Optimizing Lettuce Growth in Nutrient Film Technique Hydroponics: Evaluating the Impact of Elevated Oxygen Concentrations in the Root Zone under LED Illumination

Abstract

Evaluating different concentrations of oxygen on lettuce physiology, growth, and biochemical assays is pivotal for optimizing the nutrient film technique (NFT), boosting yields, and enhancing resource efficiency in sustainable greenhouse cultivation. Two lettuce varieties Lactuca sativa var. Lolo Bionta (Lugano) and Lolo Rosa (Carmesi), were grown using NFT in a greenhouse for two consecutive years during the months of December and January. A comparative methodology was adopted under a randomized complete block design (RCBD) to study plant growth under three different oxygen concentration levels: natural oxygen concentrations (NOC); elevated oxygen concentrations (EOC); and elevated oxygen concentrations under LED light (380–840 nm) (LED + EOC). The plants were exposed to EOC levels of 8.1–8.7 mg L⁻¹ in December and 8.7–9.0 mg L⁻¹ in January. Under LED + EOC conditions, the levels were 8.2–8.3 mg L⁻¹ in December and 8.8–9.0 mg L⁻¹ in January. The NOC levels were 6.8–7.1 mg L⁻¹ in December and 7.2–7.8 mg L⁻¹ in January for Lugano and Carmesi, respectively. The applied light intensity, measured as photosynthetic photon flux density (PPFD), ranged from 463 to 495 µmol m⁻² s⁻¹ for the Lugano and from 465 to 490 µmol m⁻² s⁻¹ for the Carmesi. The dissolved oxygen concentration and LED light exposure under greenhouse conditions had significant effects (p < 0.05) on the plant growth parameters. The biochemical and physiological attributes, including transpiration rate, stomatal conductance, nitrate, chlorophyll, sugar contents, net photosynthesis, and respiration rates, varied significantly across different oxygen concentrations. Data were analyzed using a two-way ANOVA with post hoc Tukey’s HSD tests for significance (p < 0.05) using IBM SPSS Statistics (version 29.0.2.0). Both EOC and LED + EOC treatments significantly improved growth attributes compared to NOC in Lugano, with increases in plant height (16.04%, 0.85%), fresh mass (110.91%, 29.55%), root length (27.35%, 29.55%), and root mass (77.69%, 34.77%). For Carmesi, similar trends were observed with increases in plant height (5.64%, 13.27%), fresh mass (10.45%, 21.57%), root length (37.14%, 47.33%), and root mass (20.70%, 41.72%) under EOC and LED + EOC. In the intertreatment analysis, the effect of LED + EOC was more pronounced compared to EOC. In view of the intertreatment response, Lolo Bionta (Lugano) appeared to have a high overall horticultural performance (growth and yield in both EOC and LED + EOC compared to Lolo Rosa (Carmesi). The practical significance of these results lies in their potential to inform strategies for optimizing greenhouse environments, particularly through the manipulation of oxygen levels and light exposure. The significant increases in growth metrics, especially under the LED + EOC conditions, suggest that targeted environmental adjustments can lead to substantial improvements in lettuce yield and quality. The findings also contribute to the advancement of sustainable agricultural technologies aiming to enhance food security and sustainability.

1. Introduction

The global food crisis demands innovative strategies in the agricultural sector, making it imperative for horticulturists to implement advanced methodologies for fruit and vegetable production. In the face of food scarcity driven by climatic and economic challenges, sustainable and efficient agricultural practices have become crucial. Hydroponics is an environmentally friendly and sustainable agricultural technique that allows plants to grow profitably without the use of soil [1]. The hydroponic system mainly relies on cultivation techniques such as the deep flow technique (DFT), deep water culture hydroponics (DWC), and the nutrient film technique (NFT). The NFT provides higher oxygen access to plant roots compared to the DFT and floating technique, promoting healthier growth [2]. It allows efficient water and nutrient use with a thin, continuous film, requires less space, and is ideal for small or urban farming operations due to its ease of setup and management. A nutrient solution is another crucial component of hydroponics as it delivers water, oxygen, and essential minerals directly to plant roots in a soluble form, thereby supporting optimal growth and development [3]. Ensuring the right balance of water, oxygen, and minerals, along with effective nutrient solution management and suitable hydroponic technique selection, is critical for plant health and high yields [4].

Lettuce (Lactuca sativa L.) is a highly favored salad crop in hydroponic cultivation due to its substantial nutritional benefits, including low levels of calories, fat, and sodium, while being rich in vitamins, folate, fiber, and essential minerals [5,6,7]. The NFT method is ideal for cultivating lettuce, allowing for up to eight harvests each year [6,8]. Lettuce, a member of the Asteraceae family within the Asterales order, is part of a group that includes over 1620 genera and 23,600 species worldwide [9]. Besides root nutrient intake, environmental conditions, light, temperature, and CO₂ play an equally vital role in lettuce growth. This study aims to delve into the intricate relationship between varying elevated oxygen concentrations (EOC) levels and LED illumination on the growth attributes, rate of biochemical processes, and biochemical composition of two lettuce varieties, Lugano and Carmesi, in NFT hydroponic systems.

To achieve optimal crop yield in hydroponic systems, a healthy root system, a balanced solution flow rate, adequate oxygen levels, and optimal greenhouse conditions are essential [10,11]. A healthy root system is essential for nutrient uptake, root growth (boosting shoot and root weights), and maintenance, as well as for guarding against root-borne illnesses. This is achieved by creating a highly oxygenated root zone environment [12]. Studies have shown that an adequate supply of dissolved oxygen in the root zone significantly improves plant growth, yield, and stress tolerance. For example, high levels of dissolved oxygen can mitigate the adverse effects of waterlogging and hypoxia by maintaining root function and promoting the formation of aerenchyma, which facilitates internal oxygen transport within the roots [13,14]. Plant and root performance can be compromised by low oxygen levels in the root zone, which can also lead to a higher risk of disease development [12,15]. Various studies have elucidated the pivotal role of oxygen in a range of plant biochemical processes—including cellular respiration, oxidative phosphorylation, and reactive oxygen species (ROS)—which are fundamental to specific growth attributes in plants [16,17,18]. Molecularly, oxygen supports ATP production via oxidative phosphorylation [19]. The presence of dissolved oxygen (DO) is critical for root formation and growth, as evidenced by [16,20]. Roots necessitate oxygen for respiration, a critical process to generate the energy required for the uptake of nutrients and water. The studies by [21,22] underscore the importance of addressing root zone oxygenation to enhance plant resilience and productivity in various agricultural and ecological contexts. The utilization of peroxides or peracetic acid in nutrient solutions boosts arugula biomass in floating hydroponic systems by enhancing dissolved oxygen, vital for root respiration and nutrient absorption [22]. Elevated dissolved oxygen (23 mg L⁻¹) at a low temperature (12 °C) and electric conductivity (1.0 mS cm⁻¹) spurred a 2.1-fold growth enhancement in lettuce compared to ambient room air [20,23].

Providing oxygen to roots in an NFT system is more critical than to the shoots because roots require oxygen for a process vital for energy production and nutrient uptake. Roots mainly obtain oxygen from dissolved oxygen (DO) in the nutrient solution, other methods can supplement the oxygen supply, such as aeroponics, where roots are suspended in air and misted with nutrient solution, and oxygenating the nutrient solution with air stones or oxygen generators. The relevance of root zone oxygenation and LED illumination in NFT hydroponic systems addresses several current challenges in hydroponic agriculture. Combining optimal lighting and root oxygenation significantly improves lettuce yield and quality in NFT systems [24]. Optimal light exposure enhances photosynthesis in leaves, while adequate root oxygenation ensures efficient energy release from sugars, promoting robust growth. Efficient LED illumination can significantly impact hydroponic growth by providing targeted wavelengths that maximize photosynthesis while minimizing energy consumption [25]. Combining advanced root zone oxygenation techniques with optimized LED lighting can mitigate common issues in NFT systems, leading to improved plant health, increased yields, and higher quality produce. Therefore, innovative approaches in these areas are essential for overcoming the limitations of traditional hydroponic practices and advancing sustainable agriculture [26,27].

Previous studies have also shown that the development of lettuce plants is strongly influenced by growing conditions, which include optimal temperature, adequate light, proper watering, and the use of fertilizers [28,29]. According to [23,30], three-dimensional cultivation (multiple layers or levels) in plant factories under light exposure increased lettuce yield by up to 35.80%, dry matter by up to 39.70%, and reduced nitrate content by up to 10.75% compared to non-complemented light. Regular maintenance and a good understanding of greenhouse microclimatic conditions are also crucial to achieving optimal results in hydroponics [31]. This ensures efficient resource utilization and creates the ideal growing environment for plants. Keeping the nutrient solution at an optimal temperature (usually between 65 and 75 °F or 18 and 24 °C) ensures efficient nutrient uptake and prevents root stress [32]. Consistent management of climate, soil fertility, gas exchange, and water flow are crucial. Adequate light is vital for healthy lettuce leaf development, affecting size, color, and biochemical composition [33]. Insufficient light results in weak, pale plants, while too much light can cause leaf burn. Studies by [34] explore the physiological, biochemical, and resource utilization effects of red and blue spectral components on lettuce plants.

The cultivation of lettuce has attracted considerable academic attention owing to its quick or rapid growth cycle. Contemporary research, both in Romania and internationally, focuses on the influence of LED-assisted lighting, nutrient solutions, and substrate techniques on lettuce’s growth, yield, and quality [35,36]. Recent research on the addition of oxygen to the root zone in NFT (nutrient film technique) systems highlights various innovative methods and their impact on plant growth [37]. Higher flow rates (1–2 L per min) in NFT systems increase the dissolved oxygen, benefiting root metabolic processes and overall plant growth [38]. Another study supports the need for maintaining near-saturation levels of dissolved oxygen to prevent root diseases and support beneficial microbes [39]. Nonetheless, the effects of dissolved oxygen and light on the growth, photosynthesis, yield, and quality of lettuce under different greenhouse conditions are not well understood. While some studies indicate that oxygen to the root zone in NFT (nutrient film technique) systems and light significantly affect the lettuce plant’s growth, these findings highlight the need for further investigation in the context of lettuce cultivation [40,41]. This study aims to investigate the synergistic effects of EOC and LED illumination on the overall growth of lettuce in NFT hydroponic systems during the months of December and January. By exploring various EOC levels, and the need of applying oxygen to roots and LED light spectra, this research seeks to identify the optimal conditions that enhance lettuce growth, yield, and quality. The findings from this study could provide valuable insights for improving hydroponic practices and contribute to the advancement of agricultural technologies aiming to enhance food security and sustainability. As the global population grows and arable land becomes increasingly scarce, innovative agricultural methods such as hydroponics offer promising solutions for sustainable food production. It is crucial to investigate how these factors interact with each other. Specifically, future research should focus on examining the combined effects of oxygen and light on plant cultivation. Ultimately, this study aims to support resilient agricultural practices amid global challenges.

2. Materials and Methods

2.1. Experimental Site and Greenhouse Management

This study aims to investigate the synergistic effects of dissolved oxygen concentrations (DOC) and LED illumination on the overall growth of lettuce in NFT hydroponic systems within a Venlo Glass greenhouse. The experimental site is situated at the Research Center of Quality Control of Horticultural Products, University of Agronomic Sciences and Veterinary Medicine, Bucharest (coordinates: 44.4710° N, 26.0656° E). The greenhouse is constructed with a unique design featuring hot-dipped galvanized steel and aluminum system profiles for external cladding, along with 6.30-meter-high cultivating gutters and a glass covering. The foundation and ground have a slope of 10 mm per section, amounting to a total of 4000 mm. The structure is divided into 19 compartments for cultivating flowers and various vegetables, with a dedicated 160.00 m² compartment for lettuce growth. The total covered area of the greenhouse is 2752.00 m². For precise data collection, the lettuce-growing compartment is equipped with advanced recording devices made in Latvia, EU. These include the Aranet CO₂ sensor (TDSPSPC005) with a range of up to 20,000 ppm and ±3% accuracy; Aranet temperature and humidity sensors (TDSPT509) with a range of −40 °C to +60 °C and ±0.3 °C accuracy; and a global radiation solarimeter (Aranet PAR sensor, TDSPAR02) with a range of 0 to 2000 µmol m⁻² s⁻¹ and ±5% accuracy. Additionally, the compartment is outfitted with state-of-the-art systems for lighting, cooling, drip irrigation, and ventilation, thereby optimizing the experimental conditions for research and cultivation.

2.2. Biological Sample, Treatments, and Cultivation Methods

The experiments were carried out over the two growing seasons during the months of January and February in 2023 and 2024 in an NFT (nutrient film technology) system. The experiment utilized two varieties of lettuce (Lactuca sativa L.)—Lugano and Carmesi—as biological material, with seeds that were certified for quality assurance. Seeds were sown in December and 8-day-old seedlings were transferred to jiffy growing blocks with 24 mm diameter peat pellets (Figure 1). Jiffy blocks are composed of steam-sterilized peat enriched with fertilizers and pH regulators, ensuring minimal seedling mortality and promoting vigorous and uniform growth for approximately two weeks. The nutrient solution was oxygenated using a SERA AIR 550 R PLUS pump (Sera, Heinsberg, Germany), (Figure 3a), which operates with low electricity consumption of 8 W, delivering an air flow rate of 9.2 L min⁻¹ and 552 L h⁻¹. Dissolved oxygen sensors (DOS) were installed at the inlet and outlet of each pipe. For continuous LED illumination (24 h) throughout the growing period, the experiment utilized a 100 W LED lighting system (Kathay Waterproof Plant Grow LED Crestere Plante 100 W) with IP67 water resistance, an AC 220 V input voltage, and an operating frequency of 50/60 Hz. It emitted full spectrum wavelengths from 380 to 840 nm.

2.3. Experimental Design and Statistical Analysis

The plants were arranged into 6 rows, each row accommodating 47 plants at a density of 3 m⁻². A spacing arrangement was diligently upheld, with a 30 cm distance between individual plants, as illustrated in Figure 1b. Rows 1 and 2 were provided with additional oxygen, while rows 3 and 4 were subjected to oxygen and light treatments. Rows 5 and 6 were assigned as the control group with natural oxygenation. A randomized complete block design (RCBD) with 4 rows (2 for each variety) as experimental and 2 rows (1 for each variety) as controlled groups was employed. Subplots consisted of 3 plants from each row, with genotypes (2 varieties and 3 replications) investigated to mitigate experimental error. The collected data were statistically analyzed by following the two-way ANOVA (analysis of variance) technique, employing post hoc and Tukey’s HSD test at the significance level (p < 0.05) among the group means (treatment groups) using IBM SPSS Statistics (version 29.0.2.0) software.

The difference between cultivars of main plots (experimental and control) was declared statistically significant at the confidence level of p < 0.05 for all parameters.

2.4. Data Collection

The study involved continuous monitoring of plant development, specifically focusing on diameter growth and leaf number progression.

2.4.1. Oxygen Concentration Calculation and Percentage Change

Dissolved oxygen sensors (DOS) were installed at the inlet and outlet of each NFT channel or gutter and the concentration was recorded accordingly (Figure 2). To determine the utilized oxygen concentration in the greenhouse hydroponic system, the following formula was adopted:

$$A=X-Z$$

where

• Utilized concentration = A;
• Oxygen concentration at inlet = X;
• Oxygen concentration at outlet = Z.

The results are expressed in milligrams per liter (mg L⁻¹).

To determine the percentage change in different attributes as a result of the applied oxygen treatments, the following formula was adopted:

$$PercentageChange={\displaystyle \frac{Finalvalue-Initialvalue}{Intialvalue}}\times 100$$

2.4.2. Growth Parameters

For accurate measurement, three well-lit plants from each group were selected to determine plant height, plant diameter, root length, plant mass, root mass, and root volume. Plant height (measured to the tallest leaf or stem) and root length were recorded from the base to the terminal point using a measuring tape. Following a 32-day cultivation period in the NFT system, the plants were harvested. The masses of different plant parts were assessed, and root lengths and volumes were measured using an EPSON Flatbed Expression 11,000X Scanner (Seiko Epson Corp., Suwa, Japan). Plant diameter was measured by selecting the two furthest points on the edges of the plant’s foliage, directly opposite to each other, with a measuring tape.

2.4.3. Physiological Parameters

The photosynthesis, respiration, and transpiration processes were assessed in a greenhouse setting using the automatic LC Pro+ analyzer (ADC Bio Scientific Ltd., Hoddesdon, UK) (Figure 3b). Results were quantified in µmol CO₂ m⁻² s⁻¹ for photosynthesis and respiration and in µmol H₂O m⁻² s⁻¹ for transpiration. Notably, a non-destructive research method was employed for determining photosynthesis intensity, utilizing a portable analyzer without leaf detachment from the plant.

2.4.4. Biochemical Parameters

Following a 32-day cultivation period, the biochemical components of the plants were quantified using the Chlorophyll Content Meter (002903) CCM-200 plus for chlorophyll contents, and the portable tester “The Greenest ECO” was used to assess the nitrate and sugar contents.

3. Results

Greenhouse microclimatic conditions play a crucial role in the overall growth of lettuce at different developmental stages. During the growing season, the average observed temperature fluctuated between 16.00 °C and 18.42 °C, carbon dioxide levels ranged from 600 to 653.47 ppm, and relative humidity varied between 56.20% and 58.50%. The observed minimum and maximum ranges were as follows: temperatures from 14.50 °C to 20.72 °C, relative humidity from 30.20% to 96.14%, and CO₂ levels from 410.60 to 858.28 ppm (

Table 1. Results of average, lowest, and highest values of temperature, relative humidity (RH), CO₂, and light in the greenhouse growing compartment.

Month	Week	Average			Minimum			Maximum
		Temperature (°C)	Relative Humidity (%)	CO2 (ppm)	Temperature (°C)	Relative Humidity (%)	CO2 (ppm)	Temperature (°C)	Relative Humidity (%)	CO2 (ppm)
December	1	17.50 ± 0.40	56.50 ± 0.40	600.00 ± 45.00	16.00 ± 1.80	35.30 ± 6.50	450.00 ± 81.00	20.40 ± 1.84	84.83 ± 3.20	768.52 ± 55.65
	2	17.30 ± 0.40	58.20 ± 3.30	650.00 ± 65.00	15.20 ± 1.85	36.50 ± 5.40	460.00 ± 75.00	20.72 ± 1.21	95.83 ± 6.21	858.29 ± 174.35
	3	16.20 ± 0.50	58.50 ± 2.70	650.00 ± 90.00	14.50 ± 0.65	30.20 ± 3.20	410.00 ± 25.00	20.46 ± 0.52	86.15 ± 3.86	825.96 ± 183.55
	4	16.00 ± 0.50	57.00 ± 2.50	640.00 ± 80.00	16.10 ± 1.92	34.50 ± 6.00	455.00 ± 135	20.53 ± 1.20	95.61 ± 5.75	817.92 ± 138.52
January	1	16.50 ± 0.47	57.90 ± 2.83	646.66 ± 78.33	15.26 ± 1.47	33.73 ± 4.87	441.66 ± 78.00	20.57 ± 0.98	95.86 ± 5.61	834.05 ± 165.47
	2	18.42 ± 0.39	56.20 ± 0.36	605.20 ± 46.69	16.14 ± 1.85	35.30 ± 6.63	455.16 ± 137	20.39 ± 1.84	81.83 ± 3.20	768.52 ± 55.65
	3	18.20 ± 0.39	58.20 ± 3.32	653.47 ± 65.63	15.37 ± 1.87	36.86 ± 5.42	463.82 ± 77.58	20.72 ± 1.21	85.82 ± 4.21	858.28 ± 174.35
	4	17.00 ± 0.54	58.50 ± 2.70	652.65 ± 88.82	14.66 ± 0.64	30.21 ± 3.24	410.60 ± 23.52	20.45 ± 0.52	96.14 ± 6.86	825.96 ± 183.55

).

Both significant and non-significant alterations in plant physiology, growth, and biochemical parameters of both lettuce varieties, Lugano and Carmesi, were observed in response to the different oxygen concentrations applied, including the natural oxygen concentration (NOC) level, the elevated oxygen concentration (EOC) level, and the light illumination + elevated oxygen concentration level (LED + EOC) (

Table 2. Response of physiological attributes (mean values ± SD) of lettuce varieties (Lugano and Carmesi) under natural oxygen concentrations (NOC), elevated oxygen concentrations (EOC), and LED + elevated oxygen concentrations (LED + EOC).

Plant Varieties	Oxygen Treatments	Photosynthetic Rate (µmol CO2 m−2 s−1)	Stomatal Conductance of (mol H2O m−2 s−1)	Transpiration Rate (µmol H2O m−2 s−1)	Temperature (°C)	Respiration Rate (µmol CO2 m−2 s−1)
Lugano	Natural oxygen concentration (NOC)	6.19 ± 0.22	0.11 ± 0.40	2.92 ± 1.37	32.13 ± 0.69	3.81 ± 1.40
	Elevated oxygen concentration (EOC)	7.16 ± 0.91	0.09 ± 0.75	2.70 ± 0.94	33.55 ± 1.13	3.46 ± 0.19
	LED + elevated oxygen concentration (LED + EOC)	7.11 ± 1.24	0.14 ± 0.72	3.96 ± 0.77	33.63 ± 1.12	4.54 ± 1.36
	Total (all treatments’ mean)	6.82 ± 0.84	0.11 ± 0.21	3.19 ± 1.39	33.10 ± 0.75	3.94 ± 0.43
Carmesi	Natural oxygen concentration (NOC)	6.25 ± 0.398	0.12 ± 0.35	3.10 ± 1.21	30.57 ± 1.11	3.91 ± 1.24
	Elevated oxygen concentration (EOC)	6.93 ± 1.18	0.05 ± 0.31	5.60 ± 1.01	34.00 ± 1.26	3.60 ± 0.62
	LED + elevated oxygen concentration (LED + EOC)	6.97 ± 1.24	0.10 ± 0.67	3.14 ± 1.13	32.35 ± 0.84	4.21 ± 0.71
	Total (all treatments’ mean)	6.64 ± 0.78	0.08 ± 0.69	3.68 ± 1.21	32.29 ± 0.54	3.75 ± 1.16
	Variety	0.182 ns	0.539 ns	0.365 ns	0.100 ns	0.9432 ns
	Treatment	0.044 *	0.432 ns	0.031 *	0.001 **	0.2335 ns
	Variety * Treatment	0.374 ns	0.813 ns	0.048 *	0.179 ns	0.8558 ns

p < 0.05 *, 0.01 **, 0.001 ***, ns = non-significant level.

). The intertreatment effect was also inconsistent, as plants reacted differently to both EOC and LED + EOC. Table 2 illustrates the mean values of various physiological parameters, photosynthesis, stomatal conductance, transpiration rate, and respiration rate along with temperature (°C), light wavelength (nm), and humidity (%) measured from the leaves of both varieties within the greenhouse. The light intensity received by the Lolo Bionta (Lugano) ranged from 463 (µmol m⁻² s⁻¹) to 495 (µmol m⁻² s⁻¹) with an average of 480 (µmol m⁻² s⁻¹), while it extended between 465 and 490 (µmol m⁻² s⁻¹) with an average of 478 (µmol m⁻² s⁻¹) in the case of Lolo Rossa (Carmesi) at all the three levels of oxygen (Figure 4). Elevated oxygen concentration treatments (EOC and LED + EOC) resulted in high photosynthetic activity, with rates of 7.16 to 7.11 µmol CO₂ m⁻² s⁻¹ in Lugano and 6.93 to 6.97 µmol CO₂ m⁻² s⁻¹ in Carmesi, compared to natural oxygen (NOC) rates of 6.19 and 6.25 mmol µmol CO₂ m⁻² s⁻¹, respectively. Stomatal conductance was found to be directly related to photosynthetic activity and inversely related to the applied oxygen concentrations. The values of stomatal conductance under increased oxygen concentrations varied between a minimum of 0.09 and 0.10 mol m⁻² s⁻¹ in Lugano, and 0.05 and 0.10 (mol m⁻² s⁻¹) in Carmesi, compared to the natural oxygen (NOC) values of 0.11 and 0.12, respectively. There was no statistically significant difference found among the groups regarding the stomatal conductance.

The transpiration rate appeared to be positively correlated in response to EOC in the case of Carmesi (5.60 µmol H₂O m⁻² s⁻¹), increasing by 80.74% compared to natural oxygen (3.10 µmol H₂O m⁻² s⁻¹). For Lugano, it increased by 35.61% in response to LED + EOC treatments. Transpiration decreased by 7.53% under EOC in Lugano. Temperature also varied among the different experimental conditions, with a slight increase observed in the EOC and LED + EOC treatments (34.00 °C and 32.35 °C, respectively) compared to NOC (30.57 °C). The respiration rate exhibited non-significant differences among the different treatments, despite a slight decrease in oxygen-treated plants, specifically in the EOC, 3.46, and LED + EOC, 3.46 µmol CO₂ m⁻² s⁻¹ conditions, compared to the NOC condition, 3.81 µmol CO₂ m⁻² s⁻¹ in Carmesi. The transpiration rate increased under LED + EOC treatment by 7.72%.

There was no significant relationship found among the varieties, treatments, or between the treatments and varieties with regard to the growth parameters. Intratreatment analysis also revealed that the effect of LED + EOC was more pronounced compared to EOC. A strong significant effect of oxygen concentrations (p < 0.001) was observed on plant total height (cm) in

Table 3. (a) Response of growth parameters (mean values ± SD) of lettuce varieties (Lugano and Carmesi) under natural oxygen concentrations (NOC), elevated oxygen concentrations (EOC), and LED+ elevated oxygen concentrations (LED + EOC). (b) Intertreatment statistical results on growth parameters of lettuce varieties” Carmesi” and” Lugano” under different oxygen treatments.

(a)
Plant Varieties	Oxygen Treatments	Plant Height (cm)	Plant Mass (g)	Plant Diameter (cm)	Number of Leaves	Root Mass (g)	Root Volume (cm3)	Root Length (cm)
Lugano	Natural oxygen concentration (NOC)	13.50 ± 0.79	47.65 ± 3.01	22.50 ± 180	10 ± 1.00	1.82 ± 0.49	1.86 ± 0.11	19.50 ± 0.56
	Elevated oxygen concentration (EOC)	15.66 ± 0.3	58.33 ± 1.95	23.66 ± 2.51	13 ± 1.15	3.24 ± 0.43	3.43 ± 0.02	24.83 ± 1.24
	LED + elevated oxygen concentration (LED + EOC)	15.80 ± 0.5	75.57 ± 2.66	26.66 ± 2.08	13 ± 1.73	4.36 ± 0.59	3.66 ± 1.15	35.93 ± 2.47
	Total (all treatments’ mean)	14.98 ± 1.22	54.23 ± 20.48	22.27 ± 2.27	12 ± 1.39	3.14 ± 1.18	2.98 ± 1.07	26.76 ± 0.57
Carmesi	Natural oxygen concentration (NOC)	18.30 ± 1.15	50.44 ± 0.65	26.36 ± 1.41	11 ± 1.61	3.20 ± 0.87	3.46 ± 0.64	21.27 ± 1.71
	Elevated oxygen concentration (EOC)	19.33 ± 1.10	55.72 ± 2.84	26.50 ± 1.32	12 ± 0.01	3.86 ± 0.44	4.30 ± 0.86	29.17 ± 0.67
	LED + elevated oxygen concentration (LED + EOC)	21.90 ± 1.20	67.74 ± 1.40	27.16 ± 2.75	13 ± 1.73	4.54 ± 2.05	4.07 ± 1.03	31.33 ± 0.17
	Total (all treatments’ mean)	19.84 + 1.89	57.38 ± 2.13	26.67 ± 1.72	12 ± 1.39	3.87 ± 1.27	3.53 ± 1.16	27.26 ± 1.41
	Variety	***	***	ns	ns	*	*	**
	Treatment	***	*	**	**	*	*	**
	Variety * Treatment	ns	***	ns	ns	ns	ns	*
(b)
All Oxygen Treatment Groups	Applied Oxygen Treatments	Plant Mass (g)	Plant Height (cm)	Plant Diameter (cm)	Number of Leaves	Root Mass (g)	Root Volume (cm3)	Root Length (cm)
Natural oxygen concentration (NOC)	× Elevated oxygen concentration (EOC)	*	***	ns	**	ns	ns	ns
	× LED + elevated oxygen concentration (LED + EOC)	***	***	**	**	**	*	***
Elevated oxygen concentration (EOC)	× Natural oxygen concentration (NOC)	*	***	ns	**	ns	ns	ns
	× LED + elevated oxygen concentration (LED + EOC)	ns	**	ns	ns	ns	ns	ns
LED + elevated oxygen concentration (LED + EOC)	× Natural oxygen concentration (NOC)	***	***	**	**	**	*	***
	× Elevated oxygen concentration (EOC)	ns	**	ns	ns	ns	ns	ns

In (a), p < 0.05 *, 0.01 **, 0.001 ***, ns = non-significant level. In (b), * Sign presented indicates how growth attributes of oxygen-treated plants are different from each other at three different levels (NOC, EOC, LED + EOC) in view of statistical approach at a confidence level p < 0.05 *, 0.01 **, 0.001 ***, ns = non-significant.

a and Figure 5a. In comparison to the NOC treatment, the plant height of both the Lugano and Carmesi species increased by 16.04% and 5.64% in response to the EOC treatment.

Additionally, under the LED + EOC treatment, the plants’ heights increased by 17.03% and 19.67%, respectively, Table 3a,b. The interactive effects between treatments and varieties on plant growth parameters appeared non-significant. The plant total mass (g) varied significantly among the varieties (p < 0.001). The total mass of the plant increased by 22.41% in Lolo Bionta (Lugano) under EOC compared to NOC. The plant diameter of both cultivars also showed dynamic behavior in response to applied oxygen concentration. Oxygen concentration accompanied by LED illumination does not appear as effective on plant diameter compared to EOC concentration. The final observation in Lugano showed an 18.48% increase in plant diameter under LED + EOC conditions compared to NOC. However, there was no significant difference in plant diameter between NOC and EOC conditions, with a slight decrease observed in the EOC-treated plants. Similarly, Carmesi showed a 0.53% increase in plant diameter under LED + EOC conditions compared to NOC but no significant difference in plant diameter between NOC and EOC conditions, with a slight decrease observed in the EOC-treated plants.

A statistically significant difference (p < 0.05) among the treatments in view of the number of leaves has been observed. Lugano exhibited the maximum increase in the number of leaves compared to Carmesi under both EOC and LED + EOC, respectively. The root mass, volume, and length also exhibited distinct and significantly different behavior in response to applied oxygen concentration levels (p < 0.05, p < 0.01, respectively). The root mass of Lolo Bionta (Lugano) increased by 77.69% and 139.48%, while that of Lolo Rosa (Carmesi) increased by 20.70% and 41.72% under EOC and LED + EOC, respectively, compared to NOC. The root volume of Lolo Bionta (Lugano) increased by 83.92% and 96.42%, while that of Lolo Rosa (Carmesi) increased by 24.03% and 28.84% under EOC and LED + EOC, respectively, compared to NOC.

The root length of Lolo Bionta (Lugano) increased by 27.35% and 84.27%, while that of Lolo Rosa (Carmesi) increased by 37.14% and 47.33% under EOC and LED + EOC, respectively, compared to NOC. A direct relationship was found among plant root length and the percentage changes in plant root mass and volume: the higher the plant root length, the higher the root mass and root volume. In terms of intertreatment response, Lolo Bionta (Lugano) exhibited a higher overall growth rate in both EOC and LED + EOC compared to Lolo Rosa (Carmesi). The treatment combining oxygen concentrations with LED light (LED + EOC) showed the highest growth performance compared to EOC and NOC. The effect of elevated oxygen concentrations alone appeared to have a non-significant impact on a few growth attributes.

The plants’ biochemical attributes also appeared with peculiar behavior as they significantly and non-significantly varied among the applied oxygen concentrations, see

Table 4. (a) Response of biochemical parameters (mean values ± SD) of lettuce varieties (Lugano and Carmesi) under natural oxygen concentrations (NOC), elevated oxygen concentrations (EOC), and LED + elevated oxygen concentrations (LED + EOC). (b) Intertreatment statistical results on biochemical parameters of lettuce varieties” Carmesi” and” Lugano” under different oxygen treatments.

(a)
Plant Varieties	Oxygen Treatments	Chl. at Leaf Base (mg kg−1)	Chl. at Leaf Center (mg kg−1)	Chl. at Leaf Upper Part (mg kg−1)	Nitrate at Leaf Base (mg kg−1)	Nitrate at Leaf Center (mg kg−1)	Nitrate at Leaf Upper Part (mg kg−1)	Glucose Contents
Lugano	Natural oxygen concentration (NOC)	2.92 ± 0.27	2.51 ± 0.25	2.18 ± 0.40	1133.26 ± 45.32	1157.83 ± 42.49	654.40 ± 141.40	3.64 ± 0.07
	Elevated oxygen concentration (EOC)	3.20 ± 0.86	2.85 ± 1.19	1.71 ± 0.75	1838.86 ± 41.92	1463.30 ± 47.3	286.63 ± 153.19	3.31 ± 0.18
	LED + elevated oxygen concentration (LED + EOC)	3.03 ± 0.48	2.14 ± 0.18	1.82 ± 0.72	2011.06 ± 49.87	1276.66 ± 41.72	531.10 ± 171.36	3.22 ± 0.01
	Total (all treatments’ mean)	3.05 ± 0.53	2.50 ± 0.68	1.90 ± 0.21	1661.06 ± 40.39	1132.93 ± 142.35	490.71 ± 121.53	3.39 ± 0.21
Carmesi	Natural oxygen concentration (NOC)	4.1 ± 0.18	6.06 ± 1.14	4.86 ± 0.35	1916.63 ± 61.61	515.53 ± 183.41	437.73 ± 138.64	3.82 ± 0.17
	Elevated oxygen concentration (EOC)	4.50 ± 1.08	4.78 ± 0.69	4.01 ± 0.31	1903 ± 52.01	675.50 ± 125.86	484.40 ± 157.12	2.91 ± 0.67
	LED + elevated oxygen concentration (LED + EOC)	4.31 ± 0.75	4.05 ± 1.78	3.59 ± 0.67	1165.53 ± 56.73	686.63 ± 165.34	677.73 ± 130.54	3.67 ± 0.23
	Total (all treatments’ mean)	4.84 + 1.05	4.96 ± 1.42	4.15 ± 0.69	1661.82 ± 43.71	625.88 ± 129.51	533.28 ± 121.16	3.47 ± 0.59
	Variety	**	***	***	ns	**	ns	ns
	Treatment	ns	ns	**	*	ns	ns	*
	Variety * Treatment	ns	ns	ns	***	*	*	*
(b)
All Oxygen Treatment Groups	Applied Oxygen Treatments	Chl. at Leaf Base (mg kg−1)	Chl. at Leaf Center (mg kg−1)	Chl. at Leaf Upper Part (mg kg−1)	Nitrate at Leaf Base (mg kg−1)	Nitrate at Leaf Center (mg kg−1)	Nitrate at Leaf Upper Part (mg kg−1)	Glucose Contents
Natural Oxygen Concentration (NOC)	× Elevated oxygen concentration (EOC)	ns	ns	*	Ns	*	ns	*
	× LED + elevated oxygen concentration (LED + EOC)	*	**	**	*	*	ns	**
Elevated Oxygen Concentration (EOC)	× Natural oxygen concentration (NOC)	ns	ns	*	Ns	*	ns	**
	× LED + elevated oxygen concentration (LED + EOC)	ns	*	ns	Ns	ns	ns	*
LED + Elevated Oxygen Concentration (LED + EOC)	× Natural oxygen concentration (NOC)	*	**	**	*	*	ns	**
	× Elevated oxygen concentration (EOC)	ns	*	ns	Ns	ns	ns	*

In (a), p < 0.05 *, 0.01 **, 0.001 ***, ns = non-significant. In (b), * Sign presented indicates how biochemical components of oxygen-treated plants are different from each other at three different levels (NOC, EOC, LED + EOC) in view of statistical approach at a confidence level of p < 0.05 *, 0.01 **, 0.001 ***, ns = non-significant.

a,b. The chlorophyll content at the base of the Lolo Bionta leaves (Lugano) increased by 9.83% and 17.47%, while Lolo Rosa (Carmesi) increased by 17.47–14.74% under EOC and LED + EOC, respectively, compared to NOC. A negative effect of oxygen has been observed on the leaf’s chlorophyll contents, measured at the center and upper part of the leaves. The chlorophyll content at the center in the case of Lollo Bionda (Lugano) increased by 13.67% under EOC and decreased by 14.74% under LED + EOC, while Lollo Rosa (Carmesi) decreased by 21.06% and 33.15% under EOC and LED + EOC, respectively, compared to NOC. No significant relationship was found between the applied oxygen concentration levels and natural oxygen concentration concerning the plants’ nitrate content at the base and upper part of the leaves. The nitrate content at the base of the leaves of Lollo Bionda (Lugano) increased by 62.26% and 77.45%, and decreased by 0.69% and 39.18% in the case of Lollo Rosa (Carmesi) under EOC and LED + EOC, respectively, compared to NOC. Meanwhile, the nitrate content at the center increased by 122.10% and 93.77% in the case of Lollo Bionda (Lugano), and by 31.02% and 33.18% in the case of Lollo Rosa (Carmesi) under EOC and LED + EOC, respectively, compared to NOC. The cultivar Lollo Bionda (Lugano) exhibited a negative response by decreasing nitrate content by 56.91% and 18.84%, while Lollo Rosa (Carmesi) showed an increase in nitrate content by 10.66% and 54.82% under both EOC and LED + EOC, respectively, compared to NOC.

The glucose content of both cultivars showed a small but significant difference (p < 0.05) to the applied oxygen concentrations. Both cultivars, Lugano and Carmesi, responded negatively by lowering the glucose concentration by 9.14% and 11.61%, and by 23.64% and 3.83%, respectively, under EOC and LED + EOC compared to NOC. The treatment effects within the groups also demonstrated a significant impact on the biochemical composition of the plants. The treatment of oxygen concentrations combined with LED light (LED + EOC) resulted in higher biochemical composition compared to the EOC and NOC.

4. Discussion

The impact of oxygen concentrations on lettuce (Lactuca sativa L.) involves a complex interplay of physiological processes that can significantly affect growth, yield, and overall plant health. Variations in oxygen levels significantly impact plant photosynthesis by intricately modulating light interception processes. There are several ways to apply oxygen to plants, including through the soil, via the roots, and through foliar application. Common methods include soil aeration, using air pumps in hydroponic systems, and applying oxygenated water. Enhanced oxygen levels in the root zone can significantly improve plant growth and productivity, support nutrient uptake, assist in respiration, reduce the risk of root diseases, and create a more favorable environment for beneficial microorganisms [42]. Oxygenation of the nutrient solution, coupled with specific light treatments, also plays a critical role in optimizing plant growth and development. It has been reported that dissolved oxygen (DO) concentrations above 6 mg L⁻¹ significantly improve root respiration and nutrient uptake efficiency [43]. In addition, a study reported an increase in net photosynthetic activity in response to oxygen concentrations of 6.5, 7.5, and 8.5 mg L⁻¹ in the lettuce variety Grand Rapids Tbr [44]. Understanding and managing oxygen dynamics in the NFT system is pivotal for optimizing plant productivity and ecological sustainability.

Stomatal conductance refers to the rate at which stomata, the tiny pores on the surface of leaves, open and close to regulate gas exchange—particularly water vapor and carbon dioxide—between the plant and its environment. In the present study, stomatal conductance exhibited a non-significant response to the applied oxygen concentration, consistently showing low values. The possible explanation for this decrease in stomatal conductance is the higher rates of respiration, which can lead to stomatal closure. According to a study, the closure of stomata—attributed to high respiration rates in response to low photosynthesis rates—is observed as a strategy that plants adopt to conserve water and mitigate excessive water loss through transpiration [45]. It has also been observed that different oxygen concentrations influence cellular metabolism and the production of hormones and signaling molecules within the plant, which in turn can affect stomatal conductance. For example, ethylene, a plant hormone involved in stress responses, can modulate stomatal behavior in response to environmental cues [46]. In the present study, applied oxygen concentrations appeared to increase the transpiration rate in both cultivars. This increase can be correlated with optimal ranges, as a study reported that under optimal oxygen levels, root cells can efficiently conduct respiration by maintaining metabolic functions, which provide energy for transpiration and other physiological processes [47,48]. Some plant species, including certain types of lettuce, develop aerenchyma tissue in their roots to facilitate oxygen transport to submerged tissues, helping to mitigate the effects of hypoxia or anoxia [49]. Also to note, light is another important factor influencing lettuce plant physiology. Higher light intensities can enhance the photosynthetic rate, leading to increased biomass and yield. For instance, a study found that light intensities of 350 to 600 µmol m⁻² s⁻¹ improved photosynthesis capacity and yield in lettuce under different temperature conditions [50]. Additionally, high light intensity applied shortly before harvest has been shown to improve the nutritional quality of lettuce, increasing dry matter, ascorbic acid, and carbohydrate content, which extends shelf life and enhances postharvest performance [51].

Oxygen and light are essential for plant growth and development, providing energy and signals. Light quality, intensity, and the photoperiod significantly influence plant morphology, photosynthesis, nutrient content, and antioxidant capacity, thereby impacting yield and quality. Elevated dissolved oxygen levels in the nutrient solution are essential for enhancing plant physiological processes. Increased oxygen availability boosts root metabolic activities and nutrient absorption, resulting in greater plant height, thicker stems, and a higher leaf area index (LAI) in species like lettuce [52]. In the present study, 17.03% and 19.67% increases in plant height have been observed in response to the EOC and LED + EOC in both species, Lugano and Carmesi, respectively. Contrary to this, a study observed no significant difference in plant fresh weight, shoot, and root dry weights among the DO concentrations: 2.1 (25% of saturated at 24 °C), 4.2 (50%), 8.4 (saturated), and 16.8 (200%) mg L⁻¹ [53]. A study reported that as light intensity increases from 60 to 400 µmol m⁻² s⁻¹, lettuce plants exhibit increased leaf numbers, shoot fresh and dry mass, and plant height; however, beyond a certain saturation point (around 400 µmol m⁻² s⁻¹), the benefits plateau and do not further enhance growth significantly [44,54]. Adequate oxygen levels are associated with higher yields and better-quality produce, characterized by improved texture, flavor, and nutritional content. A study found that lettuce grown in well-aerated conditions produced significantly higher yields with better marketable quality compared to those grown in low-oxygen environments [55]. Oxygen deficiency near the roots is a critical factor affecting plant health [21]. Insufficient oxygen can impair root system development and lead to root deterioration, potentially causing plant desiccation [50]. In hydroponic systems, maintaining high dissolved oxygen (DO) levels is essential for promoting healthy root respiration [56]. Adequate oxygen levels promote healthy root growth and function, while hypoxic conditions (low oxygen) can lead to root hypoxia, affecting nutrient uptake and overall plant growth. Optimal DO supports root function and nutrient absorption, contributing to robust plant development. Similar to the present study, refs. [57,58] found that transgenic lettuce plants exposed to higher oxygen concentrations under LED light exposure exhibited increased root mass compared to control plants. The effect of oxygen treatments on root volume and length may vary based on the specific experimental conditions (Figure 6). Healthy roots facilitate better uptake of water and essential nutrients, which are crucial for plant development and overall leaf physiology. Research by [59] demonstrated that lettuce plants grown under hypoxic conditions showed a reduced uptake of nitrate and ammonium, leading to lower biomass production and poor quality. Another study has shown that reduced oxygen levels in the root zone can impair root respiration, leading to reduced energy production, slower root growth, and compromised nutrient uptake [60]. When considering the distribution of these biochemical components (sugar content, chlorophyll levels, and nitrate accumulation) across different parts of the leaf (base, center, and top), it is important to note that the distribution may not be uniform due to variations in light exposure, vascularization, and physiological gradients within the leaf [61]. In the present study, we observed more chlorophyll content at the base of the leaf in the case of Lugano, while Carmesi appears to have the maximum chlorophyll content at the center. According to [62], lettuce plants exposed to optimal oxygen levels exhibited higher chlorophyll content and better photosynthetic performance compared to those grown under low-oxygen conditions.

Higher light intensity boosts levels of soluble sugar, soluble protein, and carotenoids in lettuce. It has also been reported that extended illumination time enhances nitrogen (N) and phosphorus (P) utilization efficiency and improves lettuce quality [63]. The level of nitrates serves as a crucial indicator of the quality of leafy greens. Improved quality is linked to lower nitrate levels [64]. Results revealed that short-term exposure of lettuce varieties to half the nitrogen supply and increased light intensity prior to harvest is suited to produce high-quality lettuce with a low nitrate content [54,65]. High light intensities should be balanced to avoid potential light-induced stress while ensuring sufficient oxygen supply in the root zone to maximize photosynthetic efficiency and plant health. For growers using NFT systems within the greenhouse conditions, managing greenhouse microclimatic conditions is crucial to grow different vegetables [66,67,68]. Dissolved oxygen levels in water affect root respiration and nutrient uptake in hydroponic systems, influencing plant metabolic processes like glucose production. The light quality from the LEDs further modulates photosynthesis and carbohydrate metabolism in lettuce, impacting glucose concentrations [69,70]. In the present study, the applied concentrations of oxygen and light treatments have significantly influenced (p < 0.01) the glucose contents of both varieties. This study unveiled the potential for greenhouse lettuce cultivation while identifying the ideal levels of dissolved oxygen and light intensity conducive to enhancing lettuce growth quality and efficiency. These findings offer a fresh perspective for researchers interested in exploring quality improvement avenues. This approach can lead to higher yields, better nutritional quality, and improved postharvest performance of lettuce.

5. Conclusions

This study conclusively demonstrates that elevated oxygen concentrations (EOC) and the combination of EOC with LED light exposure significantly influence the physiological, growth, and biochemical parameters of lettuce varieties Lolo Bionta (Lugano) and Lolo Rosa (Carmesi) under greenhouse conditions. It has been concluded that the cultivar Lolo Bionta (Lugano) appeared with maximum horticultural performance under both EOC and LED + EOC treatments as compared to Lolo Rosa (Carmesi). Lugano’s plant height increased by 16.04% under EOC and 0.85% under LED + EOC, fresh mass by 110.91% and 29.55%, total leaves by 26.66% and 2.63%, diameter by 5.18% and −12.67%, root length by 27.35% and 29.55%, volume by 83.92% and 6.79%, and root mass by 77.69% and 34.77%, respectively. Carmesi’s plant height increased by 5.64% under EOC and 13.27% under LED + EOC, fresh mass by 10.45% and 21.57%, total leaves by 15.15% and 2.63%, diameter by 0.50% and 2.51%, root length by 37.14% and 47.33%, volume by 24.03% and 28.84%, and mass by 20.70% and 41.72%, respectively.

Plant physiology and biochemical assays also displayed a dynamic nature with both significant and non-significant responses (p < 0.05) across oxygen concentrations, with LED + EOC having more pronounced effects than EOC alone. The applied light intensity, measured as PPFD (µmol m⁻² s⁻¹), had a significant impact on photosynthetic activity. Both the EOC and LED + EOC treatments led to an increase in photosynthetic rates compared to NOC, ultimately resulting in enhanced growth parameters. Transpiration rates increased significantly in Carmesi under EOC and LED + EOC, while respiration rates decreased under elevated oxygen compared to NOC. Chlorophyll content increased at the leaf base under EOC and LED + EOC but decreased at the center and the upper leaves. Nitrate content varied with oxygen concentration and leaf position, while glucose concentration decreased under elevated oxygen compared to NOC. These findings underscore the importance of optimizing oxygen levels and incorporating LED lighting in hydroponic systems to maximize crop yield and quality, thereby offering valuable insights for sustainable greenhouse cultivation practices.