Biostimulants Enhance the Growth and Nutritional Quality of Lettuce (Lactuca sativa L.)

Abstract

Biostimulants have emerged as effective tools for enhancing both the productivity and quality of crops. In this study, we assessed the impact of the two commercial biostimulant products (Kiana Earth^® and Kiana Climate^®) on the growth, yield, and quality of lettuce (Lactuca sativa L.). Eight treatments were established, comprising six different biostimulant formulations, a normal control (no fertilizer applied), and a positive control (chemical fertilizer application). Biostimulant treatments significantly improved plant and stem diameters, fresh and dry biomass, and yield (p < 0.01). The best yields and morphological performance were obtained with samples receiving T6 (Kiana Climate^® + 75:50:75 kg ha⁻¹ N:P:K) and T7 (Kiana Earth^® + 150:100:150 kg ha⁻¹ N:P:K) applications, which comprised biostimulant–fertilizer combinations. Chlorophyll a, chlorophyll b, and total chlorophyll levels were significantly higher with than without biostimulant treatment, indicating that the biostimulants enhanced photosynthetic efficiency. Biochemical analyses further identified significant increases in vitamin C levels, total antioxidant capacity, total phenolic compounds, and flavonoid contents, especially with treatments T5 (Kiana Earth^® + 75:50:75 kg ha⁻¹ N:P:K)–T8 (Kiana Climate^® + 150:100:150 kg ha⁻¹ N:P:K). Nitrogen assimilation analysis showed that leaf NO₃⁻ levels were lower with the combined treatment than with chemical fertilizer alone, suggesting that the biostimulants improved nitrogen-use efficiency. Micronutrient (Fe, Zn, Cu, Mn, Na) and macronutrient (N, P, K, Ca, Mg, S) levels were significantly increased with biostimulant-enriched treatments, alongside a rise in soil organic matter. Biostimulants, especially when combined with mineral fertilization, significantly enhanced lettuce growth, yield, and nutritional quality, while also promoting soil fertility. These findings highlight the potential of biostimulants as valuable tools in conventional, regenerative, and organic agricultural practices, offering a sustainable approach to enhancing agricultural productivity while ensuring long-term soil fertility.

1. Introduction

Sustainable food production and agricultural practices are closely tied to global future well-being. Healthy plants and soil are the foundation of efficient and sustainable agriculture. However, with the onset of climate change, the frequency of extreme events such as droughts and floods has increased, which diminishes crop yields and negatively impacts the income of farmers [1,2]. Soil health and productivity rely on the functioning of soil ecosystems and the harmonization of nitrogen and carbon cycles. Nevertheless, the increasing intensity of agricultural practices has reduced plant productivity and resulted in a net decline in soil organic matter (OM), leading to over-reliance on synthetic fertilizers [3].

The excessive use of mineral fertilizers and pesticides leads to the degradation of soil physicochemical properties, undermines biological activity, and contributes to groundwater depletion and contamination [4,5]. These practices increase the risk of chemical residues accumulating in both the environment and agricultural products, posing significant threats to environmental and human health [6,7]. Consequently, there is growing interest in extending the “do no harm” principle of sustainable agriculture to regenerative approaches that restore ecosystem function [8]. In parallel, adverse environmental impacts and rising fertilizer costs provide impetus for adopting alternative or complementary practices. Consequently, given the challenges of land degradation and shifting climate patterns, the application of biostimulants in plant production has gained increasing relevance [9].

Biostimulants include substances of both organic and inorganic origin, as well as beneficial microorganisms [10]. Regulation (EU) 2019/1009 defines a plant biostimulant as a type of fertilizer that stimulates nutrient uptake by plants, irrespective of the plant’s nutrient content. Importantly, its function is limited to improving one or more of the following aspects in the plant or its rhizosphere: nutrient use efficiency, tolerance to abiotic stress, qualitative features, or the availability of immobilized nutrients in the soil or rhizosphere [11]. Examples of biostimulants include amino acids, algae, nitrogen compounds, humic and fulvic acids, chitosan and chitin-like polymers, seaweed and plant extracts, inorganic compounds, and beneficial bacteria and fungi [12,13,14,15]. They are applied to plants, leaves, seeds, or root environments to stimulate plant growth, acting as both nutrients and soil enhancers [16,17]. Their inherent molecular diversity and complexity contribute to their value in sustainable agriculture, fostering potential synergistic effects [13,18]. Mixtures of organic agricultural biostimulants, such as seaweed and humus extracts, have the potential to enhance regenerative agriculture by rejuvenating natural soil processes, thereby boosting productivity [8,19,20].

Humic substances contribute to the modulation of plant and microbial growth, carbon and nitrogen cycles, the presence and movement of anthropogenic compounds and heavy metals, and the stabilization of soil structure [21]. Humin, humic acid, and fulvic acid are distinguished based on their solubility under varying pH levels. Under low-pH conditions, humic acid precipitates, whereas fulvic acid remains soluble [22,23]. Humic acid improves soil structure and buffers pH, thus increasing the water-holding capacity and nutrient availability of soil, supporting growth and yield, modulating soil biota, and selectively promoting specific rhizobacteria (e.g., Acidobacteria, Actinobacteria, Bacteroidetes) [24,25,26,27]. Under drought stress at 50% field capacity, lettuce treated with a humic-substance-based biostimulant applied to either the roots or foliage showed improved growth, photosynthesis, and water-use efficiency, accompanied by a reduction in reactive oxygen species (ROS) production and lipid peroxidation, irrespective of the application mode [28]. Fulvic acid promotes plant growth and development by accelerating cell division, preventing the accumulation of nitrate (NO₃⁻) compounds through its effects on plant metabolism, facilitating vitamin transport within cells as a catalyst, and enhancing plant resistance against a range of biotic and abiotic stresses [29,30]. Amino acids are essential primary metabolites in plants, impacting a variety of physicochemical properties of cells, tissues, and organs [31,32]. Moreover, they contribute to protein and carbohydrate synthesis, facilitate cell division, and aid in the production of natural growth hormones, ultimately leading to increased yield and improved quality [33]. Amino acids influence numerous plant processes, such as the detoxification of toxins and heavy metals; nutrient uptake, translocation, and metabolism; vitamin synthesis; and tolerance to environmental stresses [34,35,36]. Plant growth-promoting rhizobacteria (PGPR) form beneficial relationships with plant roots, encouraging growth and development while increasing the plant’s ability to tolerate a variety of environmental stresses [37]. PGPR enhance plant health by fixing nitrogen, producing essential hormones, improving water and mineral uptake, and strengthening root growth [38,39,40,41]. Ultimately, by enriching the root zone with nitrogen and facilitating the mineralization of key nutrients such as potassium (K) and phosphorus (P), these bacteria markedly improve overall plant development [42]. A study on tomato seedlings demonstrated that the application of biostimulants containing microorganisms, such as Azospirillum, Bacillus megaterium, and Bacillus subtilis, optimized the organic acid content in seedlings, thereby increasing their resistance to drought stress [43]. In lettuce, biostimulant application increased plant growth and photosynthesis, while improving nutritional quality and antioxidant capacity. A lower dose reduced leaf nitrate levels, whereas a higher dose increased nutrient uptake (especially nitrogen), leading to reduced fertilizer requirements [44]. Also in lettuce, fulvic acid- and seaweed extract-based biostimulants generally improved biomass and nutrient uptake and reduced leaf nitrate contents, while gamma-poly-glutamic acid (γ-PGA), despite increasing root growth, made only a limited contribution to nutrient uptake [45].

Lettuce (Lactuca sativa L.) is a widely consumed green vegetable globally, serving as a significant source of fibre, iron, and vitamin C [46,47]. Lettuce plants rely on a balanced supply of essential micronutrients and macronutrients for optimal growth [48]. The quantity of these nutrients directly influences lettuce crop quality and yield, underscoring the need to provide a balanced nutrient supply to ensure high-quality produce [49]. Although chemical fertilization is crucial in lettuce farming, the overapplication of nitrogen fertilizers immediately before harvest can lead to the accumulation of NO₃⁻ in the leaves, posing a health risk to humans [50].

Although studies have demonstrated the benefits of various biostimulants in lettuce, the comparative efficacy among formulations based on humic substances, amino acids, microorganisms, enzymes, and algae in improving morphological and key biochemical quality parameters remains insufficiently resolved. Therefore, the objective of this study was to investigate the effects of biostimulant formulations based on humic substances, PGPR and amino acids on the morphological (plant and stem diameter, fresh and dry weight), biochemical (antioxidant capacity, vitamin C levels, chlorophyll content), and nutritional quality (plant nutrient elements, ammonium [NH₄⁺], NO₃⁻, soil OM content) of lettuce.

2. Materials and Methods

2.1. Experimental Area, Plant Materials and Applications

This study was conducted during the 2021–2022 growing seasons in the experimental area of the Horticulture Department at Atatürk University, Erzurum, Türkiye. The location is situated at an elevation of 1900 m (39°54′ N, 41°15′ E) and is characterized by a terrestrial climate with long, cold winters and cool, dry summers. The soil is generally slightly alkaline (pH 7.825) and has a low OM content (2.083%).

Table 1. Physicochemical properties of the soil samples.

Properties	Value	Property	Value
pH	7.825	Mg (cmolc/kg)	22.806
EC (μS/cm)	130.704	Na (cmolc/kg)	2.255
CaCO3 (%)	7.40	P (mg/kg)	1.187
Organic Matter (%)	2.083	Fe (mg/kg)	0.792
Total N (%)	0.065	Cu (mg/kg)	0.246
NH4-N (mg/kg)	12.669	Mn (mg/kg)	0.098
NO3-N (mg/kg)	1.849	Zn (mg/kg)	0.176
K (cmolc/kg)	39.708	B (mg/kg)	0.030
Ca (cmolc/kg)	248.896

presents some of the soil properties in the experimental area. Meteorological data for the experimental periods (April to September) in 2021 and 2022 included 175 and 186 mm of total rainfall, and average air temperatures of 19.3 °C and 18.7 °C, respectively. The long-term (30-year) average rainfall and temperature for this period are 205 mm and 17.5 °C, respectively, indicating that the experimental seasons were close to normal.

The plant material used in the study was the lettuce variety Lactuca sativa var. crispa L. cv. Summer Kıvırcık 010. The cultivar Summer Kıvırcık 010 is widely cultivated under greenhouse and open-field conditions in Türkiye, particularly during the summer season. It is well adapted to local climatic conditions but is also known to respond distinctly to nutrient management and stress factors, making it a relevant and representative choice for studies aiming to improve nutrient-use efficiency and sustainability in regional production systems. Seeds were sown in 216-cell seedling trays filled with a peat: perlite mixture (2:1 v/v). After approximately 30 days, seedlings at the 2 to 3-leaf stage were transplanted into the field (25 May in both years), with a row spacing of 30 cm. The experiment was organized as a Completely Randomized Design (CRD) with three replicates, each containing 10 plants, and eight treatments in total (Figure 1).

The experiment consisted of eight treatment groups (T1–T8)—a control group (T1), a chemical fertilizer group (T2; 150 kg N ha⁻¹, 100 kg P₂O₅ ha⁻¹, 150 kg K₂O ha⁻¹), and six biostimulant-based treatment groups (T3–T8), designated as follows: T3 = Kiana Earth, T4 = Kiana Climate, T5 = Kiana Earth + T2 (50%), T6 = Kiana Climate + T2 (50%), T7 = Kiana Earth + T2, and T8 = Kiana Climate + T2 (

Table 2. The biostimulant applications used in the study.

Chemical Fertilizer					Composition of Kiana Products
Treatment Number	Treatment	N (kg/ha)	P2O5 (kg/ha)	K2O (kg/ha)	Humic Substance * (%)	Amino Acids ** (%)	Microorganisms *** (cfu/mL)	Enzymes **** (U/g)	Algae ***** (%)
T1	Control	–	–	–	–	–	–	–	–
T2	Chemical fertilizer	150	100	150	–	–	–	–	–
T3	Kiana earth	–	–	–	18	–	1 × 109	–	–
T4	Kiana climate	–	–	–	18	15	1 × 109	2750	2
T5	Kiana earth	75	50	75	18	15	1 × 109	–	–
T6	Kiana climate	75	50	75	18	15	1 × 109	2750	2
T7	Kiana earth	150	100	150	18	15	1 × 109	–	–
T8	Kiana climate	150	100	150	18	15	1 × 109	2750	2

* Humic acid: 18–22%, K₂O 2.5–3.0%, dry matter 20–22%, organic substance 14–15%, pH 10–11, density 1.11–1.12 g/cm³. ** Amino acids: Free amino acids 14%, organic nitrogen 7.0–7.5%, dry matter 50–52%, pH 5.0–6.5, density 1.25–1.30 g/cm³. *** Microorganisms: Bacillus pumilus, Bacillus licheniformis, Azotobacter choroococcum, Azosiprillium brasselience, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus megaterium. **** Enzymes: proteinase, alpha-amylase, xylanase, cellulase, hemicellulase, phytase. ***** Algae: Ascophyllum nodosum.

). The Kiana earth was administered three times, one week apart, via drip irrigation of the root zone of the seedlings. Kiana Climate was foliar-sprayed three times after transplantation. Both biostimulants were applied at 10 kg/ha. The study was terminated 45 days after the seedlings were planted, at which point the plants were harvested for subsequent analysis (Figure 2).

The two commercial biostimulant products, Kiana Earth^® and Kiana Climate^®, were applied to boost soil health and improve plant tolerance to environmental stress (Table 2). Kiana Earth^®, which contains humic substances, amino acids, organic acids, enzymes, and beneficial microorganisms, was applied to the soil. This formulation aims to improve soil fertility, boost microbial activity, and increase nutrient uptake efficiency. Kiana Climate^®, containing organic acids, polyphenolic compounds, amino acids, enzymes, microorganisms, phytohormones, and natural minerals, was applied as a foliar spray. It is formulated to mitigate the effects of temperature extremes by improving plant physiological resilience and promoting growth and productivity. The product provides thermal protection within a temperature range of approximately −14 °C to +55 °C, depending on the application frequency. Both biostimulants were used according to the manufacturer’s recommendations.

2.2. Measurements

In this study, several morphological, physiological, and biochemical characteristics were measured, including plant diameter, stem diameter, plant fresh and dry weights, yield, leaf colour values (L*, a*, and b*), chlorophyll a and b contents, and total chlorophyll. The yield of lettuce was also determined as g/m². Samples used for the determination of total antioxidant capacity and total phenolic and flavonoid contents were stored at −80 °C until analysis.

2.2.1. Measurements of Morphological and Yield Indicators

Morphological characteristics were assessed 45 days after seedling establishment to evaluate the effects of the treatments on vegetative growth. From each replicate, four representative plants were carefully uprooted and gently washed to remove soil particles. The following properties were recorded: plant diameter, stem diameter, and plant fresh and dry weights. Plant diameter was measured as the average of two perpendicular measurements across the widest point between the tips of the outermost leaves using a digital calliper. Stem diameter was measured at the base of the main stem, just above the soil surface, with a digital calliper. After washing, plant fresh weights were recorded. For dry weight determination, plant samples were oven-dried at 70 °C to constant weight (approximately 48 h) and then weighed. Yield was determined by weighing the marketable fresh aboveground parts of each lettuce plant at harvest.

2.2.2. Physiological Measurements

Leaf colour was measured using a colorimeter (Konica Minolta CM-700, Osaka, Japan) to obtain the colour values (L*, a*, and b*) of two randomly selected leaves. For the analysis of chlorophyll a, chlorophyll b, and total chlorophyll contents, samples were treated with an 80% acetone solution. The absorbance of the filtered extracts (663 nm for chlorophyll a and 645 nm for chlorophyll b) was measured using a Multiskan GO spectrophotometer (Thermo Scientific, Ratastie, Finland). The respective contents were then calculated using the formulas provided by Lichtenthaler and Wellburn [51], as follows:

Total chlorophyll (mg/g) = (A652 × 27.8)/g

Chlorophyll a (mg/g) = [(11.75 × A663) − (2.35 × A645)] × V/g

Chlorophyll b (mg/g) = [(18.61 × A645) − (3.96 × A663)] × V/g

2.2.3. Biochemical Measurements

The vitamin C content was quantified as mg/100 g fresh weight using an ascorbic acid kit; colour intensity was measured with a Merc reflectometre (Merck, Darmstadt, Germany). The chlorophyll levels of the lettuce plants were determined using a portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Osaka, Japan) [52].

Five grams of each sample was mixed with 100 mL of methanol, shaken in a Stuart shaker (SSL1) in the dark at room temperature overnight, and filtered through Whatman No. 1 filter paper (Merck KGaA, Darmstadt, Germany). The filtrate was then mixed with 50 mL of methanol, shaken for 2 h, and again passed through filter paper. Some of the methanol in the total extract was evaporated in a rotary evaporator at 45 °C, with the volume adjusted to 50 mL. The resulting extract was used for the determination of total phenolic and flavonoid contents and total antioxidant activity [53].

Total antioxidant activity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which measures the reduction of the stable DPPH radical by antioxidants. A DPPH radical solution was prepared by dissolving 39 mg of the radical in 100 mL of ethyl alcohol. Test tubes containing 10, 20, 30, and 40 mg of the sample extracts were prepared. To each tube, 0.5 mL of the DPPH solution was added, and the final volume was adjusted to 3 mL using ethyl alcohol. After vortexing, the mixtures were placed in a dark environment for 30 min. The absorbance of each reaction mixture was measured at 517 nm to determine the ability of the sample extracts to scavenge the DPPH free radical. IC50 values were calculated based on these absorbance readings [54,55], as follows: %inhibition = [(A DPPH − A extract)/A DPPH] × 100, where A DPPH is the absorbance value of the DPPH blank sample, and A extract is the absorbance value of the sample extract.

A 0.1 mL volume of each prepared extract was mixed with Folin–Ciocalteu reagent and Na₂CO₃ in a test tube, and the volume was adjusted to 10 mL with distilled water. After incubation for 1 h at room temperature, absorbance was measured at 760 nm. The total phenolic content was expressed as mg Gallic Acid Equivalents (GAE)/100 g of extract, based on a gallic acid standard curve prepared daily [54].

To determine the total flavonoid content, 0.5 g of each sample was first mixed with 2 mL of distilled water and then with 0.15 mL of a 5% NaNO₂ solution. The mixture was left to stand for 6 min before the addition of a 10% AlCl₃ solution (0.15 mL). After being allowed to stand for an additional 6 min, 2 mL of a 4% NaOH solution was added to the mixture, and the volume was adjusted to 5 mL with distilled water. After 15 min, the absorbance of the resulting mixture was measured at 510 nm. Quercetin and rutin were used as standards [56].

2.2.4. Determination of NH₄⁺, NO₃⁻, Mineral, and Soil OM Contents

Lettuce leaves were dried at 68 °C for 48 h in an oven and then ground. The total nitrogen (N) content was determined using the Kjeldahl method with a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Konigswinter, Germany) [57]. Mineral content analysis was performed using an inductively coupled plasma optical emission spectrophotometer (Optima 2100 DV, ICP/OES; Perkin Elmer, Shelton, CT, USA) [58,59]. For the determination of NO₃-N levels, the absorbance of the complex formed by the nitration of salicylic acid under highly acidic conditions was measured at 410 nm in basic solutions (pH > 12). The soil OM content after harvest was determined using the Smith-Weldon method as outlined in Nelson and Sommers [60].

2.3. Statistical Analysis

The experiment was arranged in a CRD with three replications, each consisting of 10 plants. The experiments were conducted over two consecutive years, and as the year effect and year × treatment interaction was not statistically significant, the data were combined for pooled analysis. All data obtained from the determination of morphological, physiological, and biochemical parameters were subjected to one-way analysis of variance (ANOVA) to determine the significance of treatment effects. The significance of the differences among treatment means was evaluated using Tukey’s Honestly Significant Difference (HSD) test at the p < 0.05 probability level. For a holistic visualization of treatment-related patterns and cumulative responses across all measured variables, a multivariate approach was employed. A polar heat map was constructed using the combined dataset. To facilitate the objective grouping of treatments, Hierarchical Clustering Analysis (HCA) was performed within the heat map. Clustering was performed using Ward’s minimum-variance method, with Euclidean distance serving as the dissimilarity metric. ANOVA and Tukey’s HSD test were executed using Minitab 22 (Minitab, LLC, State College, PA, USA). Graphical visualizations, including the polar heat map, were generated using OriginLab 2025 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Morphological and Yield-Related Parameters

ANOVA results confirmed that all morphological and yield-related variables, namely plant diameter, stem diameter, yield, plant fresh weight, and plant dry weight, were significantly influenced by the applied treatment (p < 0.01) (

Table 3. Analysis of variance (ANOVA) results for morphological, physiological, biochemical, and nutrient parameters under different treatments.

Variables	Treatment Mean Square	Error Mean Square
PD (cm)	108.883 **	1.613
SD (cm)	62.196 **	0.479
Yield (g/m2)	552,330.429 **	289.896
PFW (g)	4564.714 **	2.396
PDW (g)	45.544 **	0.111
Colour-L*	48.675 ns	21.690
Colour-a*	6.449 **	1.060
Colour-b*	9.934 ns	6.460
Chlo-a (mg/g)	0.793 **	1.960
Chlo-b (mg/g)	0.151 **	0.013
Total Chlo (mg/g)	1.403 **	0.002
Vitamin C (mg/L)	828.280 **	0.019
Total AOC (µg/mL FW)	4,423,793.333 **	89.667
Total Phenolic Content (mg GAE/100 g FW)	202.423 **	16,521.917
Total flavonoid (mg/100 g FW)	143.024 **	2.792
NH4+ (ppm/g FW)	3295.661 **	1.833
NO3− (ppm/g FW)	6,600,223.976 **	171.458
N%	0.379 **	15,346.250
P (%)	0.008 **	0.020
K (%)	0.298 **	0.000
Ca (%)	0.606 **	0.008
Mg (%)	0.039 **	0.003
S (%)	0.011 **	0.001
Mn (mg/kg)	32.033 **	0.000
Fe (mg/kg)	1313.442 **	2.716
Zn (mg/kg)	26.270 **	15.018
Cu (mg/kg)	13.504 **	1.831
Na (mg/kg)	839.578 **	1.049
Soil OM (%)	0.054 **	51.541

“**” indicates statistically highly significant differences (p < 0.01) and “ns” denotes non-significant differences (p > 0.05). PD: Plant Diametre, SD: Stem Diameter, Yield: Yield per square meter, PFW: Plant Fresh Weight, PDW: Plant Dry Weight, Colour-L: Lightness (L* value), Colour-a: Red–Green Axis (a* value), Colour-b: Yellow–Blue Axis (b* value), Chlo-a: Chlorophyll a, Chlo-b: Chlorophyll b, Total Chlo: Total Chlorophyll, Vitamin C: Ascorbic Acid Content, Total AOC: Total Antioxidant Capacity, Total Phenolic Content: Total Phenolic Compounds (as Gallic Acid Equivalent), Total Flavonoid: Total Flavonoid Content, NH₄⁺: Ammonium Ion Content, NO₃⁻: Nitrate Ion Content, N: Nitrogen Content, P: Phosphorus Content, K: Potassium Content, Ca: Calcium Content, Mg: Magnesium Content, S: Sulphur Content, Mn: Manganese Content, Fe: Iron Content, Zn: Zinc Content, Cu: Copper Content, Na: Sodium Content, Soil OM: Soil Organic Matter.

). Biostimulant application led to a statistically significant increase in plant diameter. The greatest diameter (29.00 cm, vs. 15.13 cm in the control group [T1]) was recorded with treatment T5. Similarly, treatments T6, T7, and T8 also yielded significantly larger plant diameters compared with that observed with the control treatment (

Table 4. Effects of different treatments on morphological traits.

Treatments	PD (cm)	SD (mm)	PFW (g/plant)	PDW (g/plant)
T1	15.13 ± 1.25 g	21.93 ± 0.76 e	58.17 ± 3.26 f	10.37 ± 0.51 e
T2	21.40 ± 0.49 f	25.47 ± 1.11 d	138.79 ± 1.31 c	22.13 ± 1.28 b
T3	23.13 ± 1.02 e	28.60 ± 0.91 c	71.17 ± 4.16 e	15.87 ± 0.71 d
T4	25.40 ± 0.28 d	34.73 ± 1.03 b	76.70 ± 4.55 e	16.35 ± 0.59 d
T5	29.00 ± 0.44 a	36.60 ± 0.38 ab	127.83 ± 3.89 d	21.41 ± 0.89 c
T6	28.23 ± 0.26 ab	36.00 ± 0.27 ab	141.67 ± 1.03 b	24.11 ± 1.22 ab
T7	27.33 ± 0.18 bc	36.93 ± 0.68 ab	156.32 ± 6.14 a	26.37 ± 1.75 a
T8	26.20 ± 0.45 cd	37.47 ± 0.87 a	131.80 ± 5.78 cd	21.39 ± 1.13 c

Plant diameter (PD), Stem diameter (SD), Plant fresh weight (PFW), and Plant dry weight (PDW). Data are presented as mean ± SE (n = 3). Different letters indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05.

). Stem diameter significantly increased with biostimulant applications, rising from 21.93 mm in the control group (T1) to a peak of 37.47 mm in the T8 treatment group. Notably, treatments T5, T6, and T7 also promoted substantial increases in stem diameter. These values were significantly higher than those obtained with the chemical-only (T2: 25.47 mm), organic-only (T3: 28.60 mm), and humic/amino acid-only (T4: 34.73 mm) treatments (Table 4). Plant fresh weight was lowest with the control treatment, peaking with treatment T7, followed by T6 (Table 4). Plant dry weight was also lowest in the control group, and increased with biostimulant treatments, attaining the highest value with the T5 treatment, followed by T7 and T6 (Table 4). Furthermore, yield responded positively to biostimulant applications. The lowest yield was seen in the T1 (control) group, while the highest was observed in the T7 group, significantly exceeding that recorded for all other treatments. Treatments T6 and T2 also produced high yields, followed by T5 and T8, with treatments T3 and T4 yielding only moderate increases (Figure 3).

3.2. Leaf Colour Characteristics and Photosynthetic Pigment Content

ANOVA results indicated that most colour and chlorophyll-related traits were significantly affected by the treatments (p < 0.01), the exceptions being colour-L* and colour-b* (p > 0.05) (

Table 5. Effects of treatments on external colour parameters and chlorophyll content.

Treatments	Colour-L*	Colour-a*	Colour-b*	Chlo-a (mg/g)	Chlo-b (mg/g)	Total Chlo (mg/g)
T1	50.83 ± 5.25 ns	18.40 ± 0.84 ab	36.40 ± 2.41 ns	3.25 ± 0.10 f	1.14 ± 0.02 e	4.39 ± 0.01 e
T2	54.50 ± 6.38	18.80 ± 0.68 ab	38.33 ± 2.46	3.50 ± 0.14 e	1.25 ± 0.05 d	4.75 ± 0.02 d
T3	42.80 ± 5.11	15.93 ± 0.54 c	35.27 ± 1.28	4.29 ± 0.11 b	1.69 ± 0.12 ab	5.98 ± 0.11 b
T4	45.40 ± 4.59	15.30 ± 0.49 c	33.27 ± 3.11	3.72 ± 0.17 d	1.73 ± 0.10 a	5.45 ± 0.08 c
T5	46.63 ± 4.45	16.93 ± 1.02 bc	32.80 ± 4.12	4.44 ± 0.11 b	1.61 ± 0.14 b	6.05 ± 0.12 b
T6	49.33 ± 3.89	18.53 ± 0.89 ab	36.73 ± 3.87	4.02 ± 0.10 c	1.32 ± 0.08 d	5.34 ± 0.12 c
T7	50.13 ± 4.01	17.97 ± 1.11 ab	35.20 ± 3.33	4.38 ± 0.14 b	1.48 ± 0.01 c	5.86 ± 0.11 b
T8	53.67 ± 3.94	19.47 ± 1.01 a	36.13 ± 2.84	4.75 ± 0.12 a	1.65 ± 0.02 ab	6.40 ± 0.20 a

Colour-L* (lightness), Colour-a* (red–green axis), Colour-b* (yellow–blue axis), Chlorophyll a (Chlo-a), Chlorophyll b (Chlo-b), Total chlorophyll (Total Chlo). Data are presented as mean ± SE (n = 3). Different letters indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05. ^ns: Non-Significant.

). Leaf colour parameters were variably affected by the treatments. For lightness (colour-L*) and the yellow–blue spectrum (colour-b*), no statistically significant differences were observed among the treatments. In contrast, the green–red spectrum (colour-a*) was significantly affected by treatments. The lowest colour-a* values, which reflect the most intense green colour, were recorded with treatments T4 and T3. The highest colour-a* value (least intense green) was noted with treatment T8, which also corresponded to the highest total chlorophyll content (Table 5).

Chlorophyll content was significantly affected by the treatments (p < 0.05) (Table 5). The chlorophyll a content markedly increased with biostimulant applications, reaching the highest level in the T8 group, followed by the T5 and T7 groups, while the lowest value was recorded in the control group. Treatments combining chemical fertilizer with Kiana products (T5–T8) significantly outperformed the control and chemical-only treatments regarding chlorophyll a accumulation. Chlorophyll b levels showed a similar pattern, with the most significant values observed with the T4, T3, and T8 treatments, and the lowest with the T1 treatment. Consequently, total chlorophyll content was highest with the application of T8, followed by T5 and T3. These data indicated that biostimulant-based treatments enhanced pigment biosynthesis and photosynthetic potential relative to the control treatment (Table 5).

3.3. Antioxidant Compounds, Phenolics, and Flavonoids

ANOVA results showed that all measured biochemical and nitrogen-related parameters—vitamin C content, total antioxidant capacity, total phenolic content, and total flavonoids—were significantly affected by the treatments (p < 0.01) (

Table 6. The effects of the application of different biostimulants on vitamin C, total antioxidant capacity, and total phenolic and total flavonoid contents of lettuce.

Treatments	C Vit (mg/100 g)	Total AOC (µg/mL FW)	Total Phenolic Content (mg GAE/100 g FW)	Total Flavonoid (mg/100 g FW)
T1	85.33 ± 2.11 d	819.33 ± 26.82 g	22.00 ± 1.18 e	8.67 ± 0.91 d
T2	94.67 ± 5.43 cd	1959.33 ± 97.85 f	37.67 ± 1.22 d	16.67 ± 0.57 c
T3	112.67 ± 3.45 b	4033.33 ± 115.64 b	38.33 ± 0.37 d	19.00 ± 1.13 c
T4	133.33 ± 2.15 a	3794.67 ± 59.87 cd	43.67 ± 1.67 bc	25.67 ± 1.37 b
T5	119.67± 2.11 ab	3432.67 ± 43.12 e	42.67 ± 0.44 c	28.67 ± 0.49 a
T6	106.00 ± 5.11 bc	3942.33 ± 98.25 bc	47.67 ± 0.61 a	25.33 ± 1.02 b
T7	130.67 ± 1.86 a	3694.33 ± 55.46 d	46.33 ± 1.46 ab	27.00 ± 1.12 ab
T8	106.67 ± 5.68 bc	4312.00 ± 120.12 a	44.67 ± 1.28 abc	27.00 ± 0.94 ab

Effects of treatments on antioxidant-related biochemical traits; Vitamin C, Total antioxidant capacity (AOC), Total phenolic content, Total flavonoid content. Data are presented as mean ± SE (n = 3). Different letters indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05.

). Vitamin C levels varied significantly among the treatments. The lowest concentration was observed in the control group (T1), while the highest was detected in the T4 group. Treatments T7 and T5 also yielded markedly higher values than the control treatment. These findings suggested that the application of biostimulants, particularly in combination with chemical fertilizers (treatments T4–T7), promoted ascorbate accumulation in lettuce leaves. Total antioxidant capacity exhibited a similar trend (Table 6). The lowest value was observed with T1 application, while the highest value was recorded with the T8 treatment, followed by treatment T3. Plants grown under treatments T6 and T4 also demonstrated significantly higher antioxidant potential than both control plants and plants treated with the chemical fertilizer alone (Table 6).

Regarding total phenolic content, as shown in Table 6, the highest levels were observed with treatment T6, followed by T7 and T8. The respective values for the control (T1) and chemical fertilizer-only treatments (T2) were 22.00 and 37.67 mg GAE/100 g FW, respectively. The total flavonoid content markedly increased with biostimulant treatments. The lowest content was observed in the T1 group, while the highest was noted in the T5 group, followed by the T4 and T6 groups (Table 6).

3.4. Nitrogen Assimilation

Ammonium ion (NH⁴⁺) levels varied considerably among treatments (Figure 4). The highest concentration (375.33 ppm FW) was recorded with the application of T2, likely due to elevated nitrogen absorption from the chemical fertilizer. Treatments T6 and T7 also exhibited increased NH₄⁺ levels relative to the control treatment (T1), though these values were not significantly different from the average of 310.71 ppm FW. The control treatment (T1) yielded the lowest NH₄⁺ concentration, 268.33 ppm FW, indicating that nitrogen availability or uptake was limited in the absence of external inputs. NO₃⁻ levels also greatly varied among treatments (Figure 4). The highest NO₃⁻ content was observed with treatment T2, reflecting the high NO₃⁻ input from the applied fertilizer. Treatments T5 and T6 also resulted in high NO₃⁻ contents. Conversely, the lowest NO₃⁻ level was found in the T1 (control) group. Interestingly, the application of some biostimulant combinations (such as T3, T4, and T8) led to only moderate increases in NO₃⁻ levels despite achieving high yield and biomass, potentially attributable to greater nitrogen utilization efficiency and processing under organic-enhanced conditions (Figure 4).

3.5. Macronutrient Accumulation

As shown in

Table 7. The effects of the application of different biostimulants on macro-element contents of lettuce.

Treatment	N	P	K	Ca	Mg	S
(%)
T1	2.19 ± 0.34 c	0.21 ± 0.02 e	1.67 ± 0.21 f	0.85 ± 0.10 e	0.24 ± 0.06 cd	0.17 ± 0.01 de
T2	3.47 ± 1.23 a	0.33 ± 0.01 b	2.68 ± 0.10 a	1.22 ± 0.08 c	0.27 ± 0.05 d	0.15 ± 0.01 e
T3	2.91 ± 0.67 b	0.25 ± 0.01 d	2.12 ± 0.11 cd	1.12 ± 0.05 d	0.22 ± 0.07 d	0.18 ± 0.01 cd
T4	2.95 ± 0.71 b	0.32 ± 0.01 b	1.86 ± 0.12 e	1.08 ± 0.04 d	0.28 ± 0.07 c	0.21 ± 0.01 b
T5	2.79 ± 0.73 b	0.34 ± 0.02 ab	2.25 ± 0.20 bc	1.05 ± 0.05 d	0.21 ± 0.06 d	0.18 ± 0.01 cd
T6	2.72 ± 0.77 b	0.29 ± 0.01 c	2.03 ± 0.01 d	1.25 ± 0.08 c	0.20 ± 0.04 d	0.22 ± 0.01 b
T7	2.70 ± 0.81 b	0.36 ± 0.02 a	2.39 ± 0.11 b	2.18 ± 0.12 a	0.51 ± 0.04 a	0.35 ± 0.04 a
T8	2.70 ± 0.91 b	0.24 ± 0.01 d	2.01 ± 0.11 de	1.85 ± 0.08 b	0.44 ± 0.034 b	0.21 ± 0.01 b

Macronutrient accumulation in plants under different treatments; Nitrogen (N), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg), and Sulphur (S). Data are presented as mean ± SE (n = 3). Different letters indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05.

, the contents of all the examined macronutrients, including N, P, K, calcium (Ca), magnesium (Mg), and sulphur (S), were significantly affected by the applied treatments (p < 0.01), as determined by ANOVA. The N content (%) was highest in the chemical-only treatment group (T2) and lowest in the control group (T1). Biostimulant-integrated treatments such as T3, T4, and T5 resulted in moderately high N levels. Interestingly, the N content with treatments T6–T8 remained stable (≈2.70%), indicating that N uptake was consistent under the combined inputs (Table 7).

In parallel with N dynamics, the P content (%) also responded significantly to treatments (Table 7). The highest value was recorded with treatment T7, followed by treatments T5 and T2; the lowest P accumulation was observed with the T1 treatment (Table 7). Applications involving Kiana biostimulants (T4–T8) generally resulted in improved P nutrition, likely due to enhanced root activity and microbial facilitation of P solubilization. The K content (%) also showed substantial variation among treatments (Table 7). The peak value was found with the application of T2), followed by T7 and T5, all exceeding the value seen in control plants (T1). Biostimulant-only and combination treatments (T3–T8) performed well, indicating that they contributed to K uptake efficiency (Table 7).

This pattern of nutrient enhancement was also reflected in Ca accumulation. The Ca content (%), shown in Table 7, was noticeably increased by the T7 and T8 treatments, far surpassing that in plants grown under the T1 and T5 treatments (Table 7). This suggested that biostimulants enhance Ca transport or retention in plant tissues. Moreover, the Mg content (%) was lowest in the T5 and T6 groups, and highest in the T7 and T8 (0.44%) groups (Table 7). The increase in Mg contents with T7 and T8 application may be attributed to enhanced enzymatic activity and improved chlorophyll biosynthesis, both processes in which Mg plays a central role. The S content (%) followed a similar pattern. The lowest values were detected in T2 and T1-treated plants, and the highest in T7-treated plants (Table 7). Treatments T4–T6 also increased S levels relative to the control treatment, suggesting that the presence of biostimulants, in conjunction with mineral fertilizers, promotes S uptake and metabolism.

3.6. Micronutrient Uptake and Soil OM

ANOVA results revealed that the treatments had statistically significant effects on the concentrations of all the evaluated micronutrients (Mn, Fe, Zn, Cu, Na) as well as on the soil OM content (p < 0.01,

Table 8. The effects of the application of different biostimulants on micro-element and organic matter contents of lettuce and the organic matter content of soil.

Treatment	Mn	Fe	Zn	Cu	Na	Soil OM
(mg/kg)						(%)
T1	33.55 ± 0.67 ab	68.59 ± 2.22 e	13.44 ± 0.69 e	6.48 ± 0.73 d	102.74±4.77 d	2.08 ± 0.02 b
T2	33.33 ± 0.98 ab	101.06 ± 3.46 c	22.60 ± 0.18 a	8.11 ± 0.97 cd	128.02±5.06 c	2.11 ± 0.02 b
T3	31.61 ± 1.21 ab	73.32 ± 3.58 e	22.02 ± 0.87 ab	6.97 ± 0.94 d	137.79±4.43 bc	2.16 ± 0.03 b
T4	34.13 ± 1.16 a	101.46 ± 2.28 c	19.87 ± 0.16 bc	7.56 ± 0.55 cd	152.35±5.37a	2.09 ± 0.02 b
T5	25.80 ± 2.01 c	59.51 ± 2.38 f	18.25 ± 0.78 cd	6.95 ± 0.76 d	148.77±4.67 ab	2.24 ± 0.02 b
T6	31.91 ± 1.45 ab	118.27 ± 4.15 a	17.44 ± 0.33 cd	10.93 ± 0.49 ab	153.84 ± 3.55 a	2.27 ± 0.03 b
T7	30.82 ± 0.84 b	108.45 ± 3.43 b	19.36 ± 1.01 c	12.38 ± 1.12 a	144.04±5.24 ab	2.49 ± 0.11 a
T8	26.02 ± 1.34 c	87.78 ± 4.11 d	16.76 ± 0.77 d	9.36 ± 0.75 bc	141.08±6.89 ab	2.17 ± 0.02 b

Micronutrient and sodium content along with soil organic matter under different treatments; Manganese (Mn), Iron (Fe), Zinc (Zn), Copper (Cu), Sodium (Na), and Soil organic matter (Soil OM). Data are presented as mean ± SE (n = 3). Different letters indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05.

). Mn contents varied between 25.80 mg/kg with the T5 treatment and 34.13 mg/kg with the T4 treatment (Table 8). Treatments T1–T4 generally resulted in higher Mn values (>31 mg/kg), while the application of T5, T7, and T8 led to significantly lower Mn concentrations (<30 mg/kg) (Table 8). This pattern was mirrored by changes in Fe levels, suggesting that an inverse relationship existed between Mn uptake and Fe uptake under certain treatments. Fe accumulation showed a sharp increase in biostimulant-supported treatments. The lowest Fe value was seen for T5, while the highest Fe concentrations were observed with T6 and T7 (Table 8). Mirroring Fe dynamics, Zn contents were significantly elevated with the T2 and T3 treatments relative to those observed with the T1 treatment (Table 8). Although lettuce treated with T6–T8 recorded moderate values, the observed trend suggested that Zn accumulation was influenced by both the presence and proportion of chemical and biostimulant inputs. Similarly, as shown in Table 8, Cu levels were significantly increased in the T6 and T7 groups compared with those in both the T1 group, which displayed the lowest Cu contents, and the T4 group. This pattern of Cu accumulation suggests that a direct relationship exists between biostimulant-enhanced enzymatic activity and Cu homeostasis, especially in antioxidant systems involving Cu/Zn-SOD enzymes.

The overall enhancement of micronutrient uptake noted with biostimulant application was accompanied by a consistent increase in Na content. The concentrations of Na rose steadily across treatments, with the highest levels observed with T6 and T4, and the lowest with T1 (Table 8). Although elevated Na levels can be detrimental under stress, the moderate increase observed here likely reflects an improvement in the ionic balance under enhanced nutrient uptake. Supporting this, soil OM, a key indicator of soil fertility and microbial activity, increased significantly under biostimulant-enriched treatments. The highest OM content was found with T7, followed by T6 and T5, while plants treated with T1 or T4 recorded the lowest OM levels (Table 8). These results indicated that improvements in micronutrient uptake are likely accompanied by enhanced soil quality and biological activity under biostimulant treatment regimes.

3.7. Polar Heatmap and Cluster Analysis of Treatments and Traits

The polar heatmap presented in Figure 5 provides a comprehensive overview of how the eight treatments (T1–T8) influenced a wide range of traits, including morphophysiological characteristics (e.g., plant diameter, stem diameter, yield), biochemical markers (such as vitamin C, total antioxidant capacity, and phenolics), pigment concentrations (chlorophyll a and b), and micro- and macronutrient levels (Figure 5). This radial layout enables a quick visual comparison of treatment performance. Notably, treatments T5 and T6 are associated with deep red tones for several key parameters, including yield, plant fresh weight, vitamin C levels, total antioxidant capacity, and Ca contents, implying that they exert a strong impact on plant growth and stress-related responses. These treatments are also positioned close together in the dendrogram, suggesting that they elicit similar physiological and biochemical responses, most likely due to the combined effects of the chemical fertilizer and biostimulants. In contrast, the control treatment (T1) is consistently associated with dark blue tones across almost all parameters, particularly NO₃⁻ levels, total antioxidant capacity, and chlorophyll contents, indicating that plants perform poorly under this treatment, which lacks external nutrient support (Figure 5).

An interesting observation arises from the NO₃⁻ pattern: while T2 and T5 are associated with high NO₃⁻ accumulation, as reflected in the strong red colouring in the polar heatmap, they are not necessarily among the top performers regarding pigment-related traits such as total chlorophyll or chlorophyll b contents. This discrepancy may point to differences in how efficiently each treatment supports nutrient assimilation versus mere accumulation (Figure 5). The results of the HCA further support this possibility. Treatments such as T5, T6, and T8 cluster closely together, reinforcing their similar and superior impacts across most traits. In contrast, treatments T1 and T2 form more distant branches, confirming their distinct and often less favourable effects. In summary, the clustering and colour distribution patterns clearly indicate that the integration of biostimulants with chemical fertilizers (in this study, particularly so for treatments T5 through T8) consistently enhances plant performance by improving growth, nutrient uptake, and antioxidant metabolism (Figure 5).

4. Discussion

The world’s population is projected to reach 9.8 billion by 2050, necessitating an estimated 70% rise in global food demand to sustain this growth [61]. The rapid increase in global food consumption and urbanization continues to amplify the environmental impact of agriculture, highlighting the urgent need for the development of sustainable and resilient food production systems [62]. Under these conditions, the agricultural sector must address the challenges of improving productivity and total output while simultaneously reducing adverse environmental effects throughout its value chain [63]. Over the past decades, the increasing use of fertilization practices aimed at boosting crop yields has unintentionally caused several global environmental and geopolitical problems, including nutrient imbalances, the leaching of nutrients into ecosystems, and rising fertilizer prices, which threaten economic stability and food security, especially in developing countries [64].

The agricultural sector is increasingly adopting organic and eco-friendly approaches [65]. The primary goal of this transition is to reduce agricultural inputs while maintaining high yield and quality. Strategies involving the use of sustainable fertilizers are emerging as a key solution for simultaneously achieving energy efficiency and agricultural sustainability [66]. These innovations include bio-based fertilizers, slow-release formulations, and plant biostimulants that sustainably enhance nutrient use efficiency, crop resilience, and tolerance to biotic and abiotic stresses [67,68]. Biostimulants provide a sustainable, non-pesticidal approach for enhancing crop performance and hardiness, ultimately reducing dependence on synthetic fertilizers and chemicals [18].

In the present study, the use of biostimulants resulted in a notable enhancement in the productivity and marketable yield of lettuce plants. The greatest plant and stem diameters, along with the highest biomass values, were obtained with treatments T6 and T7, indicating that combined or higher dose biostimulant applications significantly enhanced plant growth and productivity (Table 4). These findings confirm the promotive effect of biostimulants on shoot growth and cell expansion [69]. Several studies have recently demonstrated that biostimulants, such as those based on humic substances and amino acids, positively influence growth and yield in several plant species [52,70,71,72]. The application of biostimulants significantly increased yield (by 86–270%) compared with that observed with the control treatment (T1). The highest yield was obtained with T7, followed by T6, with the yields from both treatments being significantly higher than that obtained with the chemical fertilizer-only treatment (T2). Our findings indicate that applying the T5, T6, T7, and T8 treatments may represent a viable approach for achieving higher marketable yields per plant and enhancing the overall market value of lettuce (Figure 3). Building on these findings, it is evident that the treatments can be clearly differentiated according to their primary production objectives. The combinations employing biostimulants with half the mineral fertilizer dose (T5 and T6) stand out as a sustainable strategy, as they maintain robust plant growth and physiological performance while substantially reducing fertilizer inputs, reflecting improved nutrient-use efficiency and environmental benefits. In contrast, the application of biostimulants alongside the full fertilizer rate (T7 and T8) mainly maximizes yield and enhances physiological traits, but without contributing to a reduction in mineral fertilizer use. This distinction highlights that T5 and T6 are better suited for systems aiming to lower inputs and improve sustainability, whereas T7 and T8 are more appropriate for production systems prioritizing maximum yield and crop performance. In addition, the biostimulant-only treatments (T3 and T4) warrant brief consideration from a practical standpoint. Although these treatments resulted in moderate improvements in growth and physiological parameters compared with the control, their effects were generally lower than those observed when biostimulants were combined with mineral fertilization. This suggests that, under the conditions of the present study, biostimulants alone may not be agronomically sufficient to fully support optimal plant performance. However, it remains possible that their effectiveness could be enhanced through higher application frequencies, adjusted doses, or integration with other management practices. Discussing these aspects helps to place the biostimulant-only treatments in context, indicating their potential role as complementary tools rather than complete substitutes for mineral fertilizers. Soil and foliar applications of humic acid significantly improved lettuce growth and yield, with the highest total yield (47.86 t/ha) achieved through soil application at 1.5 mL/L, while foliar application at 4.5 mL/L notably increased plant height without influencing N, P, K, or NO₃⁻ contents [73]. The upward trend in yield observed across treatments indicates that biostimulants effectively boosted productivity, likely by enhancing nutrient uptake, photosynthetic efficiency, and stress tolerance [74,75,76].

Colour in lettuce is primarily associated with the biosynthesis of carotenoids and, in red cultivars, anthocyanins, a process that is influenced by genetic factors, temperature, water status, and N fertilization [77,78,79]. Statistically, biostimulant treatments significantly increased colour-a* (redness) values (p < 0.05), while colour-L* (lightness) and colour-b* (yellowness) values remained unaffected (p > 0.05), with the T8 treatment yielding the highest colour-a* value. These observations indicate that biostimulant application enhanced leaf pigmentation (Table 5). The results further suggest that biostimulant treatments, especially Kiana Climate combined with chemical fertilizers, may enhance reddish tones, likely by increasing anthocyanin levels or pigment formation. Bioactive compounds in plant-based biostimulants can enhance primary metabolism by increasing photosynthetic activity, which influences root growth, augments water and nutrient absorption, and ultimately improves productivity [80,81,82]. Chlorophyll levels in leaves are an indicator of a plant’s photosynthetic capacity, reflecting its ability to absorb light and turn it into chemical energy, and, ultimately, biomass [83,84]. Biostimulant treatments significantly increased the chlorophyll content in lettuce, with the highest chlorophyll a, chlorophyll b, and total chlorophyll contents observed with the T8 treatment, followed by treatments T5 and T3. This indicates that integrated applications promote chlorophyll synthesis and boost photosynthetic pigment accumulation (Table 5). Humic substance was shown to increase chlorophyll synthesis, thereby maintaining high chlorophyll levels and promoting photosynthetic activity [85]. Studies have shown that amino acid application enhances photosynthesis by promoting stomatal opening and increasing CO₂ assimilation [35,86]. In lettuce, amino acid supplementation reportedly positively influences chlorophyll buildup, with methionine boosting chlorophyll production by improving nitrogen metabolism and photosynthetic efficiency [87]. Also in lettuce, Cozzolino et al. [88] demonstrated that biostimulant applications increased chlorophyll, carotenoid, K, Ca, and total ascorbic acid contents. Leafy vegetables, such as lettuce, exhibit rapid leaf appearance and growth rates. For such vegetables, fertilization, particularly with N, is crucial for leaf formation, expansion, and the maintenance of greenness [89,90]. In lettuce, reduced N levels decrease chlorophyll, β-carotene, and xanthophyll levels, indicating that N deficiency negatively affects the plant’s green leaf pigments [77]. Conversely, microbial inoculants based on PGPR are highly effective at boosting plant growth through phytohormone production, improved nutrient accumulation, and enhanced chlorophyll stability [91].

Lettuce is a rich natural source of bioactive phytochemicals, including glycosylated flavonoids, phenolic acids, carotenoids, ascorbic acid (vitamin C), tocopherols (vitamin E), vitamin B groups, and sesquiterpene lactones [46]. Given that high vitamin C concentrations constitute an essential nutritional quality attribute of lettuce, several studies investigating the effects of biostimulants on lettuce quality have specifically focused on reporting vitamin C levels [92,93]. Al-Rikabi et al. [94] and Halshoy and Sadik [95] demonstrated that biostimulant applications significantly affected the vitamin C content (Table 6). A separate study similarly revealed that biostimulant application significantly increased lettuce plant height compared to the control treatment and foliar fertilizer applications, while also leading to higher vitamin C and dry matter contents, along with lower NO₃⁻ levels [96].

In our study, biostimulant treatments markedly enhanced the antioxidant capacity of lettuce, with the highest total antioxidant activity observed following T8 application (4312.00 µg/mL FW). This improvement relative to the control treatment indicated a substantial improvement in the plant’s oxidative defence system relative to that seen with the control application (Table 6). The result obtained with the T8 treatment is consistent with the literature. The application of various biostimulants such as humic substances [97], amino acids [98], microorganisms [99], enzymes [100], and algae [101] was shown to significantly augment the total antioxidant capacity of different plants. Moreover, our results showed that biostimulant applications significantly increased total phenolic and flavonoid contents compared to the control treatment. The highest total phenolic content was observed with T6 (47.67 mg GAE/100 g FW), while the highest total flavonoid content was recorded with T5 (28.67 mg/100 g FW) (Table 6). This indicates that different biostimulants effectively promote the accumulation of antioxidant-related secondary metabolites [81]. Giordano et al. [102] demonstrated that biostimulants increase the antioxidant capacity of lettuce by promoting the accumulation of phenolic and flavonoid metabolites. Fajdetić et al. [103] reported that biostimulants used in combination with organic fertilizers increased phenolic and flavonoid contents and antioxidant capacity in lettuce. Amino acid-based biostimulants can boost both the accumulation of total phenolic and flavonoid compounds and antioxidant activity in plants such as yarrow [104] and peach [105]. Al-Karaki and Othman [106] attributed the improvements in phenolic contents and antioxidant activity observed in plants treated with biostimulants to the presence of bioactive compounds, such as hormones, antioxidants, and amino acids, in these formulations.

Among the treatments investigated in this study, chemical fertilizer treatment resulted in the highest NH₄⁺ and NO₃⁻ concentrations. Despite being lower than those observed with chemical fertilizer treatment, the levels of these nutrients were still higher with biostimulant application than under the control treatment (Figure 4). There is a tendency to overfertilize, particularly in the production of leafy vegetables. However, excessive N fertilization can lead to high NO₃⁻ concentrations in fresh vegetables, which poses risks to human health [107]. Plants take up N in the form of NH₄⁺ and NO₃⁻. While the latter is readily absorbed, it can also leach into the soil through water runoff, polluting groundwater, rivers, and streams [108]. As carbon-rich biostimulants, humic acids can serve as crucial nutrient reservoirs. Fulvic acid enhances nitrogen absorption by supporting the activities of proteins involved in NO₃⁻ uptake and assimilation, and can even influence gene expression in plants [109,110]. At the molecular level, biostimulant-induced enhancement of nitrogen metabolism may be associated with the upregulation of key genes and enzymes such as glutamine synthetase (GS) and nitrate reductase (NR), which facilitate efficient NO₃⁻ assimilation and NH₄⁺ incorporation into amino acids [111,112,113]. Meanwhile, amino acids can hinder NO₃⁻ uptake and accumulation in plant root cells. The foliar application of amino acids has been shown to enhance nitrite reductase enzymatic activity in lettuce, thereby reducing NO₃⁻ accumulation [114]. Microbial modulation of root N uptake not only improves N assimilation efficiency but also helps regulate NH₄⁺ and NO₃⁻ accumulation in plant tissues, thereby mitigating potential N imbalances and toxic buildup [115].

Biostimulants enhance the uptake and assimilation of micro- and macronutrients by promoting changes in root architecture, such as increasing root length, density, surface area, and lateral root formation [69,116,117]. They also stimulate the exudation of low-molecular-weight compounds that activate microbial activity and increase the availability of nutrients [118,119]. In the present study, compared to the control treatment, biostimulant applications enhanced the uptake of essential nutrients, with the highest N and K contents detected with T2 application, while treatment with T7 resulted in the superior accumulation of Ca, Mg, and S. This indicated that different treatments selectively improved the assimilation of specific nutrients (Table 7). Given that fertilizers, particularly N-based ones, constitute a significant input cost for many crops due to the energy intensiveness of their production, high-input agricultural systems have become unsustainable from both environmental and energy perspectives [120]. Enhancing nitrogen use efficiency continues to be a primary goal in crop breeding and agronomic practices, necessitating the formulation of strategies that improve this parameter without sacrificing yield or crop quality [121]. Although the T7 and T8 treatments incorporated the same chemical fertilizer rate as the T2 treatment, they yielded lower N contents. This suggests that the biostimulants enhanced NUE by increasing N uptake efficiency, assimilation, or utilization within the plant [84]. Extracts of naturally occurring humic acid contain over 60 different mineral elements, including trace elements bound to humic acid molecules in forms readily usable by living organisms [122]. In maize, humic acids were shown to improve the effectiveness of mineral fertilization by boosting the uptake and balance of essential macroelements, particularly Mg, P, Ca, and K, thus influencing nutrient regulation and overall soil fertility [123]. Amino acid-based biostimulants can improve N assimilation and use efficiency, allowing for reduced N inputs without compromising yield [124]. Furthermore, amino acids aid in the mineralization of OM by balancing soil microorganisms [33]. Notably, in our study, biostimulant treatments led to an overall improvement in the micronutrient profile and soil quality, as reflected by elevated Fe, Cu, and trace Na concentrations and OM levels. In particular, the Fe content peaked under the T6 treatment, while T7 application yielded the greatest Cu concentration and soil OM enrichment (Table 8). These increases suggest that biostimulants increase micronutrient mobility and transport, likely by altering rhizospheric pH and cation exchange capacity [125]. Applications of both microbial and non-microbial biostimulants significantly increased the levels of essential micronutrients (Fe, Zn, Mn, Cu, Se) and macronutrients (Ca, K, Mg, P) in the tissues of tomato and spinach compared with those observed in untreated controls [126]. Similarly, the combined application of fulvic acid and arbuscular mycorrhizal fungi (AMF) significantly improved N uptake and utilization efficiency in tomato by enhancing root growth and rhizospheric microbial enzyme activity [127]. The long-term application of biostimulants improved soil quality by enhancing carbon sequestration, increasing humic acid stability, and intensifying humification processes, thereby contributing to greater soil fertility and the formation of more stable OM [128]. This improvement in soil OM likely enhances cation exchange capacity and nutrient buffering, contributing to sustained soil fertility and microbial activity [129].

5. Conclusions

In the present study, we demonstrated that biostimulant formulations combined with chemical fertilizers significantly enhanced the growth, yield, and nutritional quality of field-grown lettuce. Among the treatments, T6 and T7 elicited the greatest increases in marketable yield, confirming that biostimulants and mineral fertilization display synergistic interactions. The combined use of biostimulants and fertilizers enhanced fertilizer use efficiency, thus supporting optimal plant development, while concurrently reducing dependence on synthetic fertilizers and mitigating potential environmental risks. Physiological assessments revealed that biostimulant applications increased chlorophyll a, chlorophyll b, and total chlorophyll contents, indicating that photosynthetic activity and pigment biosynthesis were enhanced. This enhancement was coupled with increases in vitamin C concentrations, total antioxidant capacity, phenolic compound levels, and flavonoid contents, particularly with the T5 and T8 treatments, demonstrating that antioxidant-related metabolic pathways had been activated, contributing to the plant’s oxidative stress defence. In addition, biostimulant treatments improved the uptake and accumulation of essential micro- and macronutrients while reducing excessive NO₃⁻ levels in leaves. The treatments also increased the soil OM content, supporting long-term soil fertility and microbial activity. Overall, these findings confirm that formulations based on humic substances, amino acids, microorganisms, enzymes, and algae can serve as eco-efficient tools for enhancing lettuce productivity, nutrient efficiency, and soil health, while minimizing the environmental impact of conventional fertilization. Future studies should focus on the molecular mechanisms and long-term field validation of biostimulant–fertilizer synergies under variable environmental conditions.