Impact of Zeolites on Growth Dynamics of Medicago sativa and Lactuca sativa in Hydroponics

TL;DR

Natural zeolite improves hydroponic plant growth by enhancing moisture and nutrient retention compared to commercial substrates.

Contribution

Demonstrates natural zeolite's efficacy as a hydroponic substrate through growth and metabolic analysis of specific plant species.

Findings

Zeolite has a significantly higher specific surface area (21.80 m2/g) than the control (0.49 m2/g), improving moisture and cation exchange.

Zeolite promotes better root development and seedling viability in Medicago sativa compared to artificial substrates.

GC–MS analysis shows zeolite induces metabolic changes in Lactuca sativa, including increased fatty acids and genotype-specific osmoregulation.

Abstract

This study evaluates the effectiveness of natural zeolite (Shankhanai deposit, Kazakhstan) as a functional hydroponic substrate compared to a commercial foamed-glass control (GrowPlant). Using the Nutrient Film Technique (NFT), we assessed the growth and metabolic responses of Medicago sativa L. and three cultivars of Lactuca sativa L. Brunauer–Emmett–Teller (BET) analysis confirmed that zeolite (particle size 3.70 ± 1.20 mm) possesses a high specific surface area (21.80 m2/g), significantly exceeding the control (0.49 m2/g). This structure ensured superior moisture retention and cation exchange, even after a moderate decrease in surface area to 16.66 m2/g post-cultivation due to organic pore-filling. In M. sativa experiments, zeolite increased seedling viability and promoted a more branched root system compared to the artificial substrate. Gas chromatography–mass spectrometry (GC–MS) metabolic profiling of L. sativa revealed a significant substrate-driven reprogramming: zeolite increased the relative proportion of fatty acids and their derivatives (up to +51.27% in May King variety roots), suggesting membrane-protective adaptation. Genotype-specific responses were observed, with the Yeralash cultivar showing increased polyol synthesis (+2.93%) for osmoregulation. The results demonstrate that natural zeolite is an efficient, stable substrate for intensive hydroponics, optimizing root development and physiological stability through enhanced nutrient and water management.

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1. Introduction

Food security in arid and semi-arid regions, such as Kazakhstan, faces critical challenges due to climate change, water scarcity, and soil degradation [1,2,3,4]. Under these conditions, the transition to closed hydroponic systems—specifically vertical farming—has become economically and technically viable, supported by advancements in automated nutrient management and LED (light-emitting diode) lighting [5,6,7,8,9]. While hydroponics optimizes water use and eliminates soil-borne pathogens, the environmental footprint of conventional substrates like mineral wool or peat remains a concern due to disposal issues and ecosystem disruption [10,11].

The search for sustainable, functional substrates has led to increased interest in natural minerals, particularly zeolites. Zeolites are three-dimensional aluminosilicate frameworks composed of SiO_4_^4−^ and AlO_4_^5−^ tetrahedra, forming a system of micropores with a high specific surface area and significant cation-exchange capacity [12,13,14,15,16,17,18,19,20,21]. Unlike inert synthetic media, zeolites act as active nutrient carriers and buffers. They enhance the retention of ammonium and nitrate ions, stabilize substrate pH and electrical conductivity (EC), and optimize water availability in the root zone [18,19]. By effectively adsorbing ammonium ions, this substrate acts as a nutrient reservoir, improving the overall availability and assimilation of minerals for the developing plants [16,17]. These ion-exchange properties make zeolite an ideal candidate for Nutrient Film Technique systems, where maintaining a stable nutrient concentration in a thin flowing film is critical for plant performance [18,19,20,21,22]. However, further research is required to evaluate their potential in intensive and organic cultivation technologies [23].

In this study, alfalfa (Medicago sativa L.) and lettuce (Lactuca sativa L.) were selected as model crops to evaluate the efficiency of natural zeolite. M. sativa was utilized as a model legume to evaluate the ion-exchange influence [24,25] of clinoptilolite on nitrogen-fixing species in a controlled hydroponic environment. Lettuce is a globally significant leafy vegetable known for its sensitivity to nutrient imbalances and salinity [26,27,28,29,30,31]. By comparing a natural zeolite substrate (Shankhanai deposit, Kazakhstan) against a commercial foamed-glass benchmark (GrowPlant), this research aims to determine the effect of substrate mineralogy on growth parameters and the qualitative composition of volatile metabolites.

2. Materials and Methods

2.1. Water-Holding Capacity Test

Natural sorbents utilized in this study included zeolite and plant-based activated carbon. To assess moisture capacity and select the optimal substrate, a water-holding capacity test was conducted on three materials: fine-fraction zeolite (FZ, average particle size 0.50 ± 0.20 mm), coarse-fraction zeolite (CZ, 3.70 ± 1.20 mm), and plant-based activated carbon (AC, 2.50 ± 0.50 mm) [32,33]. Testing was performed at temperatures of 22 °C and 50 °C. A controlled volume of 5 g of distilled water was applied to 20 g of each substrate, ensuring even distribution across the surface.

2.2. Granulometric Analysis

The particle size distribution of the CZ was determined through mechanical dry sieving using a set of standard laboratory sieves. The analysis followed the Kachinsky method classification [34,35,36,37]. A representative sample of the substrate was separated into three fractions: large (4.5–5.0 mm), medium (3.0–4.5 mm), and micro-fraction (2.5–3.0 mm). The mass of each fraction was measured using an analytical balance with a precision of 0.01 g, and the percentage distribution was calculated relative to the total sample mass.

2.3. Hydroponic System

This study utilized a Reogen Systems Nutrient Film Technique (NFT) (Reogen Systems, Kirov, Russia) hydroponic system, providing continuous circulation of the nutrient solution. The solution flows from a reservoir through inclined channels, ensuring a thin film of nutrients in direct contact with the roots while maintaining high aeration [38]. This system ensures a uniform supply of nutrient solution through inclined channels in which the plants are placed. Such a design promotes continuous hydration of the root system while maintaining a high level of aeration. An inverted NFT configuration may increase the risk of nutrient imbalance due to the influence of gravity on solution flow. A controlled system helps maintain balanced nutrient concentrations in the root zone, preventing issues such as nutrient lockout or toxicity [32]. A schematic representation of the Reogen Systems NFT hydroponic unit is presented in Figure 1.

The setup includes six horizontal racks (150 × 50 cm) under artificial illumination provided by five linear LED modules (110 W, 11,000 Lm each) per rack. The lamps were mounted on aluminum radiators at a fixed height of 30 cm above the substrate surface. The total luminous flux was 55,000 Lm (≈73,334 Lx) per shelf, providing a high-intensity broad-spectrum light (5000–6500 K). This spectral composition, characterized by a high blue-light fraction, was chosen to ensure robust vegetative growth and representative physiological responses in both L. sativa and M. sativa. The photoperiod and irrigation schedule were adjusted according to the requirements of the cultivated plants using a programmable timer.

Each rack accommodates 20 planting cups with a volume of 100 mL. Nutrient balance was maintained using a two-component commercial N-P-K fertilizer, Hydro A and Hydro B (Bertels B.V., Ospel, The Netherlands), designed for hydroponic growth and flowering phases. Fertilizers were applied at a 1:400 ratio (2.5 mL of each component per 1 L of distilled water). The solution was applied every time during watering according to the instructions. The chemical composition of Hydro A (3-0-1): N—3.1%, K_2_O—2.0%, CaO—6.7%, pH—5.0–5.5. The chemical composition of Hydro B (1-3-6): N—1.5%, P_2_O_5_—3.4%, K_2_O—6.2%, M_9_O—1.6%, pH 2.6–3.2.

The experiment was conducted under controlled laboratory conditions with an air temperature of 22–24 °C, atmospheric pressure of 760 mmHg, and humidity of 40–60%. The hydroponic system utilized a 90 L reservoir. To compensate for evapotranspiration and maintain stable nutrient concentrations, the water level was replenished weekly to its initial volume. Throughout the growth cycle, solution parameters were strictly monitored: for alfalfa, the pH was maintained within the range of 5.5 to 6.5 with an EC of 1.5 to 2.5 mS/cm [25,31]; for lettuce, the pH was maintained between 6.0 and 7.0 with an EC of 1.4 to 1.8 mS/cm [30].

2.4. Experimental Plants and Substrates

Plants. To evaluate the effect of zeolites on plant growth, two experiments were conducted using two types of agricultural crops—alfalfa (M. sativa) and lettuce (L. sativa). The seeds were pre-treated in a 1% potassium permanganate solution for 30 min, then rinsed with distilled water.

For the 2nd experiment, three lettuce cultivars with distinct morphological characteristics were selected:

• (1)American Brown (AB): a loose-leaf Batavia variety known for its bronze-red, crinkled leaves during ripening.
• (2)Yeralash (Y): a looseleaf type with strongly wavy/frilly edges, forming a dense rosette.
• (3)May King (MK): a butterhead variety with soft, light- to mid-green leaves.

Lettuce seeds were initially germinated in Petri dishes. Healthy seedlings were then transplanted into individual containers and subsequently thinned to a final density of 3–4 plants per pot to ensure uniform growth conditions.

The photoperiod was adjusted according to the specific physiological requirements and commercial cultivation guidelines provided by the seed suppliers for each crop and growth stage. Specifically, a 16/8 h (light/dark) cycle was maintained during the initial germination phase to prevent etiolation, followed by a gradual reduction to 14/10 h and 12/12 h to simulate natural seasonal transitions and optimize biomass accumulation. The lighting regime and watering schedule are demonstrated in Table 1.

Substrates. The experimental substrates are shown in Figure 2. To evaluate the properties of natural zeolites (Shankhanai deposit, Kazakhstan) as a growing medium (Figure 2a), an artificial foamed-glass substrate (GrowPlant, ICM Glass, Moscow, Russia) was selected as a control (Figure 2b). The choice of GrowPlant (GP) was determined by the technical recommendations of the Reogen Systems NFT hydroponic unit manufacturer. Utilizing the recommended medium ensured optimal system performance and mitigated the risk of clogging irrigation pipes—a common technical failure associated with more friable substrates such as perlite or coconut fiber in this specific equipment. GP is commercially recognized as a highly efficient alternative to perlite, expanded clay, and pumice.

Preparation and Sterilization. Prior to the experiment, both substrates were thoroughly washed with distilled water to remove fine dust and impurities. Subsequently, the materials were thermally disinfected in a drying oven at 100 °C for 1 h to ensure a sterile growing environment and eliminate potential pathogens.

2.5. Brunauer–Emmett–Teller Analysis Methodology

The porous structure and surface characteristics of the substrates were analyzed using the Brunauer–Emmett–Teller (BET) method. BET analysis is commonly applied to determine the surface area of nanoporous materials, such as metal–organic frameworks and zeolites. Despite some limitations, mainly related to the quadrupole moment of the nitrogen molecule, nitrogen adsorption measurements provide valuable insight into the pore architecture of zeolites and remain a widely used approach for characterizing synthetic zeolite materials [39]. The BET theory is based on the physical adsorption of gas molecules on solid surfaces, which occurs through van der Waals interactions formed by an adsorbate film composed of atoms, ions, or molecules [40].

Prior to analysis, all samples were dried and ground using a Kubo X1000 (Beijing Biaode Instruments Co., Ltd., Beijing, China) mill to ensure uniform particle size. Approximately 0.60 g of each substrate was weighed and degassed at 300 °C for 3 h under vacuum to remove moisture and volatile impurities. Nitrogen was used as the adsorbate (molecular cross-sectional area: 0.162 nm^2^). Adsorption–desorption isotherms were recorded, and the resulting data were processed to determine the BET-specific surface area.

2.6. Gas Chromatography–Mass Spectrometry Analysis Methodology

In this study, gas chromatography–mass spectrometry (GC-MS) was used for semi-quantitative screening of volatile compounds.

Sample Preparation. The aerial biomass was air-dried at room temperature for seven days until a constant weight was achieved. The dried plant material was then homogenized into a fine powder using a porcelain mortar and pestle. Subsequently, 96% ethanol was used for the extraction of volatile and semi-volatile compounds.

The GC-MS analysis was conducted at the Center of Physicochemical Methods of Research and Analysis of Al-Farabi Kazakh National University (CFKhMA, Al-Farabi KazNU, Almaty, Kazakhstan). Organic compounds were analyzed using a 7890A/5975C GC-MS system (Agilent Technologies Inc., Santa Clara, CA, USA). A 0.5 µL sample was injected in splitless mode at an injector temperature of 280 °C. Separation was achieved on a DB-17ms column (30 m × 0.25 mm × 0.25 μm) with helium as the carrier gas at a constant flow rate of 1 mL/min. The oven temperature program was initiated at 40 °C, increasing by 5 °C/min to 300 °C (10 min hold). Mass spectra were recorded in SCAN mode in the m/z range of 34–750. No derivatization was performed. Despite this, the chosen GC-MS parameters allowed for a robust semi-quantitative comparison of major volatile and semi-volatile chemical classes between the experimental and control groups.

Compounds were identified by comparing their mass spectra and retention times with the Wiley 11th Ed. and NIST 2002 libraries (totaling 550,000 spectra). The chromatograph–mass spectrometry system was controlled, and data were collected and processed using Agilent MSD ChemStation software (version 1701EA). Post-run, the identified compounds were categorized into functional chemical classes (lipids, carbohydrates, etc.) based on their chemical structures to evaluate the overall metabolic profile of the plants. Compounds were categorized into functional classes based on the PubChem and MeSH database ontology. GC–MS tests were performed on plant leaves and roots separately.

2.7. Statistical Analysis and Biometric Measurements

Statistical analysis was performed using Microsoft Excel 2021. The data obtained were subjected to statistical analysis using Student’s t-test and one-way analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05 (marked as * in tables and figures), p < 0.01 (), and p < 0.001 (*).

For substrate physical properties (water-holding capacity), the variability was expressed as Standard Deviation (SD). All substrate physical property tests, including water-holding capacity, were performed in triplicate (n = 3).

For the plant biological quality indicator (germination rate) and biometric indicators (stem height, root length, and leaf count), the data were presented as Mean ± Standard Error of the Mean (SEM) to account for the precision of the estimated means. For plant growth experiments, each treatment group consisted of 20 independent replicates (20 individual containers per substrate type). Monitoring of germination was conducted at regular intervals. For alfalfa, observations were recorded on days 3, 5, 6, 10, 12, and 14. For lettuce varieties, measurements were performed on days 3, 5, 7, 9, 12, and 15.

To maintain a consistent plant density and minimize the ‘edge effect’ during the experiment, biometric parameters were assessed using a random sampling method. In each measurement period, 7–10 representative plants were randomly selected from each group for destructive and non-destructive analysis. Plants were carefully removed from the substrate to preserve the integrity of the root system. Stem height and root length were determined using the grid-paper method: specimens were placed on standardized graph paper (1 mm precision grid) and photographed for subsequent analysis.

3. Results and Discussion

3.1. Substrate Characterization

3.1.1. Physical Properties of Zeolites

The evaporation dynamics (Figure 3) revealed that at 22 °C, all tested materials maintained similar moisture retention for the initial 30 min. By the 120th min, divergent trends emerged: the CZ (3.80 ± 0.02 g) demonstrated superior moisture evaporation compared to the FZ (4.22 ± 0.01 g) and AC (4.53 ± 0.04 g). At 50 °C, the evaporation behavior changed: AC showed the highest evaporation rates at 50 °C (1.39 ± 0.01 g), while the CZ maintained a rate similar to its performance at 22 °C (3.69 ± 0.01 g). In contrast, the retention of FZ at 50 °C was 26% lower compared to its room temperature parameters (3.12 ± 0.01 g)

By the end of the experiment, CZ exhibited moderate moisture loss (1.94 ± 0.04 g at 50 °C and 3.73 ± 0.04 g at 22 °C) among all three substrates. While AC maintained a relatively stable water-holding capacity at room temperature (4.42 ± 0.01 g), it showed the most rapid evaporation at 50 °C (1.02 ± 0.01 g). Nevertheless, given its balanced moisture characteristics, availability, and structural properties, the coarse zeolite was selected for further hydroponic seed germination experiments. In this study, AC served primarily as a reference material to evaluate the influence of zeolite particle size (coarse vs. fine).

The selection of CZ was further justified by granulometric analysis using the Kachinsky method [40], which demonstrated an optimal particle-size distribution: large fraction—30%, medium—40%, and micro-fraction—30%. This composition is a key factor in substrate performance, significantly influencing the air-thermal and water-physical properties required for effective hydroponic cultivation.

3.1.2. BET Surface Area

The results of the BET analysis, shown in Figure 4a–c, confirmed the distinct structural differences between the media. The artificial GP substrate exhibited a very low specific surface area (0.49 m^2^/g). This is attributed to its macro-porous, closed-cell structure, which, while providing aeration, offers minimal internal surface for ion exchange. Furthermore, the mechanical grinding required for BET analysis may lead to the collapse of these macropores, further reducing the measurable area. In contrast, the natural zeolite was characterized by a considerably higher surface area (BET 21.80 m^2^/g) due to its inherent micro-porous crystalline lattice. This provides a significant area for water retention and ion adsorption. Even after the experiment, despite partial pore occupancy by root exudates or biofilm (where the BET area decreased to 16.66 m^2^/g), the zeolite retains a significant capacity to maintain moisture and nutrients for the plants.

This partially filled pore structure acts as a buffer: the zeolite retained excess water and prevents stagnation, while also ensuring the gradual release of nutrients into the root zone, reducing the frequency of watering and preventing sudden fluctuations in humidity. This provides plants with a more stable water supply, which is especially important in conditions with limited water resources. There seems to be a uniform porosity, which prevents substrate compaction and ensures oxygen flow to the roots, promoting more vigorous growth. As a result, zeolite simultaneously reduces water consumption and improves the physiological stability of plants in hydroponic systems [40,41].

3.2. Experiment 1: Growth Dynamics of M. sativa

Both sample sprouts emerged on the 6th day. Zeolite demonstrated significantly higher growth rates compared to GP. Seed germination rate is presented in Figure 5.

Owing to GP’s high porosity and low water-holding capacity, the substrate dried out rapidly, requiring frequent, abundant irrigation. The low specific gravity facilitated faster seedling emergence (the average height on the day of emergence was 3.47 ± 0.40 cm) but also caused partial seed washing during watering. Germination by day six was only 15.57 ± 1.22%, markedly lower compared to zeolite. Visual abnormalities in the plants’ condition were noted: the leaves were a less intense green color, with some yellowing. Due to the low weight of the artificial substrate and its macroporous structure, the stems developed more convoluted root systems that penetrated deeply into the material, which would complicate the subsequent reuse of the substrate.

Zeolite demonstrated a significantly high germination rate from the start of the experiment. Physical properties of the zeolite substrate, specifically its high bulk density and mechanical resistance, influenced the initial growth dynamics of the seedlings. The average height of the plants in the zeolite group was 1.66 ± 0.21 (p < 0.05), which was significantly lower compared to the control group. This indicates that this difference is statistically significant, confirming that the substrate density exerted a measurable limiting effect on vertical elongation during the early stages of development.

The surface layers of the zeolite quickly lost moisture, but the lower portion of the pot, located in the shade, retained it longer, providing more stable moisture conditions compared to the artificial substrate. This avoided overwatering and drying out, typical of GP. Watering every two hours for 5 min proved to be most suitable for this environment.

Plant growth indicators are presented in Table 2 and Figure 6. Detailed photographic documentation of seedling growth, including shoot height and root and stem length measurements, are presented in Supplementary Materials Section S1.1 Figure S1.

The development of M. sativa seedlings showed distinct phenotypic variations depending on the substrate type. Visual observations on the final day of monitoring (Figure 6) highlighted a deeper green coloration in the zeolite-grown seedlings, correlating with the metabolic shifts observed in the later stages. Detailed photographic records of the intermediate growth stages can be found in Supplementary Materials Section S1.1 Figure S2.

3.3. Experiment 2: Growth Dynamics of L. sativa

Three L. sativa varieties showed different responses to zeolite addition. Seed germination rates are shown in Figure 7.

The lettuce cultivar ‘American Brown’ (AB) exhibited a distinct germination pattern characterized by a prolonged lag phase, with no emergence recorded until day 9. This suggests a higher dormancy threshold or a more sensitive response to the initial moisture-tension conditions of the substrate. However, once germination commenced, the zeolite treatment showed a statistically superior performance, reaching 33.15 ± 2.50% (p < 0.001) compared to 29.90 ± 1.50% in the GP. This significant improvement indicates that the Shankhanai zeolite may mitigate early-stage germination stress. The porous structure of the zeolite likely creates a more favorable micro-environment by optimizing water-holding capacity and providing a stable mineral interface, which is particularly beneficial for ‘slow-starting’ cultivars like AB. The data suggest that while the genetic traits of the cultivar dictate the timing of germination, the substrate quality can effectively modulate and enhance the final germination percentage even in less vigorous varieties.

The cultivar ‘May King’ (MK) exhibited the highest germination vigor among all studied varieties, with emergence exceeding 32% by day 3 in both treatments. Starting from day 5, zeolite demonstrated a highly significant stimulatory effect on the germination percentage (p < 0.001). This rapid ‘start-up’ effect suggests that the mineral interface of the zeolite may optimize the seed-to-moisture contact area, accelerating the transition from imbibition to radicle emergence. For instance, it has been demonstrated that the application of zeolite significantly influences the micro-morphology of plants, reaching as far as the perimeter of exine perforation zones in Brassica napus L. pollen grains [41].

The cultivar ‘Yeralash’ demonstrated an intermediate germination pattern. On the third day, both groups showed comparable emergence rates of approximately 25%. However, a distinct divergence was observed starting from day 5, with the zeolite substrate maintaining a highly significant lead over the control (p < 0.001). By day 7, the germination percentage reached 56.10 ± 6.90% (p < 0.001) in the zeolite group versus 53.26 ± 6.60% in the GP group. The significance level starting from the fifth day underscores the reliability of the zeolite’s stimulatory effect on this genotype, reinforcing its role as an active growth modulator in NFT systems.

Interestingly, the stimulatory effect of the zeolite substrate was predominantly observed in the germination dynamics and final emergence percentage (p < 0.001 for all three cultivars). In contrast, the subsequent morphometric parameters, such as stem height and root length, did not show statistically significant differences (p > 0.05). This divergence suggests that the Shankhanai zeolite acts primarily as a catalyst for seedling establishment. Once the plant transitions to the autotrophic phase, the growth rates stabilize and align with the genetic potential of the cultivar.

Observations of average stem, root, and leaf heights and lengths are presented in Table 3.

Visual assessment on the final day revealed notable phenotypic differences between the treatment groups (Figure 8). While the L. sativa seedlings in the GP group showed typical elongation, the plants grown on the Shankhanai zeolite exhibited a more compact architecture and more intense leaf coloration.

3.4. GC–MS Analysis of Metabolites in L. sativa

Distribution of metabolic classes in leaves and roots of L. sativa is presented in Figure 9a–c. A more detailed breakdown of individual compounds is provided in the Supplementary Materials Section S2 Table S1.

For the American Brown cultivar, the difference in metabolic profile between the GP and zeolite substrates is most pronounced in lipid and terpenoid metabolism, especially in the leaves. Zeolite is associated with a shift in the relative proportion of metabolites, notably increasing the lipid component. In the leaves of the American Brown variety grown on zeolite, a relative increase in fatty acid (FA) content (+15.37%) and their derivatives (+5.46%) was observed. According to recent studies on lettuce extracts [40], these lipid components are essential functional ingredients. This shift potentially suggests a physiological response involving the reinforcement of cell membranes or a modification in energy balance. Simultaneously, a decrease in the relative area of sugars (–4.84%) was recorded, which may indicate a diversion of metabolic resources toward lipid synthesis under zeolite conditions. As established by Lin et al., carbohydrate composition is a primary determinant of vegetable quality, where the proportion of soluble sugars directly influences the taste profile of hydroponically grown lettuce [42,43]. The observed shifts in sugar-to-lipid ratios in our study suggest a potential modification of the edible quality under zeolite conditions. This suggests that resources in the substrate that were directed toward the synthesis of these classes were redirected to the lipids in the zeolite. A similarly critical increase in FA content (+17.31%) and a drop in sugar content (from 7.70% to 2.72%) were observed in the roots, which may reflect their intensive use for energy or the construction of fatty acids.

Zeolite, as an aluminosilicate mineral, can affect the availability of ions and water, which could hypothetically induce a specific osmotic or ionic environment in the root zone. This is particularly critical for lettuce, which is established as being relatively sensitive to elevated salinity [44]. In this context, the modified membrane compositions and the accumulation of polyols in the Yeralash variety (+2.93%) likely serve as osmoregulatory adaptations to maintain physiological stability. As suggested by Gould et al., phytochemical shifts in secondary metabolites often serve as protective mechanisms against environmental fluctuations [44,45,46].

Furthermore, the alteration in the terpenoid-to-sugar ratio may potentially influence the organoleptic properties. Sesquiterpene lactones impart bitterness [47], while Fillion and Kilcast highlight that consumer perception of quality is deeply linked to the biochemical balance of the matrix [43]. While we observed a decrease in the relative area of the terpenoid class in some cultivars, this shift might hypothetically lead to a more neutral or “buttery” flavor, though sensory evaluation is needed to confirm if these specific shifts cross the human detection threshold.

In the May King (MK) variety, the dramatic shift in the root system—where FA (+51.27%) replaced terpenoids as the dominant class—indicates a strong adaptive response. This substantial metabolic reprogramming suggests that the plant may prioritize structural lipid synthesis for root system stabilization under zeolite conditions. While sugars are known to play a key role in ‘sweet immunity’ and pathogen resistance [48,49,50], their reduction here might indicate that the plant prioritizes the synthesis of structural lipids over carbohydrate-based defense signaling when grown on zeolite.

As in other varieties, Yeralash shows a relative increase in FA (+7.25%). However, the most distinctive feature is the notable increase in polyols (+2.93%), which are widely recognized as osmoregulators that protect plants against desiccation and salinity-induced stress. Given the known sensitivity of lettuce to saline conditions [42,43], the accumulation of polyols in Yeralash suggests a superior adaptive strategy for osmotic balance compared to other varieties. The content of terpenoids remained high (from 37.90 to 31.67%), indicating the preservation of a significant protective function in the leaves. Sugar content remained virtually unchanged, indicating a lesser influence of zeolite on the primary energy metabolism in the leaves. Similarly, in roots, unlike other varieties, Yeralash maintains the dominant role of terpenoids (from 68.95% to 68.94%). This may indicate that Yeralash is less susceptible to the effects of zeolite.

An important observation is that γ-tocopherol was detected only in this variety. γ-tocopherol is the most common form of vitamin E in plants and seeds. Its primary function is antioxidant protection, particularly in membranes and chloroplasts. The decrease in γ-tocopherol concentration in zeolite (from 0.41% to 0.35%) may indicate that zeolite provides more favorable and stable conditions (e.g., better moisture retention or a more balanced, slow release of nutrients) than in the artificial substrate, leading to the plant experiencing less oxidative stress [51]. As a result, the need for antioxidant synthesis is reduced.

One potential confounding factor in the metabolic profiling was the discrepancy in developmental stages at the time of sampling. Since the zeolite-based substrate significantly accelerated seed germination compared to the control group, plants in the zeolite treatment reached specific phenological stages earlier. Sampling on a fixed calendar day thus captured plants at slightly different points of their ontogenesis [52]. While this may influence the absolute concentration of certain metabolites, we argue that the observed shifts reflect the cumulative ‘booster effect’ of the zeolite substrate [49]. This acceleration of development is, in itself, a key performance indicator of the Shankhanai zeolite’s efficiency in NFT systems.

4. Conclusions

This study demonstrated that Shankhanai zeolite is a functionally effective substrate capable of simultaneously improving the physicochemical properties of the medium and modifying plant metabolic reactions.

(1)Structural Advantages. According to BET analysis, zeolite had a significantly higher specific surface area compared to the commercial substrate: 21.80 m^2^/g versus 0.49 m^2^/g. Even after the plant cultivation cycle, the surface area remained high (16.66 m^2^/g). The microporous structure ensured enhanced water retention and a stable ion-exchange interface, providing a more resilient root-zone environment.(2)Biometric Impact. In the NFT system, the zeolite substrate acted as a developmental catalyst. While no significant differences were observed in final seedling height for both types of plants (p > 0.05), the zeolite treatment led to highly significant improvements in germination energy and final emergence percentage (p < 0.001) across all studied cultivars.(3)Metabolic Reprogramming. GC-MS analysis revealed cultivar-specific responses. Varieties MK and AB showed a significant shift toward FA and their derivatives, coupled with a decrease in terpenes and sugars. This suggests a potential adaptive shift in secondary metabolism and lipid biosynthesis in response to the zeolite’s unique ionic environment.(4)Practical Implications. The structural and chemical nature of zeolite (high cation-exchange capacity and moisture stabilization) creates a controlled environment that directs plant metabolism toward increased synthesis of structural lipids.

Consequently, the investigated natural zeolite is a promising and sustainable substrate for high-efficiency hydroponic and semi-closed agricultural systems.