Using Plasma-Activated Water Generated by an Air Gliding Arc as a Nitrogen Source for Rice Seed Germination

Abstract

This research aimed to understand the use of air gliding arc (GA) plasma to generate plasma-activated water (PAW) for fixing nitrogen in water and the chemical properties of PAW on the germination of rice seeds. The N₂, NO, and OH molecules in GA plasma led to NO₃⁻, NO₂⁻, and H₂O₂ formation in the PAW. The NO₃⁻, NO₂⁻, and H₂O₂ contents in PAW rapidly decreased after 5 days of storage. The experiment was arranged in a completely randomised design using GA plasma discharged above the surface of deionised (DI) water with different airflow rates (2, 3, 4, 5, and 6 L/min) compared to the control (DI water). The NO₃⁻ and NO₂⁻ contents increased, resulting in an increase in total nitrogen (N) and gibberellic acid (GA₃) accumulation in rice seeds. The PAW at an airflow rate of 5 L/min was optimal for enhancing radicle emergence at 48 and 72 h, germination, germination index, shoot length, fresh weight, and dry weight of seedlings. Therefore, air GA plasma to generate PAW is an efficient method for producing nitrogen in a soluble form, which can support the germination processes and early growth of rice seedlings.

1. Introduction

Nitrogen (N₂) fixation is the process by which atmospheric N₂ is converted into chemically accessible forms, such as ammonia (NH₃) and nitrate (NO₃⁻). This process is essential for producing proteins, certain types of hormones, and other compounds in plants. N₂ is the most abundant element in the Earth’s atmosphere, making up 78% of its composition [1]. However, in its gaseous state, N₂ is relatively unreactive and not readily available to most organisms. N₂ fixation transforms this plentiful resource into a form that organisms can use. There are two main types of N₂ fixation: biological and industrial. Biological N₂ fixation involves bacteria converting N₂ into NH₃, a process that requires a significant amount of energy. However, industrial N₂ fixation involves converting N₂ into NH₃ using high temperature and high pressure in the Haber–Bosch (H-B) process. However, the H-B process is energy intensive and results in the release of significant amounts of carbon dioxide (CO₂) into the environment [2,3].

At present, there are modern techniques and technologies that can fix N₂ directly from N₂ gas in the ambient air that is ionised by an electric field discharge called “plasma”, often referred to as the fourth state of matter, which is an ionised gas comprising a dynamic ensemble of free radicals, electrons, ions, and excited particles [4]. Plasma is typically classified into two main types: cold plasma (low temperature or non-thermal) and hot plasma (high temperature or thermal) based on the temperature level of its components [4,5]. The main biologically active component of plasma is reactive oxygen and nitrogen species (RONS), such as an oxygen atom (O), ozone (O₃), superoxide anion (O₂^•−), hydroxyl radical (^•OH), nitrogen oxides (^•NO), or nitrogen dioxide (^•NO₂). RONS radicals produced from these plasmas induce a variety of beneficial effects [5,6].

Plasma N₂ fixation is generated from cold plasma in the air and manifests in the form of plasma-activated water (PAW). Plasma breakdown produces a variety of active species from RONS [7]. When these species contact the water surface, they dissolve and instigate various chemical reactions in the water. Ultimately, long-lived radicals, such as NO₃⁻, NH₃, nitrite (NO₂⁻), ammonium (NH₄), and hydrogen peroxide (H₂O₂), emerge in the PAW. However, the concentration of N₂ species dissolved and stabilised in PAW depends on numerous factors, such as the plasma device, the power of the plasma system, the duration and proximity of plasma to the water surface, and the water volume. Furthermore, the storage period of PAW before its use also impacts the concentration of dissolved N₂ species and the reactions that occur in PAW [7,8,9,10].

Several plasma devices discharge air to immobilise N₂ species in water, such as pinhole plasma jets [11], plasma jets [12,13], and dielectric barrier plasmas [14]. A recently advanced and efficient plasma discharge technique is gliding arc (GA) plasma, which increases the discharge volume and leads to the separation and formation of high-density N₂ ions, in which the obtained concentrations of NO₃⁻ and NO₂⁻ are higher than those of earlier techniques [15].

GA plasma is a type of hybrid plasma in which discharge occurs when a high-voltage electric field crosses a pair of electrodes in the gas. This electric field ionises molecules, turning them into plasma that conducts electricity. GA plasma is generated by heating an arc in a low-resistance gap. The convective plasma moves and forms a glowing arc that ‘glides’ between the electrodes following the path of least resistance [15]. As the convection gas flow pushes the plasma column down the length of the electrode, the plasma cools down at the column end because of the lack of the capability of the high voltage to continue further breakdown. Accordingly, the primary plasma column goes away, and the travel cycle for the new plasma column is repeated [16]. Therefore, in this experiment, the GA plasma device was selected to produce inorganic N₂ species in the form of a plasma solution, known as PAW, that plants can immediately utilise as a source of nutrients for germination and early growth.

The properties of PAW, which generate RONS, also change in both physical and chemical reactions during its formation, such as acid–base balance (pH), oxidation–reduction potential (ORP), and electrical conductivity (EC). These changes result from the effects of plasma activation [7,10,17]. The application of PAW in agriculture has arisen with various uses such as seed treatment [18,19,20,21], crop growth improvement [11,22,23,24,25], and disease management [26,27,28]. In the plant growth process, the uptake of N₂ in the form of NO₃⁻ or NH₄⁺ is facilitated by the transport of specific proteins to the cell plasma membrane. In plants, NO₃⁻ is first reduced by NO₃⁻ reductase (NR) to NO₂⁻, and then, NO₂⁻ is further reduced by NO₂⁻ reductase (NiR) to NH₄⁺. This NH₄⁺ can be combined with amino acids, serving both as nutrients and signalling molecules that stimulate germination and plant growth [29].

The cultivation of rice holds exceptional significance, providing sustenance to billions of people worldwide [30]. To ensure the prosperity and sustenance of this staple crop, a comprehensive understanding of the key macronutrients governing rice seed growth and development is fundamental [31]. Among these essential nutrients, nitrogen (N) is a primary factor that significantly influences rice quality and productivity [32,33]. N serves as a fundamental macronutrient for rice, playing a pivotal role in various critical physiological processes. It actively participates in the synthesis of amino acids, proteins, and enzymes essential for cell division, photosynthesis, and energy metabolism [33,34,35]. As a result, the availability of N governs critical aspects of rice development, such as germination, leaf expansion, panicle formation, and grain yield [32,34,35].

The concentrations of NO₃⁻ and NO₂⁻ in PAW are believed to be major factors contributing to the improvement of plant germination and growth, as they can act as substitutes for traditional N sources [11].

This study aimed to understand the characteristics of air GA plasma in water by investigating the chemical properties of PAW on rice seed germination, radicle emergence, germination index, fresh weight, dry weight, and shoot and root lengths. It also included measurements of total N and gibberellic acid (GA₃) accumulation. The storage period of PAW determines the viability of long-lived RONS as a stable and effective N source for evaluating their feasibility for future use in agriculture.

2. Materials and Methods

2.1. Gliding Arc Plasma Device and PAW Generation

The GA plasma device was created by the Plasma and Beam Physics Research Facility and Department of Chemistry, Faculty of Science, Chiang Mai, Thailand. The GA plasma array device consisted of four GA plasma units fixed at the top of an aluminium frame. Each GA plasma unit was equipped with a curved-sharp copper electrode, which served as the high-voltage electrode for a 20-watt radio frequency (RF) self-resonance power supply, and the power supply was measured using an oscilloscope. The power supply was capable of delivering a sinusoidal alternating current (AC) output at a frequency of 700–900 kHz, with a peak voltage (Vp) ranging from 6 to 10 kV (Figure 1a,b).

In these experiments, PAW was generated using a GA plasma discharge at ambient air above the surface of deionised (DI) water at a volume of 50 mL. The airflow rates were fixed at five levels (2, 3, 4, 5, and 6 L/min) for 15 min. The distance between the plasma tip and the DI water was maintained at 5 mm. A schematic diagram of PAW using air GA plasma discharge is shown in Figure 1c,d.

2.2. Optical Emission Spectra (OES) Measurement and Vibrational Temperature of N₂

OES was a useful tool for investigating the type of reactive species generated in plasma. The OES was measured using a wide-spectrum spectrometer (Exemplar-LS Spectrometer, B&W Tek, Newark, DE, USA), which covers the range between 200 and 900 nm, and a short-range spectrometer (Avaspec-ULS3648 Starline Spectrometer, Avantes, The Netherlands), which covers the range between 265 and 430 nm. The entrance and exit spectrometer slits were set to 100 μm, and the grating had a value of 1200 g/mm. The integration time and resolution of the spectrometer were 500 ms and 0.1 nm, respectively. The OES measurements were performed three times at each airflow rate and averaged to ensure stable plasma and consistent free radical emissions.

The vibrational temperature (T_vib) of N₂. It was assumed that part of the electron temperature (T_e). The calculation was based on the light emission spectrum from the intensities of the lines at 370.9 and 380.4 nm [36], as follows:

$$\frac{{\mathrm{I}}_{370.9 \mathrm{nm}}}{{\mathrm{I}}_{380.4 \mathrm{nm}}}={1.126}^{\mathrm{e} -0.465/{\mathrm{T}}_{\mathrm{vib}}}$$

(1)

where I_{370.9 nm} and I_{380.4 nm} are the measured intensities of the emission lines at 370.9 and 380.4 nm, respectively.

2.3. Analysis of PAW Chemical Properties

2.3.1. The NO₃⁻ and NO₂⁻ Concentrations

The concentrations of NO₃⁻ and NO₂⁻ were determined using the colourimetric method with a nitrate test kit (HI3874-0, weight 156 g, cadmium, potassium disulphate, and sulphanilic acid) and nitrite test kit (HI3873-0, weight 169 g, potassium disulphate) (HANNA Instruments Inc., Smithfield, RI, USA) and measured using a UV/Vis spectrophotometer (Shimadzu UV–1800, Kyoto, Japan). Absorbance peaks were detected at wavelengths of 372 and 507 nm to determine the concentration of NO₃⁻and NO₂⁻, respectively, in the solution.

To create a standard curve for NO₃⁻, DI water treated with a nitrate test kit of the solution in the concentration range of 0 to 100 mg/L was used. The relationship of the absorbance value with the NO₃⁻ concentration was as follows:

Y = 0.0082X + 0.137    (R² = 0.998)

(2)

where Y is the absorbance of NO₃⁻ at 372 nm, and X is the NO₃⁻ concentration in mg/L.

To create a standard curve for NO₂⁻, the same steps were taken as for the NO₃⁻ standard curve. However, for NO₂⁻, a solution with a concentration range of 0 to 1 mg/L was used. The relationship of the absorbance value with the NO₂⁻ concentration was as follows:

Y = 0.5165X − 0.0091    (R² = 0.997)

(3)

where Y is the absorbance of NO₂⁻ at 507 nm, and X is the NO₂⁻ concentration in mg/L.

The PAW sample was 10 mL and was combined with a nitrate or nitrite test kit. They were mixed using a vortex mixer and left at room temperature for 10 min. Subsequently, their absorbance was measured at a wavelength of NO₃⁻ at 372 nm and NO₂⁻ at 507 nm. The recorded absorbance was substituted into the Y variable of the standard in Equations (2) and (3) to determine the NO₃⁻ and NO₂⁻ concentrations (mg/L), respectively.

2.3.2. The H₂O₂ Concentration

The concentration of H₂O₂ was measured using the iodometric titration method [37] with slight modification. First, a 3 mL PAW sample was combined with 1 mL of 2% potassium iodide (KI) solution and 1 mL of 2 M hydrogen chloride (HCl) in a tube. The mixture was shaken vigorously using a vortex until a yellow colour was observed. Then, it was kept in the dark for 15 min. After 15 min, 0.1 M sodium thiosulphate (Na₂S₂O₃H₂O) solution was added until the solution turned lighter yellow, and 30 µL of a starch indicator was added to the solution, which turned blue in colour. Finally, the solution was titrated with 0.1 M of Na₂S₂O₃H₂O with a drop of 10 µL until the solution turned from blue to clear, and the volume of 0.1 M of Na₂S₂O₃H₂O used in the titration was recorded.

2.3.3. The pH Level, EC and ORP Value

The pH level and EC value were measured using a pH/conductivity metre (SevenCompact^TM Duo S213, Mettler Toledo International Inc., Greifensee, Switzerland). The ORP value was measured using an ORP metre (Mettler Toledo International Inc., Switzerland).

2.4. Storage Period of PAW

The GA plasma was discharged at an airflow rate of 5 L/min above the surface of the DI water in a volume of 50 mL for 20 min. The PAW chemical properties were then immediately analysed (Section 2.3). The samples were stored in an ambient room in clear plastic tubes, which were then capped for storage. The temperature and relative humidity (RH) were monitored using a datalogger (Testo 174H, Testo, Guangzhou, China) for a 20-day storage period. The chemical properties of PAW were analysed every 5 days on days 0, 5, 10, 15, and 20.

2.5. Rice Seed Sample

Rice seed (Oryza sativa L.) cv. San-pah-tawng 1 variety belonged to the indica type and a kind of glutinous rice. Rice seed sample was obtained from the Chiang Mai Rice Seed Center, Chiang Mai province, Thailand. The rice seed had germination of 95%, purity of 98%, and initial seed moisture content of 11%.

2.6. PAW Treatment of Rice Seed

PAW was generated using GA plasma, as detailed in Section 2.2. The PAW airflow rates were 2, 3, 4, 5, and 6 L/min, and the pH was optimised for rice seed growth using potassium hydroxide (KOH). The three replications in each treatment used 3 g of rice seed (100 seeds) soaked in 5 mL PAW for 24 h, and untreated seed was used as a control (soaked in DI water) at an ambient room (temperature of 29 ± 1 °C with an RH of 66 ± 3%). Thus, in this experiment, there were a total of 6 treatments, namely:

Treatment 1 (T1) = Control (DI water);

Treatment 2 (T2) = PAW at an airflow rate of 2 L/min;

Treatment 3 (T3) = PAW at an airflow rate of 3 L/min;

Treatment 4 (T4) = PAW at an airflow rate of 4 L/min;

Treatment 5 (T5) = PAW at an airflow rate of 5 L/min;

Treatment 6 (T6) = PAW at an airflow rate of 6 L/min.

Rice seed germination characteristics were measured using the standard between-paper germination method [38]. Each treatment used 100 rice seeds per replicate with 20 × 35 cm paper and 30 mL of each PAW treatment and DI water for the control. The papers were rolled up and placed in an incubator at 25 °C. After 5 days of planting, 10 mL of each treatment was added to the paper and returned to the incubator for 14 days. For FW and DW, as well as SL and RL, 25 rice seeds per replicate were used in each treatment. Using the standard between-paper germination method, rice seed characteristics were evaluated, as detailed in Section 2.7 and Section 2.8.

2.7. Determination of Total N and GA₃ in Rice Seed

2.7.1. Total N Accumulation

The total N accumulation was determined using the combustion method [39] with slight modifications. After 14 days of planting, the samples were dried in a hot air oven at 55 °C for 48 h. Subsequently, the rice seedlings were crushed in an electric blade mill until fine, and 0.1400 ± 0.0009 g was weighed in a 502-186 tin foil cup. These samples were analysed using a nitrogen combustion device (FP-828 model, LECO^®, St. Joseph, MI, USA). The principle behind this method is to burn the sample at high temperatures to convert the substance into a gaseous state and then measure the N signal using a thermal conductivity detector. The total N accumulation derived from the analysis was then represented as a percentage.

2.7.2. GA₃ Concentration

The GA₃ concentration was evaluated using the chromatographic analysis method [40] with slight modifications. After the rice seed was soaked in each treatment for 24 h, they were placed on paper (10 × 20 cm). Each PAW treatment and control added a volume of 7 mL to the paper. The samples were kept in an incubator at a temperature of 25 °C for 48 h (rice seed began to germinate) and ground into a fine powder, with each sample weighing 1 ± 0.004 g. The ground samples were placed in plastic tubes and extracted with 10 mL of a solvent mixture of methanol and formic acid 5.0% (80:20). These samples were sonicated using an ultrasonic processor, maintaining a temperature of 29 ± 1 °C. The sonication process consisted of 10 min of sonication, a 5 min break, followed by 10 min of sonication.

The extracted solutions were then filtered through Whatman™ No. 1 filter paper with a size of 11 µm. The filtered solution was evaporated using a rotary evaporator (Hei-Vap Precision, Heidolph, Germany). The evaporated samples were reconstituted in 2 mL of methanol and subsequently filtered through a 0.2 µm nylon membrane filter syringe into a sample tube. The samples were analysed using high-performance liquid chromatography (HPLC-SPD-10A, Shimadzu, Japan) equipped with a symmetry reverse-phase C18 column (5 μm; 3.9 × 150 mm). The column temperature was maintained at 40 °C, and the mobile phase used was a 0.01% formic acid solution prepared by mixing 1000 mL of pure water (18 MΩ) with 0.1 mL of formic acid. A flow rate of 1 mL/min was set for the analysis, and 10 µL of each sample was injected. The presence of GA₃ was detected at a wavelength of 195 nm. The GA₃ concentration results from the HPLC analysis were compared based on retention time and the standard curve of the GA₃ solution with the area under the curve. In the concentration range of 0 to 10 µL/mL, the area under the graph is replaced with the value Y. The relationship was described by the following equation:

Y = 27,785X − 1874.1    (R² = 0.997)

(4)

where Y is the area under the graph of GA₃ at 195 nm, and X is the GA₃ concentration in µg/mL.

2.8. Evaluation of Rice Seed Germination Characteristics

2.8.1. Germination (G)

G was counted on day 5 (first counted) and day 14 (final counted). The assessments for normal seedlings, abnormal seedlings, hard seeds, and dead seeds were recorded. The G percentage was calculated as follows:

$$\mathrm{G} (\%)=\frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{n}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{s}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{d}} \times 100$$

(5)

2.8.2. Germination Index (GI)

The normal seedlings were counted daily until day 14. The GI was calculated as follows:

$$\mathrm{GI}=\sum \frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{n}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{s} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{o}\mathrm{n} \mathrm{d}\mathrm{a}\mathrm{y}}{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{a}\mathrm{y}\mathrm{s}}$$

(6)

2.8.3. Radicle Emergence (RE)

After 24, 48, and 72 h of planting, the length of the RE that exceeded 2 mm was measured and recorded. The percentage of RE was calculated as follows:

$$\mathrm{RE} (\%)=\frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s} \mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h} \mathrm{a} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \mathrm{o}\mathrm{f} \mathrm{m}\mathrm{o}\mathrm{r}\mathrm{e} \mathrm{t}\mathrm{h}\mathrm{a}\mathrm{n} 2 \mathrm{m}\mathrm{m}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}} \times 100$$

(7)

2.8.4. Fresh Weight (FW) and Dry Weight (DW) of Seedling

Fourteen days after planting, seedlings that germinated normally were counted. Subsequently, the shoots and roots were cut from the seed. They were then immediately weighed as FW. For DW, the cut shoots and roots were packed in paper bags and dried in a hot air oven at 80 °C for 24 h. Subsequently, they were weighed to determine the DW. The unit of seedling weight was measured in mg/seedling and calculated as follows:

$$\begin{gathered} \mathrm{FW} \mathrm{and} \mathrm{DW} \mathrm{of} \mathrm{seedling}=\frac{\mathrm{F}\mathrm{W} \mathrm{o}\mathrm{r} \mathrm{D}\mathrm{W} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t}\mathrm{s} \mathrm{a}\mathrm{n}\mathrm{d} \mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t}\mathrm{s}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{s}} \\ (\mathrm{m}\mathrm{g}/\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}) \end{gathered}$$

(8)

2.8.5. Seedling Moisture Content (SMC)

The SMC was measured as the amount of water removed from the seedlings by taking the weight of the seedlings from Section 2.8.4. The percentage of SMC was calculated as follows:

$$\mathrm{SMC} (\%)=\frac{\mathrm{F}\mathrm{W}-\mathrm{D}\mathrm{W}}{\mathrm{F}\mathrm{W}} \times 100$$

(9)

2.8.6. Shoot Length (SL) and Root Length (RL)

SL was evaluated by measuring from the base of the shoot to the tip of the leaf. RL was measured from the base of the taproot to the tip of the root, determined after 14 days of planting, and using a ruler with units of centimetres (cm).

2.9. Statistical Analysis

The experiment was arranged in a completely randomised design (CRD). Data were analysed using a one-way analysis of variance (ANOVA). The means were compared using the least significant difference (LSD) test, and the data were expressed as means. Statistical significance was determined at p < 0.05 (*). In terms of data on the chemical properties in PAW and pH-adjusted PAW were examined for statistical significance using a two-sample t-test at p < 0.01 (**). Statistical analysis was performed using Statistix 8 (Analytical Software, Tallahassee, FL, USA).

3. Results

3.1. Plasma Optical Emission Spectra (OES) Characteristics and Vibrational Temperature of N₂

The OES measurements were conducted at long wavelengths from 200 to 900 nm for air GA plasma discharge in an ambient room at 25 °C with an RH of 60% (Figure 2a). The emission spectrum mainly consisted of N₂, NO, OH, hydrogen atoms (Hα), and O. The N₂ molecules were identified in the range of 315–450 nm. The NO molecule bands were observed in the range of 200–300 nm. There was a peak at 308.9 nm for the OH molecules. Additionally, low intensities were observed at 656.1 nm for Hα and at 777.4 and 844.7 nm for O peaks [41,42,43].

This confirmed the generation of reactive nitrogen species (RNS) within a short wavelength from 265 to 430 nm by GA plasma at airflow rates of 2, 3, 4, 5, and 6 L/min (Figure 2b–f). The findings showed that GA plasma at all airflow rates exhibited an emission spectrum consisting of N₂ NO, and OH molecules. In particular, N₂ (second positive system; SPS and first negative system; FNS) appeared in the range of 315–410 nm [42]. The dominant peaks corresponding to N₂ (SPS) were displayed at 315.8, 337.1, and 357.6 nm. Additionally, N₂ (FNS) showed peaks at 375.4 and 380.4 nm [43]. Furthermore, NO molecules showed a low-intensity band (256–300 nm) [42], and the OH molecules had peaks at 306.3 and 308.9 nm [41]. Based on the results from the emission spectrum intensity of GA plasma at all airflow rates, significant gas phase reactions were detected, especially involving N₂ molecules. Therefore, GA plasma can effectively fix N₂ from the air into water.

The emission spectral intensity of N₂ (FNS) was given to the emission spectra derived from the line intensities at I_370.9 and I_380.4 nm of the GA plasma (Figure 3). The vibrational temperature (T_vib) of N₂ was in the range of 2800 to 3200 K. This energy value could be utilised to determine the efficiency of air GA plasma in generating plasma radicals across various species.

3.2. PAW Characteristics

3.2.1. Effect of Storage Period on PAW Chemical Properties

The DI water had a pH level of 6.04, EC value of 1.47 µS/cm, ORP value of 263 mV, NO₂⁻ content of 0.07 mg/L, and no detected content of NO₃⁻ or H₂O₂. Consequently, GA plasma was discharged at an airflow rate of 5 L/min for 20 min. The results indicated that PAW had a significantly increased NO₃⁻ (464.59 mg/L), NO₂⁻ (81.14 mg/L), and H₂O₂ content (64.25 mg/L), ORP value (542.67 mV), and EC value (330.27 µS/cm) but a decreased pH level (2.98) (Figure 4).

PAW storage at 26.5 ± 2 °C and 62.5 ± 8% RH showed a significant decrease in the content of NO₃⁻ from 464.59 to 237.97 mg/L, NO₂⁻ from 81.14 to 12.41 mg/L, and H₂O₂ from 64.25 to 25.52 mg/L from day 0 to 5 of storage. Afterwards, the contents of NO₃⁻, NO₂⁻, and H₂O₂ continued to decrease throughout the 20-day storage period (Figure 4a). However, the pH level ranged from 2.94 to 2.98, the EC value ranged from 330.27 to 435.23 µS/cm, and ORP ranged from 468.00 to 542.67 mV in PAW, with no significant differences (Figure 4b,c).

3.2.2. Effect of Different Plasma Airflow Rates on PAW Chemical Properties

GA plasma was discharged into DI water for 15 min at airflow rates of 2, 3, 4, 5, and 6 L/min, resulting in a significant decrease in the pH level of the control, from 6.04 to 3.05–3.19. In the preliminary experiment, PAW with a pH < 3.00 was used on rice seedlings that were 5 days after planting. The developing roots became shorter and had burn marks at the tips, resulting in stunted root growth. The appropriate pH range for rice growth is 5.5–6.5 [44]. Therefore, the pH of PAW at each airflow rate was adjusted using KOH, leading to an increase in the pH level to 5.97–6.01 in PAW at airflow rates of 2 to 6 L/min (Figure 5a).

The NO₃⁻, NO₂⁻, and H₂O₂ contents of PAW at the different airflow rates were not significantly different compared with the pH-adjusted PAW (Figure 5b–d). However, the EC and ORP values of PAW at all airflow rates were significantly decreased compared to the pH-adjusted PAW. The EC value decreased from 214.90–315.28 to 153.10–153.49 µS/cm, and the ORP value decreased from 550.50–558.78 to 512.67–515.91 mV (Figure 5e,f). Thus, adjusting the pH with KOH might lead to a decrease in the EC and ORP values but does not necessarily affect the NO₃⁻, NO₂⁻, and H₂O₂ contents in the PAW.

The PAW used in the germination seed experiment used pH-adjusted PAW, with a value of 5.94–6.04 in PAW airflow rates of 2 to 6 L/min. The PAW at all airflow rates significantly affected the NO₃⁻, NO₂⁻, and H₂O₂ content, EC value, and ORP value compared to T1 (Figure 6).

The NO₃⁻ content in T5 (465.61 mg/L) and T6 (471.10 mg/L) showed higher content compared to the other treatments (Figure 6b). In addition, the NO₂⁻ and H₂O₂ contents in T3, T4, T5, and T6 increased compared to T1 and T2 (Figure 6c,d). The values of EC and ORP in PAW at all airflow rates increased compared to T1 (Figure 6e,f).

When considering the chemical properties, specifically at T2, T3, T4, T5, and T6 in PAW, it was found that the NO₃⁻ content in T5 and T6 significantly increased compared to T2, T3, and T4. PAW at T3, T4, T5, and T6 showed a significant increase in the NO₂⁻ and H₂O₂ contents compared to T2. However, there were no significant differences in the pH, ORP, or EC values (Figure 6).

3.3. Effect of PAW Treatment on Total N and GA₃ Concentration in Rice Seed

3.3.1. Total N Accumulation

Fourteen-day rice seedlings after planting treated with the T2, T4, T5, and T6 in PAW treatments exhibited a significant increase in total N accumulation of 1.751%, 1.797%, 1.751%, and 1.806%, respectively, compared to T1 (1.1519%) and T3 (1.616%) (

Table 1. Concentration of total nitrogen (N) and gibberellic acid (GA₃) in rice seeds after treatment with PAW and the control.

Treatment	Total Nitrogen (%)	Gibberellic Acid (µg/g)
T1	1.519 c	0.708 d
T2	1.751 a	0.995 cd
T3	1.616 b	1.467 c
T4	1.797 a	7.047 b
T5	1.751 a	10.855 a
T6	1.806 a	11.327 a
F-test	**	**
CV (%)	2.29	6.79
LSD 0.05	0.028	0.544

Data are expressed as means, and different letters within a column indicate a significant difference according to the least significant difference (LSD) test. ** significantly different at p < 0.01 probability level.

). This is related to the NO₃⁻ and NO₂⁻ contents in PAW provided during rice cultivation, resulting in increased total N accumulation in rice seedlings.

3.3.2. GA₃ Concentration

The PAW at T5 and T6 demonstrated a significant increase in the GA₃ concentration of rice seeds compared to the other treatments, with values of 10.855 and 11.327 µg/g, respectively. Moreover, T1, T2, T3, and T4 displayed a GA₃ concentration range of 0.708–7.047 µg/g (Table 1). The GA₃ concentration in rice seeds increased with higher airflow rates of PAW.

3.4. Effect of PAW Treatment on Rice Seed Germination Characteristics

T5 had a significant increase in RE at 48 h, G, and GI compared to T1, with values of 94.33%, 97.00%, and 22.89, respectively. Additionally, in T3, T5, and T6, the RE at 72 h was 97.00, 97.00, and 97.67%, respectively, which were significantly increased compared to T1. Similarly, SL increased in T3, T4, T5, and T6 compared to T1 and T2. Moreover, the seedling FW and DW significantly increased at all airflow rates for PAW compared to T1. However, RE at 24 h (9.33–13.00%), RL (12.38–13.26 cm), and SMC (87.11–87.94%) showed no significant differences (

Table 2. Rice seed characteristics after treatment with PAW and the control.

Treatment	Radicle Emergence (%)			Germination (%)	Germination Index
	24 h	48 h	72 h
T1	9.33	85.00 c	94.00 b	94.00 c	19.81 c
T2	13.00	91.67 b	95.67 ab	96.67 ab	22.19 b
T3	12.00	93.33 ab	97.00 a	96.67 ab	22.13 b
T4	13.00	92.00 b	95.67 ab	96.00 b	21.86 b
T5	11.33	94.33 a	97.00 a	98.33 a	22.89 a
T6	11.33	91.67 b	97.67 a	97.33 ab	21.60 b
F-test	ns	**	*	*	**
CV (%)	11.61	1.21	12.7	1.15	1.77
LSD 0.05	-	1.97	2.18	1.97	0.68
Treatment	Length (cm)		Seedling Weight (mg/Seedling)		Seedling Moisture Content (%)
	Shoot	Root	Fresh	Dry
T1	13.44 b	12.38	76.70 b	9.89 b	87.11
T2	13.54 b	13.01	88.79 a	10.78 a	87.86
T3	14.60 a	13.02	89.51 a	10.78 a	87.94
T4	14.34 a	12.45	85.33 a	10.40 a	87.80
T5	14.76 a	13.26	87.08 a	10.80 a	87.56
T6	14.35 a	12.91	86.65 a	10.83 a	87.49
F-test	*	ns	*	*	ns
CV (%)	2.78	6.43	4.27	2.29	0.49
LSD 0.05	0.70	-	6.51	0.43	-

Data are expressed as means, and different letters within a column indicate a significant difference according to the least significant difference (LSD) test. ns = not significant; * significantly different at p < 0.05, and ** significantly different at p < 0.01.

).

The seedling growth rate was assessed by measuring SL and RL 5, 7, 9, 11, and 13 days after planting (Figure 7). The shoot growth rates 5 to 13 days after planting were significantly higher in T3, T4, T5, and T6 than in T1 and T2. Concurrently, the root growth rate in T2, T3, and T5 was significantly greater than that in T1, T4, and T6 on days 5 and 7 after planting. However, the root growth rate showed no significant difference 9 days after planting. Figure 8 illustrates the growth characteristics of rice seeds at 14 days after treatment with PAW compared to the control and the measurement of RE, SL, and RL.

4. Discussion

The investigation into emission spectral intensity detected various RONS, short-lived species, such as NO (few s) and OH (10⁻⁹–10⁻¹⁰ s), and longer-lived species. N₂ had stable half-lives [7]. The N₂ molecules appeared at wavelengths from 290 to 430 nm, with notable peaks for N₂ and an additional peak at 336 nm, which corresponds to NH formation [42,45]. Moreover, there was a low-emission spectral intensity band for NO molecules, as NO is rapidly converted to NO₂ through three-body reactions with O₂ in air plasma [46].

These species play a crucial role in PAW. The RONS from the air plasma contacted the water surface and dissolved in the water, leading to reactions of NO₂⁻, NO₃⁻, and H₂O₂ in PAW following the chemical reaction [47,48].

NO + NO₂ + H₂O → 2NO₂⁻ + 2H⁺_(aq)

(10)

NO₂ + NO₂ + H₂O → NO₂⁻ + NO₃⁻ + 2H⁺_(aq)

(11)

OH_(aq) + OH_(aq) → H₂O_{2 (aq)}

(12)

The occurrence of H⁺ causes the water to display acidity (a decrease in pH level), which can serve as an indicator of the accumulation of NO₂⁻ and NO₃⁻ contents in the water. The formation of NO₂⁻ and NO₃⁻ not only leads to a decrease in the pH level but also increases in the EC and ORP values [7,49]. They produced PAW with a distinctly low pH level. This is an important parameter in controlling the solubility of nutrients beneficial to plants. The low pH prevents the absorption of nutrients, which affects plant growth [9].

An increase in the airflow rate enhanced GA plasma discharge, resulting in longer plasma. This interacted more with the water surface, generating more bubbles, dissolving more plasma radicals in the water, and initiating various reactions [47,50].

The storage period of PAW reduced the contents of NO₃⁻, NO₂⁻, and H₂O₂ with changes in the EC and ORP values. However, the pH level remained relatively stable [7,51]. During extended storage periods, the NO₃⁻, NO₂⁻, and H₂O₂ contents may become unstable and degrade due to exposure to light, temperature fluctuations, or other environmental factors. This degradation can result in a decrease in the RONS concentration [7,10].

The seed germination process can begin when essential factors, such as moisture (water), are present in the PAW. This initiates the reaction of RONS having good solubility in water at appropriate concentrations. This results in the removal of wax and the etching of the seed coat, reducing its thickness [52,53,54,55]. Consequently, the seeds can absorb PAW-containing N₂ species more rapidly. In particular, NO₃⁻ is absorbed through NO₂⁻ into NH₄⁺, which can combine with amino acids. These compounds serve as both nutrients and signalling molecules that stimulate the embryo, affecting germination and plant growth processes [29]. Furthermore, NO₃⁻ in PAW is rapidly absorbed by free amino acids [11]. This causes an increase in the accumulation of total N and GA₃ in rice, which affects the germination process.

Seeds transport water and various nutrients through the chloroplast membrane. NO₃⁻ and NH₄⁺ can either be stored in vacuoles or transported further into the roots for distribution within the plant [56]. This process demonstrates that the accumulation of FW and DW in rice seedlings involves not only water but also the accumulation of N in cell vacuoles.

N has a clear effect on stimulating germination, leading to an increase in the concentration of the hormone GA₃ when rice seeds receive appropriate conditions for germination. It permeates into the seed, causing the embryo to signal GA₃ to stimulate the cells in the aleurone layer to synthesise hydrolytic enzymes. In particular, alpha-amylase decomposes starch or protein in the endosperm into food molecules, serving as a nutrient source for subsequent embryonic development [57,58]. When external inputs, such as PAW-containing NO₃⁻ and NO₂⁻, are received, they may be involved in signalling the embryo to stimulate GA₃ synthesis [59].

5. Conclusions

Air GA plasma consists of N₂, NO, and OH molecules, resulting in the formation of NO₃⁻, NO₂⁻, and H₂O₂ in PAW. Notably, NO₃⁻, NO₂⁻, and H₂O₂ contents in PAW decreased significantly after 5 days of storage.

The PAW at an airflow rate of 5 L/min enhanced the RE at 48 and 72 h, G, GI, SL, FW, and DW of seedlings. However, there were no significant differences in RE at 24 h, RL, and SMC. The NO₃⁻ and NO₂⁻ contents in PAW had a positive effect on the total N and GA₃ accumulation in rice seeds.

Therefore, the utilisation of air GA plasma to generate PAW is an effective method for producing a nitrogen source. Rice seeds receive the appropriate nitrogen content to stimulate and support the germination process. This can be used to prepare rice seedlings for cultivation.