PAW) [2,6,7]. PAW obtained from discharges operating in air (or mixtures of similar gases) contains reactive species of oxygen and nitrogen (RONSs). These RONSs are generated by the diffusion of species generated in the gas phase into the liquid. Key long-lived species in PAW include nitrate (NO₃⁻), nitrite (NO₂⁻), and hydrogen peroxide (H₂O₂). These RONSs play a crucial role in agriculture, influencing germination and plant growth [2,7,8]. Notably, these species are produced into water without the addition of chemical compounds. Therefore, PAW presents itself as a promising and eco-friendly alternative for agricultural applications.

Air-based plasma technology offers a low-complexity and potentially energy-efficient method for nitrogen fixation in water compared to conventional processes. The Haber–Bosch process remains the most important industrial method in terms of production capacity and energy efficiency [2,9,10]. Its current energy efficiency is approximately 0.5 MJ/mol of nitrogen, close to its theoretical limit (≈0.4 MJ/mol). From a technological perspective, this process is only viable on an industrial scale, as it requires high temperatures (≈500 K) and pressures (~100 atm). It accounts for 1–2% of the world’s total energy consumption and emits over 300 million tons of carbon dioxide annually [9]. The energy efficiency of nitrogen fixation processes mediated by non-thermal plasmas varies widely. However, it is worth noting that the theoretical limit for this process (≈0.2 MJ/mol [9]) is lower than that of the Haber–Bosch process, highlighting its potential significance.

Tomatoes and peppers are some of the main horticultural crops worldwide. Previous studies have investigated the effects of PAW on their germination and development [11,12,13,14,15,16,17]. However, the majority of these investigations have been conducted using soil-based substrates, with only a few exceptions [12,15] exploring PAW effects in low-nutrient or nutrient-free growing conditions. PAW’s stimulating effect on plant growth is influenced by multiple factors, one of the most critical being the nutrient composition of the substrate. In nutrient-rich substrates, the effects of PAW may be masked, or even negligible [18], because the substrate itself provides substantial nutrients.

A critical aspect often overlooked in previous studies on plant growth in soil-free systems (such as hydroponics or nutrient-free substrate) is the nitrogen concentration required for optimal plant development. The primary macronutrient of plants is nitrogen, and nitrogen deficiency is a growth-limiting factor. NO₃⁻ is the primary supplier of nitrogen for most plants; however, NO₂⁻ also can be a nitrogen supplier, especially under low-NO₃⁻ situations. Nitrogen can also be supplied in the form of ammonia. For tomato plants cultivation in soil-free systems, the recommended nitrogen concentration in nutrient solutions ranges from 12 to 17 mM [2]. Studies on tomato growth in low-nutrient or nutrient-free substrates have reported PAW-derived reactive nitrogen species (RNS) concentrations as low as 0.6–2.1 mM [12,15], significantly below the optimal range for soilless cultivation. It is therefore expected that the plants were under suboptimal growing conditions. Another limitation in studies evaluating PAW’s effects in nutrient-free systems is the absence of a positive control group with high-nutrient availability. Such a control is essential to establish a benchmark for optimal plant growth and to accurately determine the extent of PAW’s influence [2]. The lack of this control in previous studies [12,15] complicates the interpretation of PAW’s efficacy in enhancing plant development under nutrient-deficient conditions. For this reason, to specifically evaluate the contribution of PAW-derived RNSs to plant growth, this study uses two contrasting substrates: one nutrient-rich and one nitrogen-free substrate, ensuring that plants grown on the nitrogen-free substrate rely solely on PAW as a source of nitrogen.

Beyond its impact on plant growth, PAW production must also be evaluated in terms of energy efficiency and RNS synthesis rates, as non-thermal plasma in contact with liquids is a promising approach for nitrogen fixation [1,9,10,19]. Despite its importance, these parameters are frequently overlooked, even though they can be readily derived from plasma characteristics and RNS concentration.

This study addresses these gaps by investigating PAW production efficiency and its impact over the growth of tomato and bell pepper plants under controlled conditions. Specifically, it evaluates the energy efficiency and production rate of RNS synthesis and examines PAW’s differential effects on plants grown in nutrient-rich versus nitrogen-free substrates. Notably, this research achieved the highest RNS concentrations, with levels 4–26 times higher than those in previous studies [11,12,13,14,15,16,17]. Furthermore, the generated volume of PAW was between 2.5 and 100 times higher than that obtained in those studies, and the estimated energy efficiency (61 nmol/J) and RNS production rate (526 μmol/min) were among the highest values reported in the literature.

In this work, PAW was generated using an upscaled (5 L capacity) plasma reactor. The reactor consisted of a glow-type discharge with a water-cathode, operating in air at atmospheric pressure in a quasi-stationary regime (50 Hz). Tap water was activated up to 75 min. The energy efficiency and production rate of RNS synthesis were quantified, along with the total mass of RNS produced. Seedling emergence was determined in a nutrient-free substrate. To assess plant growth, seedlings were transplanted into pots filled with either nitrogen-free or nutrient-rich substrate. In addition, oxidative stress was assessed by lipid peroxidation and activities of antioxidant enzymes.

2. Materials and Methods

2.1. Plasma Activated Water Reactor

An air glow-type discharge at atmospheric pressure was employed to produce PAW. The cathode of the discharge was tap water. A schematic diagram of the scaled-up reactor is shown in Figure 1. The anode was a needle-shaped thoriated tungsten rod (radius ~ 200 μm), while the tap water to be activated (5 L) was the cathode. The water was contained in two interconnected reservoirs made of AISI 304 stainless steel, both in turn grounded. The discharge was established in the smaller volume reservoir (≈1.4 L), containing about 1 L of water. The distance between the electrodes (gap) was approximately 12 mm. Inside the cylindrical reservoir, a vortex (swirling flow) was created by directing water tangentially into the base and allowing it to exit through the center. This design enhanced the gas-liquid exchange surface and improved the mixing of RONSs as they transferred from the gas phase into the liquid. In addition, on top of this reservoir, a glass cap was mounted to obtain a closed gas chamber. The objective of using a closed gas chamber was to prevent the RONSs produced by the discharge from propagating into the surrounding atmosphere, thus improving RONS diffusion in the liquid. The volume of the confined gas in the chamber was about 400 cm³. The other reservoir (with a volume of about 20 L) was used to contain water for recirculation (4 L). The water flow rate was ≈1.5 L/min. No air flow was used between the two reservoirs, i.e., the fan was off. In the same vein, and given the thermally fragile nature of H₂O₂, the recirculation water tank was cooled during the activation process so that the temperature of activated water was maintained below 20 °C [20]. There were no significant variations in water volume (<5 mL) after activation.

To ignite the discharge current, a variable autotransformer was used. This device was connected to the primary circuit of a high-voltage AC transformer (~20 kV, 50 Hz) with a dispersion reactance of (95.3 ± 0.5) kΩ. The high impedance of the system naturally introduced negative feedback between the discharge current and voltage, eliminating the need for external ballast components and preventing the transition to a high-current thermal discharge. In the transformer’s secondary circuit, a full-wave semiconductor bridge rectifier was employed to define the polarity of electrodes. The discharge voltage (V) was recorded using a high-voltage probe (Tektronix P6015A, 1000×, 3 pF, 100 MΩ, Tektronix Inc., Beaverton, OR, USA) connected to channel 1 (CH1) of an oscilloscope (Tektronix TDS 2004C, 70 MHz analog bandwidth, 1 GS/s sampling rate, Tektronix Inc., Beaverton, OR, USA). Concurrently, the discharge current (I) was measured indirectly via a 100 Ω low-inductance shunt resistor linked to oscilloscope channel 2 (CH2). The average discharge power was determined as:

$$\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{p}\mathrm{o}\mathrm{w}\mathrm{e}\mathrm{r}={\displaystyle \frac{1}{\tau }} {\int }_0^{\tau }I\left(t\right)V\left(t\right)\mathrm{d}t,$$

(1)

where τ is the period of the signals.

2.2. Plasma Activated Water Treatments

The PAW treatments applied in the seedling emergence and plant growth tests were tap waters with different plasma exposure time. Tap water was obtained from well water of Venado Tuerto city (Santa Fe province, Argentina). Tap water was activated for 30, 60, or 75 min. Treatments were labeled PAW30, PAW60, and PAW75. Non-activated tap water (C) was the control treatment.

2.3. Plasma-Activated Water Physicochemical Properties

A conductivity meter (Oakton Cyberscan Cond 610, accuracy of 1%, range of 0–500 mS/cm, Oakton Instruments, Cole-Parmer, Vernon Hills, IL, USA) and a pH meter (Ohaus ST2200-F, resolution of 0.01, range of 0–14, Ohaus Corporation, Parsippany, NJ, USA) were used to determine the electrical conductivity and pH of PAW. The conductivity meter was calibrated with a 0.01 M KI solution (electrical conductivity = 1413 μS/cm at 25 °C), while the pH meter was calibrated employing standard buffer solutions at pH 7 and pH 10. Concentrations of NO₂⁻, NO₃⁻, and H₂O₂ in PAW were determined using colorimetric methods previously described in [21], with a UV–VIS spectrophotometer (Spectrum SP-2100, Shanghai Spectrum Instruments Co., Ltd., Shanghai, China). The NO₂⁻ was measured using the Griess reaction, which involves the formation of a colored complex with Griess reagents I and II (sulfanilic acid and α-naphthylamine), followed by spectrophotometric analysis at 520 nm after a 20 min reaction period. The concentration of NO₃⁻ was determined indirectly by reducing nitrate to nitrite using hydrazine, then calculating the concentration difference before and after reduction. The H₂O₂ concentration was quantified using the peroxidase method, in which hydrogen peroxide reacts with phenol in the presence of peroxidase, producing a colored compound that absorbs at 505 nm. Calibration curves and quality controls were processed in parallel for accurate quantification of reactive species. To assess stability over time, PAW and control treatments were stored at 4 °C, and their physicochemical properties were analyzed on days 1, 7, and 14 post-activation.

2.4. Seedling Emergence

Seeds of the tomato (Solanum lycopersicum) variety VC019 (INTA, Buenos Aires, Argentina) and bell peppers (Capsicum annuum) from Ecoproductos (Buenos Aires, Argentina) were used. The experiment was conducted using plug trays, each containing 25 cells. Two trays were used per treatment, totaling 50 experimental units per treatment. One seed was sown per cell, and each cell was filled with 10 g of vermiculite. The experiment included four treatments: PAW30, PAW60, PAW75, and a control (non-activated tap water, C). Irrigation was performed every three days, applying 10 mL of the corresponding treatment directly to the substrate in each cell. The trays were placed in a germination chamber with photoperiod of 16 light/8 h dark, temperatures of (25 ± 2) °C, and a photon density for photosynthesis of 350 µE m⁻² s⁻¹. The measured parameters included seedling emergence, expanded cotyledonous leaves, and expanded true leaves. Tomato seedlings were evaluated for 14 days and bell pepper seedlings for 18 days.

2.5. Plant Growth

To assess the effect of nitrogen compounds in PAWs on plant growth, tomato and bell pepper seedlings from the seedling emergence test were transplanted into 1-L pots at the end of the experiment. One seedling was sown per pot. For each crop (tomato and bell pepper), five seedlings per treatment were transplanted into five pots filled with 120 g of the nitrogen-free substrate (vermiculite), while another five seedlings of the same treatment were transplanted into five pots filled with 120 g of the nutrient-rich substrate (GROWMIX^® MULTIPRO, Terrafertil S.A., Moreno, Buenos Aires, Argentina). Each pot represented an independent biological replicate, totaling five replicates per treatment for each crop in each substrate condition. The nutrient-rich substrate (GROWMIX^® MULTIPRO) was composed of fine fiber Sphagnum moss peat, fine bark compost, perlite, and wetting agents (250–450 ppm nitrate, 200–300 ppm potassium, 30–100 ppm phosphate, pH: 5–5.8).

For plants grown in the nitrogen-free substrate, treatments consisted of nutrient solutions generated with PAW and other compounds, without additional nitrogen beyond what was present in the PAW.

Table 1. Macronutrient and micronutrient concentrations of nitrogen-free nutrient solutions.

Macronutrients		Micronutrients
Compound	cc [mM]	Compound	cc [µM]
KH2PO4	1.5	MnSO4·H2O	1
MgSO4·7H2O	0.8	ZnSO4·7H2O	1
CaCl2·2H2O	0.8	H3BO3	12.5
KCl	2.5	H2MoO4	0.25
Fe-EDTA	5	CuSO4·2H2O	0.25

The composition of the nitrogen-free nutrient solution was the same as [22].

presents the composition of the nitrogen-free nutrient solution. The nutrient solutions derived from PAW30, PAW60, and PAW75 were designated as NSP30, NSP60, and NSP75, respectively, while the control treatment (NSC) was a nutrient solution prepared with non-activated tap water. Phosphate and potassium levels in the nitrogen-free nutrient solution (142 ppm and 156 ppm, respectively, Table 1) are similar to the corresponding levels for the nutrient-rich substrate, ensuring a comparable supply of these macronutrients. For plants grown in the nutrient-rich substrate, treatments included PAW30, PAW60, and PAW75, with non-activated tap water as the control (C). All pots (either filled with the nutrient-rich substrate or with the nitrogen-free substrate) were kept in a growth chamber (photoperiod of 16 h light/8 h dark, temperatures of (25 ± 2) °C, and a photon density for photosynthesis of 350 µE m⁻² s⁻¹). The tomato growth test was conducted in May–June 2024, while the bell pepper growth test was conducted in July–August 2024.

Irrigation was applied as needed, with about 0.1 L of solution per event. The solutions were applied directly to the substrate, and the water of irrigation (control and treatments) was refreshed every 7–14 days, according to plant requirement and stage of growth. To ensure the consistent quality of the treatments, PAW-treatments and control water were stored in a refrigerator at 4 °C and were not used more than 14 days after activation.

Tomato plants grown in pots with the nutrient-rich substrate were harvested at 32 days (14 days in the nutrient-free substrate during the seedling emergence test + 18 days in the nutrient-rich substrate). Since plant growth was slower in the nitrogen-free substrate, these plants were allowed to grow to 39 days to ensure they reached the same phenological stage (early vegetative growth, sixth true leaves). A similar approach was used for bell pepper plants: those grown in the nutrient-rich substrate were harvested at 36 days (18 days in the nutrient-free substrate + 18 days in the nutrient-rich substrate), while those in the nitrogen-free substrate were evaluated at 51 days to compensate for their slower growth rate. This method to compare plants growth, which prioritizes the phenological stage over chronological age, has been applied in other agronomic studies [23,24,25].

The fresh weight of plants was recorded using an analytical balance (Ohaus Pioneer PX, appreciation 0.1 mg, Ohaus Corporation, Parsippany, NJ, USA). Then, the plant material was dried at 80 °C for 120 h to obtain dry weight. The total chlorophyll content of the leaves was determined with a chlorophyll meter atLEAF CHL BLUE (FT Green LLC, Wilmington, DE, USA) and expressed in arbitrary units (a. u.).

2.6. Oxidative Stress of the Plants

2.6.1. Lipid Peroxidation

Oxidative stress was evaluated by quantifying lipid peroxidation through the determination of the malondialdehyde (MDA) content. For this purpose, the method described by [26] was followed with slight modifications, which were the addition of butylated hydroxytoluene and the measurement of the non-specific absorbance at 600 nm to minimize interferences. Fresh samples were homogenized in a 20% (w/v) trichloroacetic acid (TCA) solution and subsequently centrifuged at 3500× g for 20 min. We combined 1 mL of the obtained supernatant with 1 mL of 0.5% (w/v) thiobarbituric acid and 100 µL of a 4% butylated hydroxytoluene solution. This mixture was then incubated at 95 °C for 30 min and subsequently cooled on ice to halt the reaction. Following this, the sample was centrifuged (3000× g, 15 min), and the absorbance of the supernatant was measured at 532 nm (A1) to determine the levels of thiobarbituric acid-reactive substances (TBARSs), with the non-specific absorbance recorded at 600 nm (A2). The concentration of TBARS was determined by calculating the difference between the absorbance values (A1−A2) and applying the molar extinction coefficient of MDA (155 mM⁻¹ cm⁻¹). The final results were expressed as nmol MDA per gram of tissue.

2.6.2. Antioxidant Enzymes

Extracts were prepared with a portion of plant tissue was homogenized in ten volumes of a 50 mM phosphate extraction buffer (pH 7.7) containing 0.5 mM EDTA, 0.5% (v/v) Triton X-100, and 1 mg of polyvinylpyrrolidone (PVP). The homogenates were then centrifuged at 13,000× g for 30 min at 4 °C, and the resulting supernatant was collected for further assays. The protein content in the extracts was measured following the method described by [27].

Catalase (CAT) activity was determined in a reaction mixture consisting of 0.1 mL of the homogenate, buffer of potassium phosphate (50 mM, pH 7.2), and H₂O₂ (2 mM). The activity was measured at 30 °C by recording the decrease in absorbance at 240 nm, which corresponds to the consumption of H₂O₂. The content of CAT was noted in pmol/(mg protein) employing a constant k rate of 4.7 × 10⁷ M⁻¹ s⁻¹ [28].

Activity of superoxide dismutase (SOD) was measured by observing the inhibition of nitroblue-tetrazolium (NBT) photoreduction, based on the method outlined by [29]. The assay mixture contained 200 μL of the supernatant and 3.5 mL of an O₂⁻ generating solution, which included 82.5 μM NBT, 14.3 mM methionine, and 2.2 μM riboflavin. The extract was diluted for a final volume of 0.3 mL using a 50 mM potassium phosphate buffer and 0.1 mM EDTA. Initial absorbance at 560 nm was measured, and then the tubes were incubated at 25 °C while being exposed to fluorescent light. The reaction started and stopped by alternating the light, ensuring that ambient light conditions were consistent during reagent preparation and absorbance measurements. NBT reduction was tracked by recording absorbance at 560 nm every two minutes for six minutes. Control samples without enzyme extracts served as blanks. SOD activity was defined as the enzyme amount needed to inhibit 50% of reduction of NBT under the test conditions, and the results were expressed as U/(mg protein) [30].

Guaiacol peroxidase (GPOX) activity was measured following the method described by [31], which involves monitoring the oxidation of guaiacol at 470 nm (with an extinction coefficient of 26.6 mM⁻¹ cm⁻¹). The enzyme activity was noted as μmol/min/(mg protein). The reaction medium consisted of a potassium phosphate buffer (50 mM, pH 7.2), 10 mM guaiacol, 2 mM H₂O₂, and 0.15 mL of vegetable extract.

2.7. Statistical Analysis

The statistics analyses were conducted with the R software version 4.3.1 [32]. The data variance obtained was analyzed using an analysis of variance (ANOVA). Having checked the assumptions of homogeneity of variances, normality of the residuals, and independence of observations, a Tukey test was performed. Differences were considered significant if p < 0.05. All data are shown as mean ± standard error of the mean.

3. Results

3.1. Electrical Characteristics of the Plasma Reactor and Physicochemical Properties of the PAW

Figure 2 shows voltage (V), current (I), and power discharge (P) waveforms. The measured voltage V includes not only the voltage drop in the gas gap, but the voltage drops in the electrodes non-neutral sheaths, as well as the voltage drop in the tap water acting as resistive cathode. However, given that the electrical conductivity of tap water is high (≈1600 μS/cm), the drop in voltage in liquid cathode is lower compared to the gas-gap voltage drop; therefore, the observed voltage is roughly the same as the discharge voltage drop. Voltage peaks of about 5 kV are found at the beginning of each pulse of the voltage signal. These peaks correspond to the ignition of the discharge through a high-voltage transition (streamer-to-spark) [33]. After the breakdown, the voltage drops to about 2 kV due to the high-impedance of the transformer, and the discharge stabilizes to a glow-type discharge. The current and power discharge (I and P, respectively) waveforms show an almost sinusoidal shape, controlled by the high stray impedance of the HV transformer. The maximum current pulse value is about 150 mA with an RMS value of 100 mA. For the mentioned experimental conditions, the average power was 145 W.

Figure 3 shows the physicochemical properties of PAW. As the exposure time of the water to the plasma increases, the electrical conductivity increases and the pH decreases. This is related to the formation of acids (HNO₃ and HNO₂) and ions (mainly, H⁺, NO₂⁻, and NO₃⁻) in the solution [34]. These phenomena are characteristic of water in contact with non-thermal discharges in air or similar mixtures. The decrease in pH was slight, and the liquid remained around neutral pH because of the tap water’s buffer capacity caused by the hydrocarbon present in the water solution [35]. Tap water (C) contains only NO₃⁻ (concentrations of H₂O₂ and NO₂⁻ are below the detection limit of the techniques); while the PAW contains H₂O₂, NO₂⁻, and NO₃⁻. The concentrations of these three species increase as the activation time increases, reaching the maximum values for 75 min of activation. The maximum synthesized values were: 20 mg/L for H₂O₂, 211 for mg/L NO₂⁻, and 212 mg/L for NO₃⁻. Note that this NO₃⁻ concentration synthesized at 75 min is calculated by subtracting the initial nitrate concentration in the control (tap water). It is also notable that the physicochemical properties of the PAW remained approximately constant for at least 2 weeks due to the buffer action of the tap water. It is important to mention that the nutrient solutions generated from the PAWs (NSP30, NSP60, and NSP75) for irrigation of the plants grown in the pots filled with vermiculite, also maintained their physicochemical properties over two weeks.

As non-thermal plasmas are considered a nitrogen fixation method [9,10], two key parameters for evaluating the RNS synthesis process are the average energy efficiency and the average production rate. Energy efficiency η can be determined by the slope of the plot showing RNS concentration as a function of the energy density supplied by the discharge [20]. Similarly, the average production rate γ is estimated from the slope of the graph depicting the mass of RNSs over activation time. Figure 4 shows the values of η and γ obtained in this work.

Table 2. In-water average energy efficiency η of RNS synthesis, average RNS production rate γ, and RNS mass for various discharges in contact with water.

Reference	Discharge Type	Power [W]	Activation Time [min]	Activation Volume [L]	Maximum [RNS] [mM]	η [nmol/J]	γ [μmol/min]	RNS Mass [mmol]
[11]	DBD	13	60	0.5	1.2	12	9.6	0.57
[12]	Jet	3.6	60	0.05	0.6	2.2	0.5	0.03
[14]	Jet	15	40	0.215	0.3	1.5	1.4	0.05
[15]	Glow	420	80	1.9	2.1	1.9	49.1	3.93
[16]	Arc	37	30	2	1.4	43	96.1	2.88
[17]	DBD	3	30	0.25	0.3	12	2.2	0.06
This work	Glow	145	75	5	8	61	526.0	40.00

shows values in water of maximum concentrations of RNSs, the average energy efficiency of RNS synthesis, the average generation rate of RNSs, and the generated RNS mass for some types of discharges in contact with water used in the irrigation of tomato and bell pepper plants. The average energy efficiency can be estimated following the procedure in [20] as [RNS]/ε, where [RNS] is the aqueous phase concentration of RNSs (=[NO₂⁻] + [NO₃⁻]) and ε is the energy density delivered to the discharge per liter of water. On the other hand, the average generation rate of RNS synthesis in water can be estimated as [RNS] × volume/(activation time)⁻¹.

The highest RNS concentration obtained in this study (8 mM) was between 4 and 26 times greater than those reported in other studies that irrigated tomatoes or bell peppers with PAW. Notably, this high RNS concentration (8 mM) was achieved in a large water volume (5 L). Similarly, the average energy efficiency of RNS synthesis in this study (61 nmol/J) was also the highest reported. The highest mean energy efficiencies are typically associated with glow (or arc) discharges operating at low power (≤100 W), rather than with DBD or jet discharges. Regarding the average RNS production rate, the value obtained in this study (526 μmol/min) was significantly higher—between 5 and 1000 times—than the rates reported in previous studies (Table 2). Likewise, the total mass of RNSs synthesized in water in this study (40 mmol) was the highest reported, exceeding previously published values by factors ranging from 10 to 1300.

3.2. Seedling Emergence

Figure 5 presents the seedling emergence parameters for tomatoes and bell peppers: percentage of emerged seedlings, percentage of seedlings with expanded cotyledon leaves, and percentage of seedlings with expanded true leaves. Overall, no negative effects on the evaluated emergence parameters were observed in any of the crops irrigated with PAW compared to the control. In tomato seedlings, the emergence percentage of those irrigated with PAW was generally significantly higher than that of the control plants throughout the experiment (Figure 5A). On days five and six, the percentage of emerged seedlings in the control treatment ranged between 50–60%, while in PAW-treated seedlings, it was between 75–80%. Notably, between days eight and fourteen, 100% of the seedlings irrigated with PAW60 and PAW75 had emerged, whereas emergence in PAW30- and control-irrigated seedlings was lower (around 85–90%). The percentage of tomato seedlings with expanded cotyledon leaves was also higher in PAW-treated plants compared to the control (Figure 5B). By day eight, this percentage ranged between 70–90% for PAW60- and PAW75-irrigated seedlings, whereas it was only 25–40% for those irrigated with PAW30 or the control. Regarding true leaf expansion (Figure 5C), tomato seedlings irrigated with PAW exhibited a higher percentage of seedlings with expanded true leaves. By day eleven, none of the control-irrigated seedlings had expanded their true leaves, while those irrigated with PAW30, PAW60, and PAW75 reached percentages of 10%, 50%, and 60%, respectively. By day fourteen, the percentage of seedlings with expanded true leaves was 25% in the control group and 50%, 75%, and 90% for PAW30, PAW60, and PAW75, respectively.

For bell pepper seedlings, no significant differences were observed in the percentage of emerged seedlings (Figure 5D). By days sixteen and eighteen, the percentage of emerged seedlings was similar across all treatments and the control (approximately 80–90%). Regarding the percentage of seedlings with expanded cotyledon leaves (Figure 5E), no significant differences were found between treatments and control. In terms of true leaf expansion (Figure 5F), PAW-irrigated bell pepper seedlings exhibited a higher percentage of seedlings with expanded true leaves. By the end of the emergence experiment, 25% of the control-irrigated seedlings had expanded their true leaves, while this percentage ranged between 40% and 75% in PAW-treated seedlings, increasing with PAW activation time.

3.3. Plant Growth

Figure 6 illustrates the effect of PAW on biometric parameters (fresh and dry weight, and the chlorophyll content) in tomato and bell pepper plants grown in both nutrient-rich and nitrogen-free substrates. In plants cultivated in the nutrient-rich substrate, no significant differences were observed in the biometric parameters between PAW treatments and the control in either crop, except the fresh weight of bell pepper plants, which showed a reduction in those irrigated with PAW60 compared to the control (Figure 6D). However, when considering dry weight—recognized as a key indicator of plant growth [2,35,36]—no differences were detected between treatments and control plants (Figure 6E). It is important to highlight that the plants grown in the nutrient-rich substrate, including those irrigated with the control, did not experience nutrient deficiency.

For tomato and bell pepper plants grown in a nitrogen-free substrate, a significant effect of PAW-derived nutrient solutions (NSP30, NSP60, and NSP75) was observed on plant development compared to control plants irrigated with a non-activated solution (NSC) (Figure 6). The most pronounced effects were observed in tomato plants. The fresh weight of both tomato and bell pepper plants increased significantly relative to control plants, with greater increases observed with longer PAW activation times: 2.6–13.1 times in tomatoes (a 3.7-fold increase between NSP30 and NSP75) and 2–2.6 times in bell peppers. Regarding dry weight, control tomato plants were too small for this parameter to be determined (Figure 6B), as all available fresh tissue was required for biochemical analysis. However, based on fresh weight measurements, it can be inferred that the dry biomass of control plants was substantially lower than that of PAW-treated plants. As a reference, the fresh weight of control plants (0.349 g) was lower than the dry weight of NSP75-treated plants (0.352 g), providing further evidence of PAW’s growth-promoting effect. The data also indicate that tomato biomass increases with PAW activation time as the dry weight of NSP75-treated plants was 2.7 times higher than that of NSP30- or NSP60-treated plants. In bell pepper plants, a significant 2.3-fold increase in dry biomass was observed in PAW-treated plants compared to control plants; however, no significant differences were found among the PAW treatments. The chlorophyll content in tomato plants (Figure 6C) treated PAW-derived nutrient solutions showed similar levels to control plants (NSC). As for bell pepper plants, the chlorophyll content was higher in plants treated PAW-derived nutrient solutions than in control plants (NSC).

3.4. Oxidative Stress of the Plants

Figure 7 shows the MDA content in tomato (above) and bell pepper (bottom) plants grown in both the nutrient-rich substrate (black bars) and the nitrogen-free substrate (gray bars). In plants, MDA is a subproduct of lipid peroxidation and, consequently, is usually employed as a marker of RONS-induced cellular damage. In both crops grown in the nutrient-rich substrate, the MDA content in plants irrigated with PAW is comparable to that of plants irrigated with the control treatment. In bell pepper plants, a slight increase in MDA content is observed as PAW activation time increases, although this increase is not statistically significant.

The MDA content in tomato and bell pepper plants grown in the nitrogen-free substrate decreases as the RNS content (and activation time) of PAW increases. In tomato plants, MDA levels in those irrigated with nutrient solutions derived from PAW (NSP30, NSP60, and NSP75) decrease significantly compared to the control solution (NSC) as PAW activation time increases. In bell pepper plants, the MDA content in those irrigated with NSP30 is similar to that of plants irrigated with NSC. However, MDA levels decrease significantly in plants irrigated with NSP60 and NSP75.

Figure 8 shows the activities of antioxidant enzymes CAT, SOD, and GPOX in leaves of tomato and bell pepper plants grown in either a nutrient-rich substrate (black bars) or a nitrogen-free substrate (gray bars). In both crops, plants grown in the nutrient-rich substrate exhibited no statistically significant differences in enzyme activity between the control and PAW-treated plants. Although some variability in CAT activity was observed (Figure 8A,D), these differences were not statistically significant, and no clear trend was evident. Regarding SOD and GPOX activities, all treatments showed levels similar to those of the control plants. These results align with the biometric parameters (Figure 6) and lipid peroxidation levels (Figure 7), indicating that PAW irrigation did not induce stress in tomato and bell pepper plants when grown in a nutrient-rich substrate.

In tomato plants grown on the nitrogen-free substrate (Figure 8A–C), no differences in CAT activity were observed between the treatments and the control. SOD activity, although not statistically significant, tended to decrease with increasing PAW activation time, a trend also observed for GPOX, where the decrease was significant. In contrast, in bell pepper plants grown in the nitrogen-free substrate, CAT activity significantly increased with PAW activation time (Figure 8D). SOD activity was lower in PAW-treated plants compared to control plants (Figure 8E), while GPOX activity in PAW-treated plants was similar to control plants (Figure 8F).

4. Discussion

4.1. Plasma-Activated Water and Nitrogen Fixation

The highest RNS concentrations and energy efficiencies in RNS synthesis are expected in discharges with high gas temperatures, in particular glow-type, rather than jet or DBD-type discharges (Table 2). Glow-type discharges operating in atmospheric pressure air exhibit a fairly high gas temperature (~1000 K) at currents around 100 mA. In this study, the discharge gas temperature reached approximately 3500 K, which is sufficiently high to generate substantial amounts of NO (~10²²–10²³ m⁻³) via Zeldovich reactions [37]. NO molecules are primary precursors for RNS formation, they undergo oxidation in humid air, predominantly forming NO₂, NO₃, HNO₂, and HNO₃. The presence of NO₃⁻ and NO₂⁻ in the aqueous phase is primarily due to the transfer of these species from the gas phase to the liquid [38], rather than NO diffusion, as NO is significantly less soluble in water compared to NO₂, NO₃, HNO₂, and HNO₃. Diffusion between gas and liquid at the recombination area is especially relevant in the present study, as the enclosed gas chamber impedes the RONS from leaking to the environment [39]. In addition, the effect of the vortex improves this transference to the water by breaking the boundary shell at the liquid interface, that would otherwise form under static conditions.

Nitrogen fixation using non-thermal plasma technology has been studied in recent years [10,19,40,41]. Two recent reviews report reference values for the production rate and energy efficiency (or energy consumption, defined as its inverse) in the RNS synthesis in water using a variety of devices based on non-thermal plasmas. For instance, Ref. [10] report maximum values of 12 mg/min for the production rate and 2890 mg/MJ for energy efficiency. In comparison, the results obtained in our study (28.2 mg/min and 3240 mg/MJ) significantly exceed these values.

In the recent review by [19], the reported RNS production rates range from 0.2 to 2000 μmol/min, while energy consumption varies between 8 and 2000 MJ/mol of nitrogen. Within this context, the results of our work (526 μmol/min for the production rate and 16.4 MJ/mol for energy consumption) fall within the reported ranges but outperform most of the studies reviewed, with the exception of [40].

A distinguishing feature of [40] is the use of a hybrid system combining an arc discharge and a DBD. This design aims to oxidize the RNS produced in the arc discharge through ozone generated in the DBD. A similar approach has been explored by other authors, such as [41], who implemented a cascade system consisting of one DBD and three spark discharges. In their case, the reported nitrogen production rate (15.2 mmol/h) and energy consumption (25.7 MJ/mol) are lower than those achieved in our study, which reached a production rate of 32 mmol/h and energy consumption of 16.4 MJ/mol.

At this point it is interesting to recall that the plasma reactor used in this work operates with a quasi-stationary 50 Hz power supply, unlike the devices mentioned above that use power supplies with high frequency (in the range of kHz or MHz). Our results show that a simple technological configuration, like the one used in our work, can efficiently process large water volumes (5 L) while achieving an elevated RNS production rate with high synthesis energy efficiency.

Although the efficiency values of the nitrogen synthesis process in PAW are high—in particular, energy consumption (16.4 MJ/mol)—compared to other studies, the values are far from those corresponding to the Haber–Bosch process (0.5 MJ/mol). It should be emphasized that nitrogen fixation by non-thermal plasma technology—and in particular, by PAW—is not intended to replace the Haber–Bosch process. Rather, nitrogen fixation by non-thermal plasma represents a complementary and even small-scale approach [9]. The main objective is to develop an alternative method that supplements nitrogen demand in a more flexible and eco-friendly manner, especially in regions where large-scale production is inaccessible.

4.2. Seedling Emergence

The results show that, in general, the evaluated parameters related to seedling emergence improve as PAW activation time increases (Figure 5). The most pronounced increases were observed in tomatoes, although similar trends were noted for bell peppers. By exposing seeds to PAW through discharges burning in air or similar gas mixtures, the seeds come into contact with an aqueous solution containing appreciable amounts of RONSs (mainly nitrate, nitrite, and hydrogen peroxide). The increase in seed germination upon PAW exposure is attributed to the presence of RONSs in the solution, as these reactive species may cause physiological and physical alterations in the seeds [2,7]. It has been reported that PAW can modify the seed surface structure by removing the hydrophobic wax, which changes the seed surface from hydrophobic to hydrophilic [2,8,42].

In recent years, the effect of PAW on the germination of various seed types has been extensively studied [13,17,43,44,45]. The study by [13] used a DBD to generate PAW, which was then used to irrigate aged bell pepper seeds. After eight days, the germination percentage of seeds irrigated with PAW activated for 15 min increased by 28.3% compared to the control. In our study, at nine days, increases in the emergence of bell pepper seedlings irrigated with PAW of 136–154% were obtained. Similarly, the work of [45] evaluated the effect of plasma-activated tap water on lentil seed germination, observing an increase of ~40% in germination after fourteen days. The authors suggested that H₂O₂ and NO₃⁻ were responsible for this improvement, particularly due to its synergic effect in rupturing seed latency through the production of endogenous NO.

The number of tomato seedlings with expanded cotyledon leaves (Figure 5B) was significantly increased by PAW, as well as those tomato and pepper seedlings with expanded true leaves (Figure 5C,F). Given its high content of RNSs in solution, PAW is likely to promote seedling development through these species [42]. In [18], the content of RONSs in PAW in contact with wheat seeds was evaluated for seven days. Their results showed that H₂O₂ drops rapidly (in less than one day), while NO₂⁻ and NO₃⁻ drop sharply after the first day and disappear from the second day. The authors postulate that RNSs are probably metabolized by the seeds after germination.

4.3. Growth and Oxidative Stress of the Plants

At the same phenological stage, plants in the nutrient-rich substrate exhibited greater biomass accumulation than those in the nitrogen-free substrate (Figure 6), as reflected in their higher fresh and dry weight values. Regarding nitrogen availability, the nutrient-rich substrate contained 250–450 ppm of nitrate, while the nitrogen supplied by PAW-derived nutrient solutions (in the form of NO₂⁻ + NO₃⁻) ranged from 191–465 ppm for the treatments and 42 ppm for the control (in the form of NO₃⁻). However, if only NO₃⁻ is considered, the concentrations were 42 ppm for NSC, 91 ppm for NSP30, 182 ppm for NSP60, and 254 ppm for NSP75. Except for NSP75, which had nitrate levels comparable to the nutrient-rich substrate, the NO₃⁻ values of the other PAW-derived nutrient solutions were lower than those found in the nutrient-rich substrate. Since NO₃⁻ is the primary nitrogen source for most plants, this difference in the available NO₃⁻ content could account for the reduced growth as well as the slower growth rate observed in plants cultivated in the nitrogen-free substrate.

Most of the studies evaluating PAW’s effect on tomato and bell pepper plant growth have been conducted using a soil-based substrate [11,13,16,17], with the exception of [15], where plants were grown in a low-nutrient substrate, and [12], where a nutrient-free substrate was used. While the studies by [11,17] report significant increases in the growth of PAW-treated plants in a nutrient-rich substrate, both studies measured plant height but not biomass. At this point, it is worth mentioning that, although plant length is commonly reported as an indicator of development, some authors recommend avoiding this parameter, as it can be affected by factors like temperature differential, quality of light, etc. Rather, researchers suggest evaluating plant growth on the basis of biomass (especially dry weight), which represents the most reliable parameter for evaluating plant growth [2,35,36].

In studies using a soil-based substrate where biomass is reported as a growth indicator [13,16], the authors found substantial increases in plant biomass: up to 61% in fresh weight for tomato plants [16] and up to 194.4% in bell pepper plants [13]. These results contrast with those obtained in our work, where no significant differences were observed between PAW-treated and control plants. In these studies [13,16], the plant growth period ranged from 46 to 60 days. A major difference in our experimental design is that plants grew in a nutrient-rich substrate for only 18 days. This is a considerably shorter period, representing only 30–40% of the growth duration in the other studies [13,16]. At the time of harvest, the plants in our experiment were still in the early vegetative growth stage. In a study evaluating the effect of PAW on wheat plants, no stimulating effect on plant growth was observed for plants grown in a nutrient-rich substrate. Although the reported RONS concentration was not high (≤1 mM), the authors hypothesized that the stimulatory effect of PAW on plant development would be more pronounced in the later stages of vegetative growth, where nitrogen demand is higher and PAW can supply nitrogen through its RNS content [18]. This hypothesis aligns with the results of our study and a previous study conducted by our research group [46]. In that previous study, the effect of PAW with similar RNS concentrations was evaluated on the growth of 40-day-old tomato and bell pepper plants cultivated in a nutrient-rich substrate (GROWMIX^® MULTIPRO) under controlled conditions. While the present study has not shown a PAW stimulating effect in early vegetative stage plant growth (fresh biomass of 6–8 g per plant, Figure 6), the previous study demonstrated a significant growth-promoting effect of PAW at an advanced vegetative stage (fresh biomass of 20–40 g per plant), with dry weight increases of 40–60% in PAW-treated plants. These findings suggest that the impact of PAW on plant development in nutrient-rich substrates depends on the plant’s growth stage, with the effect becoming more pronounced at later vegetative stages when nitrogen demand is higher.

Other studies have also evaluated the effect of PAW on plant growth in nutrient-poor or nutrient-free substrates. The work of [12] examined the effect of irrigation with plasma-activated deionized water on tomato seedlings grown in vermiculite for 35 days. Their experimental design did not include a group of plants growing under optimal conditions (with high-nutrient availability). Their results showed an increase in shoot and root length in plants treated with PAW activated for 15 or 30 min, compared to control plants irrigated with deionized water, while PAW activated for 60 min had no significant effect. However, the authors did not report plant biomass measurements. In [15], plants were grown for 28 days, and results showed increases of 1.7–2 times in the dry aerial biomass of tomato plants. However, their experimental design also lacked a group of plants growing with high-nutrient availability.

When assessing the effect of PAW on plant development in soil-free systems (either hydroponic or nutrient-free substrates), the recommended nitrogen concentration (as nitrate or ammonium) for tomato plants ranges from 12 to 17 mM [2]. Although these optimal nitrogen concentrations are high, the value obtained in this study for PAW75 (8 mM) is close to the recommended range. Moreover, this concentration is considerably higher (4–10 times) than those reported in previous studies (0.6–2.1 mM) that evaluated the effect of PAW on tomato growth in soil-free systems [12,15].

In studies evaluating the effect of PAW in soilless systems, it is crucial to consider a plant group with high nutrient availability to establish an optimal growth reference. In our study, one objective was to assess the effect of PAW on plant growth at the same phenological stage in two different substrates: one nutrient-rich medium and the other nitrogen-free medium. Plants grown in the nutrient-rich substrate and irrigated with non-activated water (C) served as the optimal growth control, as they did not experience nutrient deficiencies and developed under conditions similar to those of conventional greenhouse cultivation. Notably, the biomass of control plants grown in the nutrient-rich substrate (C) was similar to that of tomato plants irrigated with the nutrient solution derived from PAW75 (NSP75)—the treatment with the highest RNS concentration (8 mM), both reaching approximately 0.4 g (Figure 6B). This finding suggests that tomato plants grown in a nitrogen-free substrate and irrigated with NSP75 achieved biomass accumulation comparable to that of plants cultivated under optimal nutritional conditions.

For bell pepper plants grown in the nitrogen-free substrate, Figure 6E (dry weight) shows that the biomass of plants irrigated with nutrient solutions—NSP30, NSP60, and NSP75—(0.1 g) did not reach the levels observed in plants grown in the nutrient-rich substrate and irrigated with the control treatment (C) (≈0.4 g). Moreover, no significant differences were observed among the plants irrigated with NSP30, NSP60, and NSP75. In another study on bell pepper plants irrigated with different PAWs (activated water for 5, 10, and 15 min—PAW-5, PAW-10, and PAW-15, respectively) [13], although it was on a soil-type substrate, the results showed an increase in the fresh biomass of the treated plants with respect to the control plants. However, the plant biomass did not grow as the PAW activation time increased. In fact, the fresh weight of plants irrigated with PAW-5 and PAW-15 was similar, while with PAW-10, it was significantly lower.

Our study, along with previous research, highlights that the effect of PAW on plant growth is influenced by multiple factors, including substrate type, plant growth stage, and PAW composition. While no significant differences were observed in early-stage plants grown in a nutrient-rich substrate, PAW significantly enhanced biomass accumulation in a nitrogen-free substrate, with stronger effects at higher activation times. These findings suggest that PAW’s growth-promoting impact is more pronounced in nutrient-deficient conditions and later vegetative stages, where nitrogen demand is higher.

The results on lipid peroxidation (Figure 7) indicate that neither tomato nor bell pepper plants experienced oxidative stress due to PAW application in any of the evaluated substrates. In fact, plants irrigated with PAW-based nutrient solutions (NSP30, NSP60, and NSP75) in the nitrogen-free substrate exhibited lower MDA levels than control plants, suggesting they were exposed to less stressful growing conditions. The stress observed in plants grown in the nitrogen-free substrate was likely due to nitrogen deficiency. Previous studies have shown that the MDA content increases under nitrogen deficiency in cucumber seedlings [47] and rice plants [48]. This aligns with our observation that the highest MDA levels occurred in plants irrigated with the lowest RNS concentrations. Our findings indicate that as nitrogen concentration in the solution increases, lipid peroxidation in plants grown in the nitrogen-free substrate approaches the levels observed in plants cultivated under optimal nutritional conditions.

In [12], the MDA content was quantified in tomato plants grown in a nutrient-free substrate and irrigated with PAW. The authors showed that in the aboveground plant parts, those treated with 15 min-PAW and 30 min-PAW had lower MDA levels compared to control plants. Nevertheless, 60 min-PAW irrigated plants exhibited a higher MDA content. The authors attributed this increase mainly because of the toxic effects of PAW owing to its high RONS concentration. Although tomato plants are tolerant to nitrite, this species can be toxic to plants [2]. The authors report the total concentration of RNSs (NO_X), although it is not indicated what proportion corresponds to nitrate and nitrite. The lipid peroxidation result in [12] contrasts with our findings, in which no PAW-induced toxicity was observed, despite the fact that RONS concentrations in our PAWs were substantially higher ([RNS] = 3.8–8 mM, [ROS] = 0.15–0.6 mM) than the maximum RONS concentrations reported by [12] ([RNS] = 0.6 mM, [ROS] = 0.04 mM). It is important to note that in our experimental design, PAW was applied through nutrient solutions that provided all essential nutrients for optimal plant development. In contrast, ref. [12] supplied only the nitrogen present in PAW.

The abilities to resist oxidative damage depend to a large extent on the capacity of plants to increase the activities of antioxidant enzymes [18]. SOD reduces superoxide radicals into H₂O₂, while CAT breaks down H₂O₂ into oxygen and water, and GPOX utilizes H₂O₂ to oxidize various substrates. Studies on nitrogen deficiency have shown that CAT, SOD, and GPOX activities are altered under these conditions. Given that PAW serves as a nitrogen-rich nutritional source, it is expected to influence the plant’s enzymatic response. In our study, the most pronounced enzymatic changes were observed in plants grown on the nitrogen-free substrate (Figure 8). Overall, in plants grown in the nitrogen-free substrate, enzyme activity tended to be similar levels observed in nutrient-rich plants as PAW activation time and, consequently, RNS availability increased. For instance, under nitrogen deficiency, increased SOD and CAT activities have been reported in cucumber seedlings [47], whereas in rice plants, SOD activity increased while CAT activity decreased [48].

Some studies have evaluated the effect of PAW irrigation on CAT, SOD, and GPOX activities. A similar trend to the one obtained in our work was reported in [49] on 28-day-old maize seedlings, where plants grown in a soil-like substrate were irrigated with PAW. In that study, CAT activity increased in PAW-treated plants, while SOD and GPOX levels decreased. Likewise, ref. [18] evaluated the effects of PAW irrigation on wheat plants grown in perlite and found that CAT, SOD, and GPOX activities were lower in PAW-treated plants compared to controls. The highest enzyme activities were observed in control plants, indicating that they experienced greater stress due to nutrient deficiency.

CAT is the primary H₂O₂-scavenging enzyme in leaves [18], and PAW irrigation has been shown to increase endogenous H₂O₂ and NO_X levels [12]. The observed increase in CAT activity in bell pepper plants grown in a nitrogen-free substrate with increasing PAW activation time was likely driven by elevated endogenous RONS levels. A study on barley seedlings [50] found that increased RONS levels correlated with enhanced CAT activity, suggesting an upregulation of antioxidant enzymes to mitigate ROSs and maintain redox balance. In tomatoes in the nitrogen-free substrate, however, CAT activity appeared to be inhibited.

Several studies have reported a decrease in SOD activity following PAW irrigation [18,35,49]. In [35], the reduction in SOD activity in lettuce plants was attributed to increased nitrate levels with activation time of PAW. This agrees with the results from nitrogen-deficiency studies, which show that SOD activity decreases as nitrogen availability increases [47,48].

5. Conclusions

Plasma-activated water (PAW) was generated using an upscaled (5 L capacity) plasma reactor. The discharge employed was a glow-type with water-cathode at atmospheric pressure air, operating in quasi-stationary regime (50 Hz). Tap water was activated up to 75 min. PAW was evaluated on tomatoes and bell peppers. Seedling emergence was determined in a nutrient-free substrate. To assess plant growth, seedlings were transplanted into pots filled with either nitrogen-free or nutrient-rich substrate. Additionally, plant oxidative damage and activities of antioxidant enzymes were determined. The PAW effect was more pronounced in tomatoes than in bell peppers: the greatest increases in seedling emergence parameters and plant biometric parameters were obtained for tomatoes. The results showed that:

• PAW with 75 min of activation resulted in high concentrations of long-lived RONS: 212 mg/L of NO₃⁻, 211 mg/L of NO₂⁻, and 20 mg/L of H₂O₂. Notably, the RNS (NO_X = NO₂⁻ + NO₃⁻) concentration (≈8 mM) was 4 to 26 times greater than that previously found in other studies on tomato and bell pepper. Similarly, the total RNS mass synthesized in water (40 mmol) exceeded literature values by factors ranging from 10 to 1300.
• The PAW generation process demonstrated high efficiency, with an average RNS synthesis energy efficiency of 61 nmol/J and an RNS production rate of 526 μmol/min. These values rank among the highest reported for similar plasma-based systems.
• PAW significantly promoted seedling emergence and the expansion of cotyledon leaves in tomato, as well as the expansion of true leaves in both tomato and bell pepper seedlings.
• The growth-stimulating effects of PAW-derived nitrogenous compounds were pronounced in plants grown in a nitrogen-free substrate but were not observed in plants grown in a nutrient-rich substrate. In the nitrogen-free substrate, PAW irrigation resulted in a significant increase in the biomass of tomato and bell pepper plants: tomato plant fresh weight increasing by 2.6- to 13.1-fold and bell pepper plant fresh weight by 2- to 2.6-fold. Notably, at the same phenological stage, tomato plants irrigated with PAW activated for 75 min attained a dry weight comparable to that of plants cultivated in a nutrient-rich substrate.
• PAW has not induced oxidative damage in plants, indicated by the MDA values of PAW-irrigated plants being like or lower than control plants. In fact, in nitrogen-free conditions, MDA levels in PAW-irrigated plants decreased compared to the control, approaching those observed under optimal nutrient-rich conditions. Furthermore, biochemical analyses of key enzymes (superoxide dismutase, catalase, and guaiacol peroxidase) confirmed that plant resistance mechanisms respond adequately to irrigation with PAW.

PAW emerges as an innovative and eco-friendly option for the nutrition of crops by irrigation as it requires no chemical additives while effectively promoting germination, enhancing plant growth, and strengthening plant defense mechanisms.