Optimizing Plasma Discharge Intensities and Spraying Intervals for Enhanced Growth, Mineral Uptake, and Yield in Aeroponically Grown Lettuce

Abstract

Sustainable agriculture necessitates innovative solutions to enhance plant growth while optimizing resource efficiency. Plasma discharge generates reactive oxygen and nitrogen species (NH₄⁺, NO₃⁻, and NO₂⁻), which form plasma-activated water upon dissolution, affecting the nutritional solution pH and electrical conductivity (EC) and, consequently, plant development. Four treatments were applied, resulting from combining high or low plasma discharge intensities at 45 or 60 min spray intervals: low plasma discharge with a 45 min interval (T1), low plasma discharge with a 60 min interval (T2), high plasma discharge with a 45 min interval (T3), and high plasma discharge with a 60 min interval (T4). The experiment followed a 4 × 5 × 2 factorial design comprising the four treatments, five replications per treatment, and two independent experimental repeats, resulting in forty experimental units. Each unit contained 12 lettuce plants, for a total of 480 plants. The multivariate analysis of variance confirmed statistically significant treatment effects. The combination of high plasma intensity and a 45 min spray interval significantly increased the growth parameters and yield as compared with the other treatments. In particular, compared with T1, which produced the lowest values across all measured parameters, T3 resulted in a 97% increase in leaf area, a 72% increase in stem diameter, a 49% increase in leaf number, a 44% increase in leaf width, and a 30% increase in leaf length. Additionally, T3 increased edible yield by 210% and total biomass production by 203% compared with T1. These results demonstrate the combined effect of plasma intensity and spraying frequency in optimizing plant development in aeroponic systems. As far as mineral uptake is concerned, T3 increased the nitrogen, potassium, phosphorus, calcium, and magnesium concentrations by 18.2%, 16.7%, 32.3%, 20.2%, and 11.2%, respectively, compared with T1. The regression analysis further validated the robustness of the findings, indicating plasma intensity to be a dominant factor. Enhanced mineral uptake (N, P, K, Ca, and Mg) and consistent growth trends across treatments highlighted the significance of plasma technology in optimizing plant growth, yield, and nutrient absorption, suggesting it is a sustainable and efficient approach to modern agriculture.

1. Introduction

The growing global population demands innovative agricultural methods that boost food production, conserve resources, and minimize environmental impact. Aeroponics, a soilless farming method using air and mist, offers advantages over traditional farming, such as higher yields, reduced water and nutrient use, minimal space and labor, faster growth, and improved sustainability [1,2]. It features a root chamber, plant support system, and fertilizer supply system. The root chamber holds plant roots and provides a sterile atmosphere with the right amount of humidity, temperature, dissolved oxygen, pH, and electrical conductivity (EC), which are key factors influencing plant growth and yield [3,4,5]. Atomization nozzles spray roots with a solution possessing a uniform nutritional concentration, while reservoirs store and circulate nutrients, ensuring efficient uptake under varying conditions [6].

Supporting these advantages, NASA [7] reported aeroponics conserves 99% water, 50% nutrients, and 45% cultivation time. These benefits underscore the potential of aeroponics as a transformative approach to food production, especially when combined with the optimization of plant physiological parameters, plant density, nutrient formulations, and precise environmental control [8,9,10].

Lettuce (Lactuca sativa) is one of the most widely grown leafy vegetables in the world and holds significant economic value, with a global market worth about USD 2.93 billion and an annual growth rate of 2.52% [11,12]. Its fast growth, high market demand, and sensitivity to environmental conditions make it an ideal crop for controlled environment agriculture, including soilless systems such as aeroponics. Lettuce grown in vertical farms can fetch up to USD 7.82 per kilogram, more than twice the price of field-grown lettuce, which averages USD 3.04 per kilogram [13]. Due to its compact size and quick growth cycle, lettuce is well-suited for space-efficient systems and is often used as a model crop in research [14]. It is especially responsive to precise nutrient delivery and root-zone aeration, making it ideal for aeroponic setups. In this study, lettuce was chosen to evaluate how plasma discharge intensities and spray intervals affect plant growth, yield and mineral uptake in aeroponic systems.

However, the performance of lettuce in soilless systems is closely linked to nutrient availability and environmental management. Previous studies have demonstrated that deficiencies in essential nutrients such as potassium, nitrogen, and phosphorus significantly affect lettuce development and yield [15,16]. Moreover, environmental factors such as pest control, irrigation frequency, and nutrient concentration are critical to optimizing horticultural crop performance [17,18]. Micro-spray irrigation, for instance, has been shown to mitigate heat stress and prevent growth inhibition under high temperatures, thereby improving both plant health and productivity [19,20].

To further improve resource efficiency and plant performance in controlled environments, emerging technologies such as non-thermal plasma have gained increasing attention in recent years. Driven by its proven efficacy in sterilization and material processing, non-thermal plasma has recently been explored in agriculture as a means to enhance crop performance through plasma-activated water (PAW) and direct plasma exposure. With advantages including cost-effectiveness, minimal chemical input, and operation at low temperatures, non-thermal plasma holds promise in enhancing plant growth while maintaining nutritional quality, texture, and flavor [21,22,23]. In the context of plasma agriculture, ionized air generates reactive oxygen and nitrogen species (ROS and RNS) such as NO₃⁻, NO₂⁻, NH₄⁺, and H₂O₂, which form PAW. These reactive species improve germination, stimulate hormone activity, enhance nutrient uptake, and contribute to higher yields by promoting favorable physicochemical conditions in the root-zone [24,25].

Additionally, plasma treatment has shown antimicrobial and sterilization effects, reducing plant pathogen loads and improving root health [26,27,28]. PAW has been reported to trigger auxin and cytokine production, supporting accelerated growth and development [29,30]. Studies have demonstrated that plasma exposure not only improves early plant stages such as germination and seedling vigor but also enhances final crop yields when applied through irrigation [31,32]. The presence of high-energy radicals facilitates catalytic biochemical processes, and ROS/RNS generated through plasma interactions serve as signaling molecules that further modulate plant development [33,34,35,36]. Notably, nitrate (NO₃⁻), a key nutrient produced in plasma processes, plays a central role in protein synthesis, amino acid production, and chlorophyll formation. At controlled concentrations, hydrogen peroxide (H₂O₂) can act as a positive growth signal while also contributing to the pest-resistance properties of PAW [37,38].

Given the increasing global demand for sustainable and high-efficiency food production systems, integrating advanced technologies such as non-thermal plasma with aeroponics presents a promising solution. Lettuce, as a high-value, fast-growing, and environmentally sensitive crop, is well suited for evaluating such innovations due to its responsiveness to precise nutrient delivery and controlled root-zone conditions. While aeroponics already offers significant resource savings and yield improvements, the application of PAW may further enhance plant growth, nutrient uptake, and disease resistance through the introduction of RNS and ROS. Despite growing evidence of plasma’s agricultural benefits, there remains limited research on its optimized use in aeroponic systems, particularly regarding discharge intensity and spray interval effects. Therefore, this study aims to bridge that gap by investigating how plasma parameters influence lettuce growth, yield, and mineral uptake in a controlled aeroponic environment, thereby contributing to the development of more resilient and resource-efficient farming practices.

2. Materials and Methods

2.1. Aeroponic System

Experiments were conducted in the atomization laboratory (Room F107) at Jiangsu University’s School of Agricultural Engineering. The aeroponic system comprised a three-level vertical iron frame (2 × 1 × 2 m³). Four blue plastic cylinders (0.5 cm thickness, 20 cm width, and 100 cm length) with perforations fixed in two layers of the frame, each holding 12 lettuce seedlings. Each cylinder featured a 1 mm pressure-type atomization nozzle at its base, while a 12 V automatic fan mounted on the upper cover regulated temperature and humidity. Light-emitting diode (LED) lights positioned 15 cm above the canopy provided a photosynthetic photon flux density (PPFD) in the range of 200 to 250 μmol/m²/s, with red and blue wavelengths maintaining a 16 h light/8 h dark cycle [39]. High-pressure pumps PLD-1205 (Shijiazhuang City Plandi Electromechanical Equipment Co., Ltd., Shijiazhuang, Hebei, China), operating at 12 V, with a power rating of 25 W, 0.65 MPa pressure, and 3.2 L/h flow rate, ensured optimal droplet size and PAW circulation. The delivery and drain system used water-softened Polyvinyl chloride pipes (20 mm inner diameter, 2 mm thickness) with a bottom drainage pipe directing the water into a 10 L tank for recirculation (Figure 1A,B).

The vertical multi-layered aeroponic system includes several key components (Figure 1B). Red and blue grow lights (1) provide light for plant growth. Plants are placed in the atomization cultivation box (2), which is supported by an iron rack (3). Nutrient mist is sprayed through pressure nozzles (4), and excess liquid drains through a pipe (5). The nutrient solution is stored in the supply tank (6). A camera (7) monitors plant development. Sensors track the light intensity (8), as well as temperature, humidity, and carbon dioxide levels (9). The mist system is connected through a nozzle inlet pipe (10). The system is controlled by a lower control box (11), and a chiller (12) cools the solution. A temperature probe (13) measures water temperature, while a water pump (14) circulates the solution. A computer (15) runs the system with the help of an upper controller (16).

2.2. Materials and Experimental Setup

2.2.1. Experimental Design Overview

Expanded polystyrene 72-cell trays filled with perlite were used for lettuce seedlings (Lactuca sativa L.) obtained from Shouguang Xinxinran Horticulture Co., Ltd., Shouguang, Shandong, China. Tap water was applied four times weekly, and seedlings were transplanted into aeroponic systems on day 16. The experiment evaluated the effects of spraying intervals and plasma discharge intensity on growth, mineral absorption, and yield in an aeroponic system. Four treatments were tested: high intensity (three plasma generators, each with an output voltage of 8–10 kV) and low intensity (a single plasma generator) combined with 45 and 60 min spray intervals. A randomized complete block design (RCBD) was used based on theoretical factors, prior research, and experimental objectives [40]. A total of 40 experimental units (4 treatments × 5 replications × 2 repeats) were used to ensure result replication. Controlled environmental parameters, including humidity (45 ± 5% to 50 ± 2%), temperature (28 ± 1 °C during the day and 27 ± 1 °C at night), and nutritional solution composition, were kept constant throughout the experiments. Spray intervals were set to ensure efficient nutrient delivery without over hydration or depletion of RNS and ROS. These intervals affected the delivery and stability of reactive species. Plasma levels were chosen to generate these species while avoiding toxicity. The plasma intensity influences the concentrations of these species, which may have an impact on the spray frequency. This study followed ethical guidelines and did not involve human or animal subjects.

2.2.2. Plant Allocation and Selection for Analysis

Each aeroponic system contained 12 evenly distributed lettuce seedlings. The experiment included 480 plants (4 treatments × 5 replications × 12 plants/replication × 2 repeats).  For analysis, nine plants per treatment were randomly selected to ensure an unbiased representation of the treatment’s effects.

2.2.3. Simultaneous Cultivation

All aeroponic systems were operated simultaneously to ensure uniform environmental conditions. Nutritional solutions were sprayed at 45 and 60 min intervals for 3 min per cycle (

Table 1. Nutrient composition of Hoagland’s solution for aeroponic lettuce cultivation.

	Compound Name	Compound Formula	Concentration (mg/L)
1	Calcium Nitrate	Ca(NO3)2·4H2O	945
2	Potassium Nitrate	KNO3	607
3	Magnesium Sulphate	MgSO4·7H2O	493
4	Ammonium Dihydrogen Phosphate	NH4H2PO4	115
5	Boric Acid	H3BO3	2.86
6	Zinc Sulphate	ZnSO4·7H2O	0.22
7	Ammonium Molybdate	(NH4)6Mo7O24	0.02
8	Copper Sulphate	CuSO4·5H2O	0.08
9	Manganese Sulphate	MnSO4·4H2O	2.13
10	Iron-EDTA	Fe-EDTA	28

The concentrations (mg/L) of essential macro- and micronutrients used in the aeroponic system, including nitrate of calcium and potassium, magnesium sulphate, ammonium dihydrogen phosphate, and elements necessary for plant growth and development.

). The specifics of the test treatments are as follows: low plasma with a 45 min interval (T1), low plasma with a 60 min interval (T2), high plasma with a 45 min interval (T3), and high plasma with a 60 min interval (T4). Each aeroponic system’s plant-to-plant spacing was kept at 14 × 16 cm² (Figure 2). A nutrient solution with a pH range of 6.05–6.95 and electrical conductivity (EC) of 1.86–2.45 dS/m was applied in the aeroponic system and replaced every 4 days to maintain consistent nutrient availability.

2.3. DC Plasma Discharging in the Aeroponic System

High-output voltage (8–10 kV) direct current (DC) corona discharge plasma generators were installed within the culture cylinder of the aeroponic system to ionize the root chamber and generate substantial amounts of ROS and RNS (Figure 3A). The plasma generators operated for 5 min before and after each nutrient spraying cycle, producing nitrogen oxides (NO_x) and ammonia (NH₃), which enhanced the adsorption of plasma-generated species onto the nutrient droplets [41].

Dissociation:

(i)

N_2(g) + e⁻ → N + N + e⁻

(ii)

O_2(g) + e⁻ → O + O + e⁻

(iii)

H₂O + e⁻ → OH⁻ + H⁺ + e⁻

(iv)

H₂O → OH⁻ + H⁺ (UV irradiation)

Recombination:

(i)

O_2(g) + N → NO + O,

(ii)

O + N_2(g) → NO + N

(iii)

NO_2(g) + O + M → NO₃⁻ + M

(iv)

H + N → NH

(v)

H + NH → NH_2(g)

(vi)

H + NH_2(g) → NH_3(g)

N stands for nitrogen; M for molecule; O for oxygen; e⁻ for electron; (g) gas form; UV for ultraviolet rays.

2.4. Detection of NH₃ and O₃ in the Aeroponic Growth Chamber

The MQ137 and MQ131 gas sensors from Zhengzhou Winsen Electronic Technology Company Limited, Zhengzhou, China (Figure 4A,B) were used to detect ozone and ammonia gas in the aeroponic chamber, respectively. During plasma discharge, the two sensors were placed inside the aeroponic boxes.

Output voltage data from the sensors were recorded and plotted on a graph (Figure 5B) to analyze its trend and determine the corresponding NH₃ and O₃ based on the sensor datasheet and equations.

The relationship between the resistance ratio and the concentration of ozone (O₃) is given by the following equation [42].

$$O_3\left(\mathrm{p}\mathrm{p}\mathrm{m}\right)=A\times {\left(R_S{R}_0^{-1}\right)}^B$$

(1)

where

R_s is the sensor resistance in the presence of O₃ (output resistance);

R₀ is the sensor resistance in clean air (baseline resistance);

A and B are calibration constants derived from the sensor datasheet.

The relationship between the resistance ratio and the concentration of ammonia (NH₃) is given as follows [43]:

$${NH}_3\left(\mathrm{p}\mathrm{p}\mathrm{m}\right)=C\times {\left(R_S{R}_0^{-1}\right)}^D$$

(2)

where

R_s is the sensor resistance in the presence of NH₃;

R₀ is the sensor resistance in clean air;

C and D are sensor calibration constants from its datasheet.

The voltage divider equation was used to calculate R_s from the sensor output voltage.

The MQ sensors are typically used with a load resistor (R₁) in series as follows [43]:

$$R_s=R_1\times V_{out}{\left(V_{supply}-V_{out}\right)}^{-1}$$

(3)

where

V_supply is the supply voltage to the sensor;

V_out is the output voltage of the sensor;

R₁ is the sensor load resistor whose value is chosen based on the sensor.

2.5. Nutritional Solution pH and EC Measurement

Daily measurements were taken for the pH and electrical conductivity (EC) of the atomized nutritional solution. A ProfiLine pH 3110 m (accuracy: ±0.001), WTW, Weilheim, Germany, was used for pH measurement, while EC was measured using a ProfiLine Cond 3110 m (accuracy: ±0.1 mS/cm), manufactured by Xylem Analytics Germany Sales GmbH & Co. KG (Brand WTW), Weilheim, Germany.

2.6. Lettuce Growth and Yield Measurement

Growth parameters, including leaf area, leaf length, stem diameter, leaf width, number of leaves, edible yield, and biomass yield, were measured 40 days after transplant. The number of leaves was counted manually; the stem diameter was measured by a digital Vernier caliper; leaf length and width were measured using thread and a meter scale; leaf area was measured by a CI-203 laser (CID Bio-Science, Inc., Camas, WA, USA). The total biomass yield (stem + leaves + root) and edible yield (stem + leaves) were measured in grams using an electronic analytical balance. Samples were then oven-dried at 105 °C for 24 h, and their dry weights were recorded.

2.7. Mineral Uptake

Total nitrogen was determined using the Kjeldahl digestion technique [44]. The phosphorous concentration was measured via automated colorimetry (molybdovanadate method), potassium by flame photometry, and calcium and magnesium by Optima 5300 DV spectrometer (PerkinElmer, Shelton, WA, USA) [45,46,47].

2.8. Analytical Statistics

The effect of spraying intervals and plasma discharge intensities on lettuce was assessed using multivariate analysis of variance (MANOVA) and regression analysis. MANOVA was selected over univariate ANOVA because of its ability to simultaneously analyze multiple dependent variables (growth parameters, mineral absorption, biomass yield, and root-to-shoot ratio) while accounting for the influence of multiple independent variables (spray intervals and plasma discharge intensities). Unlike ANOVA, which examines each dependent variable separately, MANOVA considers the intercorrelations among dependent variables and evaluates them jointly, offering a more comprehensive understanding of treatment effects and potential interactions [48]. This multivariate approach reduces the risk of Type I error inflation that can result from conducting multiple independent ANOVAs and increases statistical power by incorporating shared variance among outcomes [49].

Before conducting MANOVA, the assumptions of multivariate normality and homogeneity of variance–covariance matrices were evaluated. These data met the normality requirement, which assumes that each dependent variable and all linear combinations of them are normally distributed within groups, a crucial condition for valid MANOVA results. The homogeneity of variances was confirmed using Levene’s test (p ≥ 0.05), ensuring equal error variance across groups and validating the use of MANOVA [50,51,52]. Additionally, regression analysis complemented MANOVA by quantifying the relationships between plasma intensity levels and lettuce growth responses, allowing the prediction of optimal plasma conditions. Together, these statistical methods provided a robust analytical framework to interpret both the inferential significance and predictive trends of treatment effects in a controlled aeroponic system.

2.9. Modelling Approach

Multiple linear regression models were developed to assess the effects of plasma intensity (X1) and spraying interval (X2) on growth, biomass yield, and mineral uptake. These models predict plant responses based on the specific input conditions, with R² values indicating model accuracy [53,54].

3. Results

This section details the results and analysis of the effect of spray intervals and plasma discharge intensities on lettuce growth, mineral absorption, and plant yield. The treatment effects varied significantly, with high plasma discharge treatment producing the greatest improvements. Both repetitions showed a consistent trend in growth metrics. Statistical analysis confirmed that the variation between repetitions was minimal and did not significantly affect the observed treatment effects. Therefore, the combined results from both repeats are reported. High plasma discharge treatments (T3 and T4) showed greater pH reductions and EC increases compared with low plasma intensity with a 45 min interval treatment (T1), as illustrated in Figure 6.

3.1. Plant Growth

To evaluate the overall impact of spraying intervals, plasma discharge intensities, and their interaction on the growth parameters of lettuce, a multivariate analysis of variance (MANOVA) was conducted (see

Table 2. Multivariate analysis of variance (MANOVA) results showing the effects of spraying interval, plasma discharge intensity, and their interaction on the dependent variables.

	Effect	Value	F	Hypothesis df	Error df	Sig.
Intercept	Pillai’s Trace	1.00	6472.302b	5.00	40.00	0.000
	Wilks’ Lambda	0.00	6472.302b	5.00	40.00	0.000
	Hotelling’s Trace	809.04	6472.302b	5.00	40.00	0.000
	Roy’s Largest Root	809.04	6472.302b	5.00	40.00	0.000
Spraying interval	Pillai’s Trace	0.83	40.249b	5.00	40.00	0.000
	Wilks’ Lambda	0.17	40.249b	5.00	40.00	0.000
	Hotelling’s Trace	5.03	40.249b	5.00	40.00	0.000
	Roy’s Largest Root	5.03	40.249b	5.00	40.00	0.000
Plasma discharge	Pillai’s Trace	0.96	199.550b	5.00	40.00	0.000
	Wilks’ Lambda	0.04	199.550b	5.00	40.00	0.000
	Hotelling’s Trace	24.94	199.550b	5.00	40.00	0.000
	Roy’s Largest Root	24.94	199.550b	5.00	40.00	0.000
Spraying interval × Plasma discharge	Pillai’s Trace	0.90	71.935b	5.00	40.00	0.000
	Wilks’ Lambda	0.10	71.935b	5.00	40.00	0.000
	Hotelling’s Trace	8.99	71.935b	5.00	40.00	0.000
	Roy’s Largest Root	8.99	71.935b	5.00	40.00	0.000

Multivariate tests using Pillai’s Trace indicate significant effects of the main factors and their interaction on the combined dependent variables (p < 0.001) Superscript ᵇ indicates that the F-statistic is based on the approximate F distribution.

). The analysis revealed highly significant effects for all tested factors. Plasma discharge intensity accounted for the highest proportion of variance across the dependent variables, explaining 96% (Pillai’s Trace = 0.96, F = 199.55, p < 0.001). Spraying intervals also demonstrated a strong influence, accounting for 83% of the variance (Pillai’s Trace = 0.83, F = 40.25, p < 0.001). Furthermore, the interaction between spraying interval and plasma discharge intensity contributed significantly to variability, explaining 90% of the variance (Pillai’s Trace = 0.90, F = 71.94, p < 0.001). These results underscore the importance of optimizing both parameters to enhance plant performance.

To explore which specific traits contributed to the observed multivariate effects, separate univariate analyses were performed for each growth parameter. The results are presented in

Table 3. Univariate MANOVA results showing the effects of spraying interval, plasma discharge intensity, and their interaction on lettuce growth parameters.

Source/Factor	Measure Parameters	Type III Sum of Squares	df	Mean Square	F	Sig.
Spraying intervals	Leaf Number	60.75	1	60.75	23.90	0.000
	Leaf Length	24.14	1	24.14	24.82	0.000
	Leaf Width	9.28	1	9.28	28.73	0.000
	Stem Diameter	0.23	1	0.23	164.42	0.000
	Leaf Area	1530.59	1	1530.59	43.14	0.000
Plasma discharge	Leaf Number	168.75	1	168.75	66.39	0.000
	Leaf Length	106.68	1	106.68	109.67	0.000
	Leaf Width	41.59	1	41.59	128.80	0.000
	Stem Diameter	1.19	1	1.19	852.58	0.000
	Leaf Area	4403.54	1	4403.54	124.11	0.000
Spraying intervals × Plasma discharge	Leaf Number	33.33	1	33.33	13.12	0.001
	Leaf Length	3.69	1	3.69	3.79	0.058
	Leaf Width	4.5	1	4.50	13.94	0.001
	Stem Diameter	0.48	1	0.48	346.56	0.000
	Leaf Area	5.68	1	5.68	0.16	0.691

The results indicate significant effects of both spraying intervals and plasma discharge intensity on all measured growth traits (p < 0.001). However, the interaction between spraying interval and plasma discharge was not significant for leaf length (p = 0.058) and leaf area (p = 0.691).

. Plasma discharge intensity had a particularly strong effect, with highly significant F-values across parameters, especially stem diameter and leaf area. The interaction between spraying interval and plasma intensity also contributed significantly to some traits, including stem diameter and leaf width, indicating a synergistic effect on morphological development.

Key growth differences across treatments are summarized in Figure 7 and Figure 8. These underlying values were used to calculate the percentage increases reported in the abstract. Lettuce growth was highest in T3, with 24 leaves, 1.22 cm stem diameter, 19.06 cm leaf length, 8.93 cm leaf width, and 61.73 cm² leaf area. Treatments T1 and T2 showed comparatively lower growth performance, while T4 gave intermediate results. Overall, T3 demonstrated significantly superior growth performance across all measured parameters.

The equations below represent the regression models derived from the analysis, describing the predictive relationships for leaf length, leaf width, stem diameter, leaf area, and leaf number with coefficient of determination (R²) values indicating strong correlations.

$$\begin{gathered} Y \left(leaf length\right)=9.986+2.982X1+1.418X2+46.487 R^2=0.738 \\ Y\left(Number of leaves\right)=11.167+3.750X1+2.250X2+145.16 R^2=0.613 \\ Y (Stem diameter)=0.215+0.315X1+0.138X2+0.545 R^2=0.723 \\ Y (Leaf width)=3.137+1.862X1+0.879X2+18.709 R^2=0.731 \\ Y \left(Leaf area\right)=0.488+19.156X1+11.294X2+1566.816 R^2=0.791 \end{gathered}$$

3.2. Plant Yield and Root-to-Shoot Ratio

Table 4. Univariate MANOVA results showing the effects of spraying interval, plasma discharge intensity, and their interaction on lettuce yield.

Source/Factor	Measured Parameters	Type III Sum of Squares	df	Mean Square	F-Value	Sig.
Spraying interval (min)	Shoot fresh weight	1155.029	1	1155.029	82.49	0.000
	Root fresh weight	12.679	1	12.679	32.01	0.000
	Edible yield	1350.971	1	1350.971	91.47	0.000
	Total biomass yield	1625.41	1	1625.41	95.65	0.000
	Ratio of root to shoot wet	4.75	1	4.75	0.53	0.473
	Ratio of root to shoot dry	726.185	1	726.185	19.48	0.000
Plasma discharge	Shoot fresh weight	3128.578	1	3128.58	223.45	0.000
	Root fresh weight	32.489	1	32.49	82.01	0.000
	Edible yield	3528.956	1	3528.96	238.93	0.000
	Total biomass yield	4238.648	1	4238.65	249.44	0.000
	Ratio of root to shoot wet	55.255	1	55.26	6.10	0.017
	Ratio of root to shoot dry	2509.967	1	2509.97	67.34	0.000
Spraying intervals × Plasma discharge	Shoot fresh weight	749.078	1	749.08	53.50	0.000
	Root fresh weight	10.24	1	10.24	25.85	0.000
	Edible yield	889.671	1	889.67	60.24	0.000
	Total biomass yield	1090.804	1	1090.80	64.19	0.000
	Ratio of root to shoot wet	0.227	1	0.23	0.03	0.875
	Ratio of root to shoot dry	990.992	1	990.99	26.59	0.000

Spraying interval and plasma discharge intensity significantly affected most biomass parameters (p < 0.05). However, the root-to-shoot wet ratio was not significantly influenced by the spraying interval or its interaction with plasma discharge (p > 0.05).

presents the MANOVA results assessing the influence of plasma intensity, spraying interval, and their interaction on lettuce yield parameters, including shoot and root fresh weights, edible and total biomass, and root-to-shoot ratios on wet and dry bases. The analysis reveals significant effects of both main factors and their interaction; however, unlike the other parameters, neither the spraying interval nor its interaction with plasma discharge intensity showed a statistically significant effect on the root-to-shoot ratio on a wet basis (p = 0.473 and p = 0.875, respectively).

Lettuce yield differences across treatments are presented in Figure 9. These values formed the basis for determining the percentage increases reported in the abstract. Lettuce yield was highest in T3, with 38.57 g shoot fresh weight, 4.47 g root fresh weight, 40.99 g edible yield, and 45.45 g total biomass. Treatments T1 and T2 showed comparatively lower yield performance, while T4 gave moderate results. Root-to-shoot wet ratios were similar across treatments, ranging from 11.63% to 14.4%. However, T3 had the lowest dry root-to-shoot ratio (25.69%), indicating a greater proportion of shoot biomass. Overall, T3 demonstrated significantly superior productivity and biomass accumulation across all yield parameters.

The regression models show that plasma discharge intensity and spraying intervals significantly affect both total biomass and edible yield. With high correlation values (R² = 0.761 and R² = 0.760), these factors are key in determining plant productivity.

$$Y\left(Total biomass\right)=-18.839+17.149X_1+10.610X_2+1539.544 R^2=0.761$$

$$Y(Edible yield)=-20.182+18.794X_1+11.638X_2+1838.491 R^2=0.760$$

3.3. Mineral Uptake Efficiency

Table 5. Univariate MANOVA results showing the effects of spraying interval, plasma discharge intensity, and their interaction on lettuce mineral uptake.

Source/Factor	Uptake Minerals	Type III Sum of Squares	df	Mean Square	F	Sig.
Spraying interval	Nitrogen	42.772	1	42.77	49.60	0.000
	Potassium	215.796	1	215.80	324.42	0.000
	Phosphorus	3.022	1	3.02	91.66	0.000
	Calcium	8.122	1	8.12	129.23	0.000
	Magnesium	0.245	1	0.25	90.49	0.000
Plasma discharge	Nitrogen	228.715	1	228.72	265.22	0.000
	Potassium	414.801	1	414.80	623.60	0.000
	Phosphorus	17.043	1	17.04	516.98	0.000
	Calcium	19.478	1	19.48	309.88	0.000
	Magnesium	0.697	1	0.70	257.50	0.000
Spraying interval × Plasma discharge	Nitrogen	8.546	1	8.55	9.91	0.004
	Potassium	93.896	1	93.90	141.16	0.000
	Phosphorus	3.706	1	3.71	112.41	0.000
	Calcium	1.11	1	1.11	17.65	0.000
	Magnesium	0.167	1	0.17	61.58	0.000

Spraying interval, plasma discharge intensity, and their interaction significantly influenced the uptake of all measured mineral nutrients (p < 0.05).

presents the multivariate analysis of variance (MANOVA) results evaluating the effects of spraying intervals, plasma discharge intensities, and their interaction on the uptake of essential minerals in lettuce, including nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), and magnesium (Mg). The analysis reveals significant influences of both individual and interactive factors on mineral nutrient absorption.

The levels of mineral uptake across treatments are summarized in Figure 10. These data were used to determine the percentage increases highlighted in the abstract. Lettuce grown under T3 recorded the highest mineral concentrations, with 46.90 mg nitrogen, 81.68 mg potassium, 7.99 mg phosphorus, 14.44 mg calcium, and 4.37 mg magnesium. Treatments T1 and T2 showed comparatively lower mineral uptakes, while T4 gave intermediate results. Overall, T3 demonstrated significantly superior nutrient absorption across all measured mineral elements.

The regression equation models show that plasma discharge intensity and spraying intervals significantly affected the mineral uptake with high correlation values (R² ranging from 0.788 to 0.898).

$$\begin{gathered} Y \left(Nitrogen\right)=32.948+5.041X_1+2.180X_2+36.142 R^2=0.883 \\ Y \left(Potassium\right)=59.923+6.789X_1+4.897X_2+115.182 R^2=0.846 \\ Y \left(Phosphorus\right)=4.402+1.376X_1+0.576X_2+4.761 R^2=0.808 \\ Y \left(Calcium\right)=9.422+1.471X_1+0.950X_2+3.121 R^2=0.898 \\ Y \left(Magnesium\right)=3.416+0.278X_1+0.165X_2+0.253 R^2=0.788 \end{gathered}$$

4. Discussion

4.1. Plasma Discharge Effects on Nutritional Solution’s EC and pH Levels

The electrical conductivity (EC) and pH of the Hoagland nutrient solution varied across treatments during the four-day observation period. The high-intensity plasma treatments showed a greater decrease in pH and a more consistent increase in EC compared with low-intensity treatments, indicating active plasma interaction with the nutritional solution [55]. The MANOVA analysis confirmed that both plasma intensity and spraying interval significantly affected EC and pH levels (p < 0.05). These changes are attributed to the generation of RNS and ROS, which form nitric acid and ionized compounds such as nitrate, thereby acidifying the solution (pH 6.05–6.95) and increasing its ionic strength (EC 1.86–2.45 dS/m). This plasma-induced chemical shift probably enhanced nitrate solubility and mineral availability, promoting nutrient uptake and lettuce growth. These findings align with previous studies [56,57], which highlight the importance of maintaining optimal EC and pH levels in aeroponic systems to avoid nutrient deficiency or salt-induced stress.

4.2. Effects of Plasma Intensities and Spraying Intervals on Lettuce Plant Growth

Plant growth parameters varied notably across treatments, influenced by both plasma discharge intensity and spraying intervals. The high-intensity plasma treatments (T3 and T4) led to marked improvements compared with low-intensity with a 45 min interval treatment (T1), as shown in Figure 7 and Figure 8. These results underscore the consistent positive impact of plasma intensity on vegetative growth. The MANOVA analysis (Table 3) confirmed that both plasma intensity and spraying interval had significant effects (p < 0.05) on most parameters. However, the interaction effect was not significant for leaf length (p = 0.058) and leaf area (p = 0.69), suggesting that these traits were less sensitive to the combined influence of both factors. Plasma discharge produces reactive nitrogen and oxygen species that improve nutrient availability, photosynthesis, and plant growth. These species react with water to form nitrogen compounds such as ammonium (NH₄⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻), which support nutrient absorption and key physiological processes [41,58]. The higher plasma intensity likely increased the concentration of these compounds, which may have contributed to enhanced lettuce growth, especially at the 45 min spray interval. Statistical models showed plasma intensity had a stronger impact than spraying intervals. These findings align with previous studies [59,60] on the importance of spray timing and plasma application in promoting plant development in aeroponic systems.

4.3. Effects of Plasma Intensities and Spraying Intervals on Lettuce Plant Yield

The high-intensity plasma treatments (T3 and T4) significantly boosted the edible yield and total biomass of lettuce compared with low-intensity with a 45 min interval treatment (T1). The MANOVA results confirmed that plasma discharge intensity, spraying interval, and their interaction significantly affected edible yield and total biomass (p < 0.05), with the highest values in T3 and the lowest in T1 (Table 4, Figure 9A). Plasma intensity significantly influenced both wet and dry root-to-shoot ratios (p < 0.05), while spraying intervals affected only the dry ratio. Their interaction was significant only for the dry ratio (Table 4). ROS such as H₂O₂ and OH⁻, generated by plasma, act as signaling molecules that promote root elongation, cell wall loosening, shoot expansion, and CO₂ uptake [61,62,63]. Excessive ROS, however, may cause oxidative stress, highlighting the need for controlled plasma application. RNS such as NO₂⁻ and NO₃⁻ enhance nitrogen metabolism by regulating nitrate reductase, boosting ammonium formation and nutrient uptake, and stimulating root hair growth while enhancing stress tolerance through interactions with key phytohormones [64,65]. PAW improves mineral availability and biomass. The highest yields occurred with high plasma discharge and a 45 min spray interval, consistent with optimized aeroponic cycle studies [66,67] on optimized aeroponic spray cycles. The lowest dry root-to-shoot ratio, indicating greater shoot growth, was under the same condition, while the highest, favoring root growth, was under low discharge at 60 min. These results suggest that plasma treatment may contribute to improved yield and resource efficiency, indicating its potential value in sustainable agriculture under specific conditions [68].

4.4. Effects of Plasma Intensities and Spraying Intervals on Mineral Uptake

Mineral uptake was significantly affected by plasma discharge intensity and spraying intervals. The high-intensity treatments (T3 and T4) significantly improved nutrient uptake compared with low-intensity with a 45 min interval treatment (T1), as shown in Figure 10. The highest nitrogen and potassium levels were found in T3, suggesting that plasma-generated reactive species probably enhanced mineral absorption. Plasma discharge likely contributed to improved mineral uptake by moderately acidifying the nutrient solution, as evidenced by a lowered pH and increased EC conditions that are generally associated with enhanced nutrient solubility and availability. It also appears to facilitate the conversion of atmospheric nitrogen into reactive nitrogen species (RNS), which may have supported improved nitrogen absorption. MANOVA showed that plasma intensity, spray interval, and their interaction had a significant effect on mineral uptake (p < 0.05), suggesting a possible improvement in lettuce metabolism and nutrition. These results agree with previous studies linking higher mineral uptake with increased photosynthesis and crop yield [69,70]. The regression analysis suggested that plasma intensity and spray intervals both influenced nutrient absorption with good predictive accuracy. Plasma intensity appeared more impactful, possibly because of increased ROS and RNS aiding nutrient transport. Longer spray intervals may have helped by reducing over hydration and supporting nutrient balance, contributing to improved plant nutrition and growth. This study suggests that plasma-integrated aeroponics can enhance nitrogen fixation and help regulate key growth conditions such as pH, EC, and nutrient availability. While previous studies mainly focused on plasma for seed sterilization or foliar application, our findings highlight the potential of in-system plasma treatment to produce ammonia, ozone, and other reactive species that may improve nutrient availability. Plasma discharge also appears to help maintain a slightly acidic environment and stable EC, both of which are important for mineral uptake and yield. Overall, the results support plasma-aeroponic systems as a promising method for controlled-environment agriculture, with the potential to reduce fertilizer use, increase crop productivity, and advance sustainable food production.

5. Conclusions

This study highlights the potential of high-intensity plasma discharge as a sustainable enhancement within the tested aeroponic cultivation treatments, demonstrating its relative effectiveness in promoting plant growth, improving nutrient uptake, and maintaining favorable root-to-shoot biomass distribution. The integration of plasma technology was shown to support optimal conditions within the nutrient solution, specifically, slightly acidic pH and stable electrical conductivity, thereby enhancing the solubility and availability of essential minerals. The generation of RNS and ROS contributed to improved nutrient assimilation, root development, and shoot elongation, supporting the potential role of plasma-induced nitrogen fixation in controlled environment agriculture.

This work presents plasma-integrated aeroponics as a novel and potentially beneficial approach for enhancing crop productivity while reducing dependency on synthetic fertilizers under controlled conditions. A balanced combination of plasma discharge intensity and spraying intervals is recommended to optimize plant health and yield outcomes within the scope of the treatments tested. Future research should aim to refine discharge parameters, including intensity, exposure duration, and misting intervals tailored to specific crop requirements to further advance the scalability and efficiency of plasma-aided aeroponic systems.