Exploratory Assessment of Some Regional Food Wastes as Potential Biofertilizers: Effects on Vegetative Growth and Phytochemical Profile of Phaseolus vulgaris L.

Abstract

Developing innovative green strategies regarding the integration of by-products and waste from the food industry for sustainable agriculture and a circular economy represents a current challenge of great interest. The present exploratory study evaluated four regional potential food-waste-derived biofertilizers under controlled greenhouse conditions. Eggshell powder (EGP), whey, sea buckthorn pomace powder (SBPP), and grape pomace hydroalcoholic extract (GPHAE), used separately or in combination, were tested to observe the response of a relevant crop, Phaseolus vulgaris L. var. communis Auria Bacăului, in terms of vegetative growth parameters and its phytochemical profile. Plant biometric parameters, some representative metabolites (chlorophyll, carotenoids, polyphenolic compounds, and amino acids), and antioxidant activity were investigated using appropriate analytical techniques. The results demonstrated that bean plants grown on soil amended with EGP exhibit high biometric values, validating the efficacy of this by-product as a potential biofertilizer. The mixture of water and GPHAE used on the amended soils with EGP, and with EGP and SBPP, induces a positive effect on the total chlorophyll content accumulated in the bean samples (0.204–0.212 mg/g) compared to similar samples sprayed only with water. The mixture of water, whey, and GPHAE resulted in free amino acid accumulation in beans regardless of soil amendment. The highest synergetic effect on amino acid accumulation was found between this fertigation solution and the soil supplemented with EGP. The overall results of the present work confirm the potential of these regional food by-products and waste as biofertilizers, offering a dual solution for food industry waste management and sustainable agricultural development.

1. Introduction

Globally, food processing generates significant amounts of waste and by-products [1]. As a result, there is an imperative push to valorize or convert these materials in different domains, offering multiple benefits by maximizing the utility of our natural resources, reducing environmental harm, and ensuring long-term sustainability [2,3,4,5,6,7].

Notable wastes rich in bioactive compounds include eggshells from the poultry sector [8,9,10,11], by-products such as whey from the dairy sector [12,13,14,15,16], and pomaces from the beverage industry [17,18,19,20,21,22,23]. Over the years, researchers have attempted to find numerous strategies for the recovery of these wastes precisely because of their valuable chemical composition. In alignment with the global transition towards a bio-circular economy, the food industry embraces this approach, transforming the waste and by-products into versatile reusable resources. Food waste can be upcycled into a diverse spectrum of high-value products, ranging from sustainable energy sources such as biofuels to everyday materials such as bioplastics, or could contribute to ensuring global food security due to its content of bioactive compounds [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23]. Rapid global population growth has led to a sharp increase in demand for food, creating significant obstacles in expanding agricultural production without harming the environment [24]. Although chemical fertilizers are widely used to enhance crop yields, this approach is increasingly unsustainable. Biofertilizers can provide an affordable and environmentally friendly solution by using locally sourced materials, making them a versatile and easily accessible tool [25,26,27,28,29].

Repurposing food waste and by-products into biofertilizers or biostimulants serves a dual purpose, promoting circular management while simultaneously enhancing soil vitality and crop yields [24,25,26,27,28,29,30,31]. Additionally, improvements can be expected in the metabolite content of plants grown in soil using biofertilizers. Various formulations of bio-based fertilizers have been developed using food by-products such as eggshells [32,33,34,35,36], fruit peels [34,35,36], whey [37,38,39,40,41,42], grape pomace [43,44,45,46,47], and spent coffee grounds [5,26]. The efficacy of food-derived by-products as biofertilizers often depends on their physical state and stability. For example, water-soluble fertilizers are suitable for fertigation, improving irrigation water with nutrients for optimal plant growth. Studies evaluate these matrices separately or as mixtures, either without any modification or subjected to preliminary processing, such as digestion or fermentation (

Table 1. Some food waste and by-products used as biofertilizers.

Physical State	Characteristics	Example	Refs.
Solid (powder)	Improves soil amendment Slow release of nutrients	Eggshells Fruit peels Grape pomace Whey	[32,33,34,35,36] [34,35,36] [43,44] [37]
Liquid	Appropriate for fertigation High bioavailability of nutrients	Whey Grape pomace extract	[38,39,40] [47]
Mixture	Usually subjected to digestion or fermentation Synergetic effect and multi-nutrient enrichment	Eggshells + fruit peels Different waste + spent coffee grounds Kitchen food waste	[29] [5,26] [27]

).

Eggshells represent one of the most well-known by-products used as biofertilizers, playing an important role in plant growth and yield due to their bioavailable calcium (Ca), a vital element for maintaining cell wall structural stability and cellular homeostasis balance [32,33,34,35,36]. Prior to soil integration, thermal pretreatment is recommended to reduce microbiological contamination, ensuring enhanced crop performance. It can be used alone or in combination with other food waste, such as fruit peels [29], spent coffee grounds, or coffee husks [5].

Similarly, as an important by-product of the dairy industry, whey can serve as a viable substrate for biofertilizer formulation due to its chemical profile, which is characterized by significant concentrations of minerals and organic matter, including lactose, essential amino acids, and proteins [37,38,39,40,41,42].

Extensive research has identified pomace from the alcoholic and non-alcoholic beverage industry as a high-value by-product, precisely due to its chemical composition, which is rich in polyphenols and other bioactive phytochemicals [17,18,19,20,21,22,23]. Comprehensive analyses of the available literature show that different approaches and strategies have been used to successfully apply pomace (e.g., grape pomace or sea buckthorn pomace) in different fields [17,18,19,20,21,22,23]. Composting is considered a robust strategy for stabilizing grape pomace, enhancing its agronomic utility [44,45,46]. Existing research has shown that the application of composted grape pomace not only improves soil fertility and optimizes plant nutrient assimilation but also facilitates high-value waste valorization, aligning perfectly with the principles of circular bio-economy. On the other hand, despite the significant level of bioactive compounds existing in sea buckthorn pomace, its potential as a biofertilizer is under-researched. Very few recent studies have designated this by-product as a worthy alternative carrier for nitrogen-fixing bacteria [48,49,50].

In this context, it should be mentioned that the regional food sector of Moldova, Romania is well represented by poultry, dairy, and viticulture industries. Sea buckthorn is cultivated in our region not only for fresh consumption, but particularly for its juice and oil, which are obtained by pressing the berries. All these industries generate a significant amount of waste and by-products. Collaboration between researchers and producers in order to find solutions for valorizing these materials is imperative for repurposing industrial wastes and building a more sustainable economy.

From this perspective, as a core objective of our doctoral research, the identification and development of ecological recovery pathways for regional food industry waste and by-products have materialized in several studies, such as the use of eggshells as an adsorbent for heavy metals or the effect of pomace extracts for the development and growth of plants in vitro [9,39,47].

Therefore, through collaborations with regional food producers, together with the Vegetable Research and Development Station Bacău (Bacău County, Romania), an opportunity arose, focusing on the bioconversion of food by-products into potential valorized biofertilizers. To the best of our knowledge, no greenhouse cultivation study using eggshells, whey, grape pomace, and sea buckthorn pomace as prospective biofertilizing agents has been reported thus far.

From a sustainability perspective, developing innovative green strategies regarding the agricultural integration of by-products and waste from the food industry represents a substantial challenge. Exploratory assessment usually begins with observable biological outcomes, such as biometric parameters or metabolite accumulation, and results may frequently be limited by a narrow experimental scope, necessitating multi-step validation [51].

Hence, the present study aims to investigate the biofertilizing potential of four regional food by-products—separate and combined—with respect to vegetative growth and the phytochemical profile of Phaseolus vulgaris L. var. communis Auria Bacăului. Beans represent an important vegetable crop worldwide due to their significant content of bioactive compounds with antioxidant benefits and their highly nutritious properties as sources of plant-based protein and essential amino acids [52,53,54]. The quality of seedlings and the young plant stages of Phaseolus vulgaris L. is critical for determining plant vigor, biomass accumulation, and future yield potential. Early traits such as root length, leaf area, internode development, and pigment content directly influence the plant’s ability to grow, its photosynthetic capacity, and its potential to adapt to environmental conditions [55]. At the same time, the phytochemical profile—including polyphenols, flavonoids, glucosinolates, carotenoids, and antioxidant capacity—plays a key role in stress tolerance, nutritional value, and metabolic regulation. These compounds are highly dynamic during early growth and can be shaped by seed origin, growth media, and environmental factors. Together, vegetative growth parameters and phytochemical composition provide essential indicators of plant health, resilience, and quality, making them fundamental for breeding, cultivation strategies, and improving crop performance in legumes.

In this view, in the current research, eggshell powder and sea buckthorn pomace powder were evaluated as potential soil fertilizers, and whey and grape pomace extracts were evaluated as possible biostimulating agents in fertigation solutions—separately and simultaneously—for bean crops grown under controlled greenhouse conditions. Measuring vegetative growth parameters and quantifying metabolite accumulation in bean leaves, such as chlorophyll contents, carotenoids, polyphenolic compounds, and amino acids, represent key aspects of our research to determine the effects of these specific food-waste and by-products. In addition, to emphasize the importance of the accumulation of these compounds in the plant growth process, DPPH (2,2′-diphenyl-1-picrylhydrazyl) radical scavenging activity was evaluated. This approach also aims to integrate the application of appropriate, simple, and efficient analysis methods, such as UV–Vis spectrometry and high-performance thin-layer chromatography, to support a truly sustainable working methodology. This study is intended as an exploratory step that needs to be continued in several stages in an attempt to find green solutions, contributing both to reducing waste from the food industry and to sustainable agriculture.

2. Materials and Methods

2.1. Food Waste and By-Products Used as Biofertilizers—Source and Preparation

The sources of food waste and by-products, along with the steps for their use as biofertilizers, are presented in

Table 2. Food waste and by-products used in the present study.

Waste/By-Products	Source	Preparation Details	Abbreviation
Chicken eggshells and quail eggshells (mixed in equal proportion)	Chicken eggshells from Agricola International Bacău (Bacău County, Romania) [56] Quail eggshells from a micro-farm from Petru-Vodă (Neamț County, Romania) [57]	Washing with tap water to eliminate impurities Rinsing with distilled water Drying in an air circulation oven (Memmert Universal, model UFE 500, Memmert GmbH + Co.KG, Schwabach, Germany) at 100 °C for 30 min Milling for 3 min using an electric grinder (Heinner, model HCG-150SS, 150 W, Heinner, Bucharest, Romania) Stored in a desiccator until use [9]	Eggshell powder EGP
Whey	Cow sour whey acquired from Călugăru micro-farm from Dărmănești (Bacău County, Romania)	Stored in a refrigerator Used in its crude form, without any modification, and mixed in established proportions with water	Whey
Grape pomace	Grape pomace from red variety of Vitis vinifera L. var. Fetească neagră from Moșia Panciu wine producer (Vrancea County, Romania) [58]	Stored in the freezer at −18 °C Thawed slowly before extraction Hydroalcoholic extract obtained via classical agitation using a magnetic stirrer (Nahita Blue, model 692, Auxilab S.L., Navarre, Spain) at room temperature for 12 h Solvent: ethanol:distilled water 50:50 (v/v) Grape pomace:solvent 1:5 (w/v) [47]	Grape pomace hydroalcoholic extract GPHAE
Sea buckthorn pomace	Derived from local cultivation, the sea buckthorn pomace (Hippophae rhamnoides L.) provided by a micro-farm from Lichitișeni S.C. Eco Catena S.R.L. (Bacău County, Romania) [59]	Stored in the freezer at −18 °C Dried using food dehydrator (Biovita model Deluxe-6, Biovita, Cluj-Napoca, Romania) at 70 °C for 12 h Milling for 2 × 5 min using an electric grinder (Heinner, model HCG-150SS, 150 W, Heinner, Bucharest, Romania) Stored in a refrigerator until use	Sea buckthorn pomace powder SBPP

.

2.2. Experimental Greenhouse Conditions

The research took place during the spring 2024 season at the Vegetable Research and Development Station Bacău (Bacău County, Romania) [60]. The site is located at coordinates 46°34′ N, 26°55′ E, with an elevation of 165 m above sea level. The experiments were performed in an Almería-type greenhouse protected by UV-stabilized polyethylene film where day/night temperatures were maintained at 25–30 °C/18–20 °C, and the relative humidity was 60–75%. All environmental variables were subjected to continuous monitoring throughout the duration of the experiments to maintain stability and accuracy.

Beans (Phaseolus vulgaris L. var. communis Auria Bacăului) were grown in pots of size 10.5 cm (diameter) × 8 cm (height). Bean seeds were provided by the Vegetable Research and Development Station Bacău (Bacău County, Romania).

Commercial universal potting soil from Compo Sana^® (Compo Group, Münster, Germany) was used. The soil composition, as declared by the manufacturer, is as follows: Peat H2–H7, perlite, lime, NPK fertilizer, Agrosil (silicon phosphate); salinity (KCl): <3.0 g/L; pH (CaCl₂): 5.0–6.5; nutrients: 200–450 mg/L soluble nitrogen (CaCl₂); 200–500 mg/L soluble phosphorus (P₂O₅); 300–550 mg/L soluble potassium (K₂O) [61].

The experimental design comprises four types of distinct samples with respect to soil: a control (soil without any addition) and three other amended soil samples containing eggshell powder (EGP) and/or sea buckthorn pomace powder (SBPP). EGP and SBPP were incorporated, mixed with the soil, and then added to the pots. Two bean seeds were placed on the seeding medium for each variant at an appropriate depth and distance.

All soil amendments were applied at a total weight-to-weight ratio of 10% relative to the soil, as presented in

Table 3. Fertilization strategy and experimental details.

Samples Codes	Soil	Fertigation Solution
		Water (a)	Water + Whey (Ratio 20:80 v/v) (b)	Water + Whey + GPHAE (Ratio 19:76:5 v/v/v) (c)	Water + GPHAE (Ratio 95:5 v/v) (d)
Fs1	Control (s1) (soil without any amendment) 220 g	Fs1-a (a1, a2, a3, a4)	Fs1-b (b1, b2, b3, b4)	Fs1-c (c1, c2, c3, c4)	Fs1-d (d1, d2, d3, d4)
Fs2	Soil + EGP (s2) (ratio 100:10 w/w) 200 g:20 g	Fs2-a (a1, a2, a3, a4)	Fs2-b (b1, b2, b3, b4)	Fs2-c (c1, c2, c3, c4)	Fs2-d (d1, d2, d3, d4)
Fs3	Soil + SBPP (s3) (ratio 100:10 w/w) 200 g:20 g	Fs3-a (a1, a2, a3, a4)	Fs3-b (b1, b2, b3, b4)	Fs3-c (c1, c2, c3, c4)	Fs3-d (d1, d2, d3, d4)
Fs4	Soil + EGP + SBPP (s4) (ratio 100:5:5 w/w/w) 200 g:10 g:10 g	Fs4-a (a1, a2, a3, a4)	Fs4-b (b1, b2, b3, b4)	Fs3-c (c1, c2, c3, c4)	Fs4-d (d1, d2, d3, d4)

F—“fasole” beans (in Romanian); Fs1, Fs2, Fs3, and Fs4—bean samples grown on different soils; EGP—eggshell powder; SBPP—sea buckthorn pomace powder; GPHAE—grape pomace hydroalcoholic extract; a, b, c, and d—the fertigation solution used (a—water; b—mixture of water and whey; c—mixture of water, whey, and grape pomace hydroalcoholic extract; d—mixture of water and grape pomace hydroalcoholic extract); indices 1, 2, 3, and 4 (e.g., a₁, a₂, a₃, a₄) represent the number of replicates for each sample.

. Keeping organic amendments at low percentages (10–15%) is typical practice to prevent phytotoxicity.

Regarding the experimental design of fertigation, foliar spraying was carried out with four different solutions: water as a control and three other combinations in which different proportions of whey and grape pomace hydroalcoholic extract (GPHAE) were added to the water (Table 3). Foliar watering was carried out using a manual sprayer in the morning every two days [62], ensuring a regular supply of moisture. In total, 30 mL was applied to each pot per spraying event. The pots were rotated to minimize positional effects.

As the specific combination of food waste used in this study is novel, the doses established did not previously exist in other studies, but they are based on our previous research [39,47] and some preliminary experiments carried out in the laboratory. Specifically, a 1:4 (v/v) ratio for water:whey was selected based on its proven efficacy in our previous study on different plant species, such as wheat, soybean, and broccoli; this ratio produced good results with respect to both fresh biomass and soybean root weight [39]. Regarding the GPHAE, the application ratio was established based on our in vitro tissue culture study on oregano, where media supplemented with 1% and 5% extracts yielded the highest biometric performance in explants [47]. Furthermore, the final suitability of these ratios was validated through preliminary laboratory trials conducted explicitly on bean plants.

The following coding was used: F (from “Fasole” in Romanian) for beans; s for soil (s1, s2, s3, and s4); a, b, c, and d for the watering solution used. Four replications were performed for each type of fertigation solution, resulting in a total of 64 variants. Considering that the commercial substrate already contains fertilizer and in order to observe the real effect of food-waste treatments, a control group was established, denoted as Fs1-a (a commercial substrate was used, and spraying was carried out with water only). This allowed for measurement of the baseline growth performance provided by the substrate’s pre-existing NPK content. By comparing the food-waste treatments against this control, an additional or synergistic effect can be related to the food-waste treatments. Any significant growth enhancement observed in the treatment groups relative to the control can be directly attributed to the food-waste applications. Using a standard commercial substrate reflects real horticultural practices, demonstrating that food-waste derivatives can provide supplemental benefits even in nutrient-sufficient environments.

Plant collection was carried out 30 days after the experiment was set up—during the vegetative stage of development.

2.3. Determination of Vegetative Growth Parameters and Phytochemical Screening of Collected Leaves

The growth indicators recorded include germination percentage, aerial plant average height, root weight and length, and fresh biomass (stems and leaves).

For the final measurements, both seedlings per pot were kept. Each plant was measured, and the average of the four variants for each sample was calculated.

The germination percentage (GP) represents the proportion of seeds that sprouted and is calculated using the following Formula (1):

GP [%] = (Number of germinated seeds/Total seeds sown) × 100

(1)

A ruler was used to measure aerial plant height and root length. The average height was calculated as the mean of the minimum and maximum plant height values.

The fresh biomass was weighed using a precision balance (KERN ABT220-4M, sensitivity 0.1 mg, KERN & SOHN GmbH, Bensheim, Germany).

The phytochemical response of plants to the use of the considered biofertilizers was first evaluated by scanning the obtained leaf extracts using UV–Vis spectroscopy.

Furthermore, the quantification of metabolite contents, such as total chlorophyll, carotenoids, and polyphenolic compounds, was carried out using the specific methods described in detail below. All spectrophotometric absorbance readings were recorded using a UV–Vis spectrophotometer (Shimadzu UV-1280, Shimadzu Corporation, Kyoto, Japan). Moreover, the rapid comparative estimation of amino acid content was performed using the HPTLC (high-performance thin-layer chromatography) technique and CAMAG HPTLC system (CAMAG, Muttenz, Switzerland). All solvents and reagents used for these determinations—namely, acetone, ethanol, methanol, Folin–Ciocalteu reagent, sodium carbonate, gallic acid, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), and amino acid standards (L-valine, L-leucine, L-tryptophan, and L-cysteine)—were of analytical grade and were purchased from Sigma-Aldrich Chemical Company (Darmstadt, Germany).

2.3.1. Determination of Total Chlorophyll and Carotenoid Content

In order to determine the chlorophyll content of bean leaves, the 80% acetone extraction method was used. In particular, 80% acetone (10 mL) was added to 0.5 g of fresh leaves, carried out in a low-light environment to prevent chlorophyll degradation. The samples were stirred at room temperature and 150 rpm for 30 min using a magnetic stirrer (Nahita Blue, model 692, Auxilab S.L., Navarre, Spain). The extracts were carefully filtered through Whatman paper and diluted to 50 mL with acetone. In order to determine chlorophyll contents, absorbance readings were taken at 645 and 663 nm using 80% acetone as a blank. Along with the determination of total chlorophyll in the acetone extract (ExAcet), carotenoid contents were also targeted by reading absorbance at 480 and 510 nm using the same spectrophotometer [63,64,65].

The values for chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (total Chl), and carotenoid content were calculated according to the formulas in

Table 4. Calculation formula for determination of chlorophyll and carotenoid content.

Parameters	Calculation Formula
Chl a [mg/g]	(12.7 * A663) − (2.59 * A645) * V/1000 * W
Chl b [mg/g]	(22.9 * A645) − (4.7 * A663) * V/1000 * W
Total Chl [mg/g]	(8.2 * A663) + (20.2 * A645) * V/1000 * W
Carotenoid [mg/g]	(7.6 * A480) − (1.49 * A510) * V/1000 * W

A₆₆₃ = Absorbance measured at 663 nm; A₆₄₅ = absorbance measured at 645 nm; A₄₈₀ = absorbance measured at 480 nm; A₅₁₀ = absorbance measured at 510 nm; V = volume of extract; W = fresh weight of tissue extracted.

.

2.3.2. Determination of Total Phenolic Content and Antioxidant Activity via the DPPH Method

Ethanolic extracts (ExEth) were prepared for the evaluation of these parameters. For this purpose, 0.25 g of fresh leaves was extracted with 10 mL of 80% alcohol. The samples were moderately stirred at 150 rpm for 24 h using a magnetic stirrer (Nahita Blue, model 692, Auxilab S.L., Navarre, Spain) at room temperature, after which filtration was performed through Whatman paper. The filtrate was stored in brown bottles and refrigerated in order to determine the total phenolic content (TPC) and radical scavenging activity (RSA) via the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) method.

The total phenolic content of leaf extracts was determined using the Folin–Ciocalteu method [66]. In total, 50 µL of extract was mixed with 150 µL of 80% ethanol, 400 µL of Folin–Ciocalteu reagent, and 4 mL of distilled water. After 3 min, 2 mL of sodium carbonate solution was added. The resulting mixture was left in the dark for 1 h. The absorbance of the samples at 765 nm was recorded using the same spectrometer mentioned above. A standard curve was generated using gallic acid (0–500 ppm), and TPC was expressed as mg GAE (gallic acid equivalent)/100 g.

The free radical scavenging activity (RSA) of the leaf extract was tested using the DPPH procedure and an improved protocol [67]. The stock solution was prepared from 24 mg of DPPH dissolved in 100 mL of methanol. The DPPH working solution was obtained from 10 mL of stock solution and 40 mL of methanol. Then, 100 µL of leaf extract was combined with 3 mL of DPPH working solution, and the tubes were kept for 30 min in the dark. As for the standard, it was prepared identically, with the exception that instead of the leaf extract, 100 µL of methanol was used. Absorbance was then recorded at 517 nm, and the following formula was used to calculate the percentage of DPPH radical scavenging activity or antioxidant activity:

DPPH radical scavenging activity [%] = [(A_c − A_s)/A_c] × 100

(2)

where the variables are defined as follows: A_c = absorbance measured at 517 nm for the control sample; A_s = absorbance measured at 517 nm for the tested sample.

2.3.3. Screening of Free Amino Acids Profile Using HPTLC Technique

The HPTLC technique was applied for a quick assessment of the free amino acid profile of leaf extracts [68,69]. In this view, chromatographic separation was carried out on 20 cm × 10 cm HPTLC silica gel 60 F254 plates (Merck, Darmstadt, Germany). A semi-automatic sample applicator, CAMAG^®Linomat 5(CAMAG, Muttenz, Switzerland), was used for the application of 8 mm band spots using a spray-on procedure. Before analyzing samples using the HPTLC technique, preliminary tests were performed using the TLC technique to establish the most suitable mobile phase, as well as the amino acids used as references. Four amino acids (L-valine, L-leucine, L-tryptophan, and L-cysteine) were used as standards and applied at a volume of 8 μL (1 mg/mL in distilled water). The developing step of the plates was performed in a chamber containing a mixture of butanol–acetic acid–water (40:10:50, v/v/v) as the mobile phase. After the developing set, the plate was dried using a household hair dryer. Derivatization was performed by spraying chromatographic plates with a 2% ninhydrin solution (0.2 g in 100 mL of 96% ethanol, acidified with 3 mL of acetic acid), and the plates were dried again for 3 min. The colored spots on the chromatograms were then scanned at 512 nm using CAMAG TLC Scanner 3 (CAMAG, Muttenz, Switzerland) and analyzed with the VisionCats CAMAG HPTLC software (version 3.0).

Based on a comparison of spots with the standard used and their retention factor (Rf) values, specific amino acids for each leaf extract were identified.

2.4. Statistical Analysis

All data are expressed as the mean ± standard deviation (SD) of four replicates for each sample. Pearson correlation analyses of the data generated were completed using the Microsoft EXCEL 2010 Statistical Tool Package in order to establish a relationship between different recorded parameters. Graphical representations were created using the Origin 2024 10.1.0.170 Academic software.

R-Commander (version 4.4.3) and FactoMineR plugins for multivariate analysis [70] were used to perform principal component analysis (PCA) and hierarchical cluster analysis (HCA) to emphasize the resemblances or dissimilarities between the samples.

3. Results and Discussion

3.1. Germination and Vegetative Growth Parameters of Plants Developed Under the Studied Conditions

The results of growth indicators (germination percentage, average plant height above ground, root weight and length, and fresh biomass) are presented below.

It is well known that beans germinate quickly due to their larger seeds and higher nutrient storage, often showing high germination percentages within 3–4 days.

The germination percentage reached high values, exceeding 90% within the first 3 days, in samples where water (a) and a mixture of water and whey (b) were used as the watering agent.

For the bean samples sprayed with solutions c (water + whey) and d (water + whey + GPHAE), the germination rate was slower after the first 4 days (with values ranging between 50 and 87.5%) but, ultimately, most samples reached a germination percentage of 100% after 7 days (Table S1 in Supplementary Materials). Three variants in which only one of the four replications had a germination rate of 50% (Fs1-c₁, Fs1-d₃, and Fs4-c₁) were the exceptions.

Pictures of the experimental setup and some comparative replicates of bean plants, obtained during the greenhouse experiment, are shown in Figure 1.

The biometric parameters, such as aerial plant average height, fresh biomass, and root weight and length for bean plants, are graphically represented in Figure 2. The recorded data are reported in Table S2 of the Supplementary Materials.

Analyzing the biometric results of bean samples depending on soil and fertigation solution, the following were observed:

–

In the case of samples grown on soil (s1) without any addition, the highest biometric parameter values were recorded for the control sample Fs1-a (average height: 54.37 cm; fresh biomass: 28.62 g; root weight: 2.90 g). Treatment with the water + whey + GPHAE mixture resulted in a significant decrease in fresh biomass (4.08 g) for Fs1-c variants, and a 7-fold lower value compared to Fs1-a was detected (see Table S2 in Supplementary Materials).

–

Regarding the samples developed on soil amended with EGP, the Fs2-a variant (sprayed with water) presented the highest average height values (55.50 cm), comparable with Fs1-a (54.37 cm), and it had a fresh biomass of 24.55 g. For plants watered with the GPHAE solution (Fs2-d), the average plant height reached a value of 49.37 cm. It can also be noted that bean plants grown on soils supplemented with EGP and watered with b (Fs2-b), c (Fs2-c), and d (Fs2-d) developed better than similar ones watered with the same watering solution but on different soils. For example, samples watered with Fs2-b show values of 39.63 cm/18.97 g/3.69 g (average height/fresh biomass/roots weight), in contrast with Fs1-b (36.76 cm/11.19 g/1.80 g), Fs3-b (33.08 cm/9.96 g/1.36 g), and Fs4-b (23.96 cm/9.91 g/1.39 g) (see Table S2 in the Supplementary Materials). In accordance with other studies, our results showed that soil amended with eggshell powder represents a worthy biofertilizer [32,33,34,35,36].

–

Samples grown on soil amended with SBPP (s3) showed lower fresh mass values compared to corresponding plants on soil without additions (s1) and soil with the addition of EGP (s2), regardless of the fertigation solution (with one exception; namely, sample Fs1-c, which has slightly higher biometric parameter values than sample Fs3-c).

–

The variants developed on soil with a mixture of EGP and SBPP (s4) behaved generally similarly to those grown on soil with SBPP only (s3). When linked with samples collected from EGP-amended soil (s2), it can be observed that the values of biometric parameters were lower for all fertigation variants. Particularly, in the case of Fs4-a, the fresh biomass value was doubled (23.90 g) compared to Fs3-a (12.32 g) and was very close to Fs2-a (24.55 g). Among all samples developed on soil amended with EGP and SBPP, Fs4-a stands out for its second-highest root mass value (3.03 g) (see Table S2 from Supplementary Materials).

From the perspective of fertigation solutions, it can be noted that mixtures b (water + whey), c (water + whey + GPHAE), and d (water + GPHAE) result in increased values with respect to all measured parameters when plants are grown on soil (s2) with the addition of EGP compared to variants developed on other amended soils (Figure 2).

A remarkable behavior was observed in the case of samples watered with whey-containing solutions (Fs2-b); namely, their root weight presented the highest value among all samples (3.69 g), which indicates not only a synergistic effect of whey and eggshell compounds on root development but also on fresh biomass (18.97 g). According to a previous study on soybeans (a legume plant from the same Fabaceae family as beans), the results showed that whey added to the watering solution had a positive influence on root development, as well as on the total weight of aerial parts [39]. This synergy is likely determined by the concomitant presence of calcium derived from eggshells and organic nutrients derived from whey, creating a highly favorable microenvironment that promotes both lateral root branching (mass) and overall vegetative vigor (fresh biomass).

Moreover, the fertigation solution containing GPHAE produced a good result with respect to the aerial plant average height (49.37 cm) in soil with EGP (Fs2-d).

A very good Pearson correlation (0.88) can be observed between the fresh mass of the aerial parts and the average height of bean plants. The root average length exhibited a high positive correlation with the height of plants (Pearson coefficient of 0.83) and fresh biomass (Pearson coefficient of 0.80) (see Table S3 in the Supplementary Materials). Moreover, root weight is well correlated with fresh biomass, with a Pearson coefficient of 0.63.

3.2. UV–Vis Phytochemical Screening of Bean Plant Extracts

Prior to the quantification of metabolite accumulation in plants, such as chlorophyll or polyphenolic compounds, the obtained extracts were subjected to phytochemical screening to obtain a comparative spectrophotometric fingerprint of different samples. Thus, both the acetone and hydroalcoholic extracts of bean leaves were evaluated in the range of 190–1100 nm, and their recorded profile spectra are presented in Figure 3.

It can be observed from Figure 3A that all acetone extracts presented a similar profile, with two significant peaks specific to chlorophyll at λ = 664.5 nm and λ = 434 nm, in accordance with the literature [71]. Considering that the same method was used to obtain the extracts (same proportion of plant material and solvent, same time, etc.), the slightly different intensity of the peaks could be due to the lower or higher chlorophyll content of each sample, thus reinforcing the fact that the soil and watering agent used can influence the accumulation of metabolites such as chlorophyll or carotenoids.

The ethanolic spectra (ExEth) of leaves (Figure 3B) exhibited a different phytochemical fingerprint than those extracted with acetone, and this is certainly due to the higher polarity of ethanol and, thus, the wider spectrum of compounds that were extracted. First, higher peak intensities and some additional peaks compared to ExAcet are observed. The identified peaks were as follows: λ = 667 nm, λ = 538–538.5 nm, λ = 462–463.5 nm, and λ = 440.5–441 nm. Studies on various leaf extracts, using more sophisticated detection methods, show that photosynthetic pigments, including derivatives of chlorophyll and carotenoids (e.g., lutein, neoxanthin, xanthophylls), absorb heavily in the 400–550 nm range [72]. Moreover, the broad peak between 290 and 490 nm increased its area, indicating the presence of polyphenolic compounds in the ethanolic extracts, as these molecules are known to highly absorb and overlap in this range [73,74].

Thus, the obtained spectra demonstrate the complexity of metabolites accumulated in the vegetative state of plants. Samples Fs2-a-ExEth and Fs4-d-ExEth exhibited higher peak intensities compared to other extracts.

3.3. Investigation of Metabolite Accumulation and Antioxidant Activity

In order to reveal the effect of the food-waste fertilizers on the metabolites accumulated in bean leaves, the content of chlorophyll, carotenoids, and polyphenolic compounds was determined. Additionally, DPPH (2,2′-diphenyl-1-picrylhydrazyl) radical scavenging activity was evaluated.

The results of the phytochemical parameters for bean leaf extracts are graphically represented in Figure 4 using a logarithmic scale (Table S4 in the Supplementary Materials).

Chlorophyll levels varied widely among the bean plants collected in the tested conditions, with values ranging from a minimum of 0.049 mg/g in sample Fs2-d to a maximum of 0.375 mg/g in sample Fs1-a.

Thus, for the variants watered with the same fertigation agent but grown on different soils, it can be seen that the most homogeneous results occur in the case of whey, where the total chlorophyll content is between 0.143 and 0.279 mg/g. In the series sprayed with water (a) and mixture c—containing water, whey, and GPHAE—the lowest chlorophyll values were recorded for soil s3; namely, Fs3-a (0.071 mg/g) and Fs3-c (0.079 mg/g).

In the case of samples sprayed with solution d, the level of accumulated chlorophyll was reasonably high (0.204–0.362 mg/g), with the exception of sample Fs2-d (grown on soil supplemented with EGP), where the value was surprisingly low (0.049 mg/g) despite the fact that the biometric parameters were the best for this variant. Good chlorophyll content values were observed for Fs1-d, Fs3-d, and Fs4-d, reinforcing that GPHAE can constitute a sustainable biofertilizer if added in an appropriate proportion to the fertigation solution, in accordance with our in vitro results on oregano [47].

The series of bean plants grown on the same soil accumulated different chlorophyll levels depending on the fertigation agent. Thus, for the variants on soil without amendment (s1), it can be observed that the Fs1-d variant presented a total chlorophyll content value (0.362 mg/g) very close to that of the control sample Fs1-a (0.375 mg/g). The lowest chlorophyll content was accumulated for samples grown on soil supplemented with SBPP Fs1-c (0.093 mg/g). Chlorophyll levels in sample Fs2-c (0.281 mg/g) were significantly higher than in the other s2 soil treatments: twice the amount found in Fs2-b and approximately six times that of Fs2-d.

When examining samples grown on the s3 soil amended with SBPP, the most efficient watering agent in terms of chlorophyll accumulation was the mixture of water and GPHAE (d), followed by whey (b). In the case of these variants, the level of accumulated chlorophyll pigment was 0.212 mg/g for Fs3-d and 0.153 mg/g for Fs3-b.

The chlorophyll content in samples developed on soil s4 presented values ranging between 0.122 and 0.249 mg/g. Of all fertigation options, the replicates sprayed with whey (Fs4-b) proved to be the best in terms of chlorophyll accumulation. It can be observed that the results of the quantitative chlorophyll analysis were consistent with the phytochemical profile of the acetone spectra (Figure 3A), with the highest peaks corresponding to samples with the richest chlorophyll content (e.g., Fs1-a-ExAcet, Fs1-d-ExAcet).

Carotenoid content is strongly related to total chlorophyll content, with the Pearson correlation coefficient being 0.86, as shown in Table S5 of the Supplementary Materials.

Regarding the total phenolic content (TPC) of bean plants, values between 114.27 mg GAE/100 g (sample Fs3-d) and 274.55 mg GAE/100 g (sample Fs1-b) were recorded.

In addition to Fs1-b, samples Fs3-b and Fs1-c also exhibited notably high TPC values that were very strongly correlated with high DPPH radical scavenging activity values (Pearson coefficient: 0.91; see Table S5 in the Supplementary Materials). In contrast, samples Fs3-a and Fs3-d showed the lowest TPC and DPHH% values compared to Fs1-b. Samples sprayed with a mixture containing GPHAE accumulated a moderate polyphenol content, probably due to the low proportion of GPHAE in the solution mixture. Conversely, the use of solution d as a fertigation agent resulted in high chlorophyll and carotenoid accumulation in soils s1, s3, and s4 and in good aerial part height development in bean plants.

3.4. Free Amino Acid Screening Using the HPTLC Technique

Beans are well known for their nutritional value. Common beans are widely recognized as one of the best plant-based sources of protein [52,53,54]. Beyond their essential role in the synthesis of proteins, free amino acids serve as precursors for the biosynthesis of secondary metabolites [75,76,77].

To further evaluate the compositional profile of bean extracts, the HPTLC test was completed. This accessible and reliable chromatographic technique can provide important information regarding amino acid accumulation in plants developed in greenhouse conditions. The standard amino acids used were as follows: L-valine (Val), L-leucine (Leu), L-tryptophan (Trp), and L-cysteine (Cys). The retention factor values for the standard amino acid solution are in accordance with the literature [78]: Rf_Val = 0.450, Rf_Leu = 0.585, Rf_Trp = 0.614, and Rf_Cys = 0.384 (see HPTLC digital scanning profiles of chromatograms at 512 nm—Figure S1 from Supplementary Materials).

After the completion of derivatization with a ninhydrin solution, the chromatographic plates were scanned at 512 nm, and their isometric profile are shown in Figure 5 (see also Figure S2 in the Supplementary Materials). The effect of soil formulation and the fertigation solution on amino acid accumulation in the vegetative phase of bean plants is reflected in the different qualitative profiles of the leaf extracts. Beans are considered a good source of lysine, and leucine is considered a good source of essential amino acids, but both contain limited amounts of methionine and tryptophan [53].

Analyzing the chromatograms of bean leaf extracts, it can be observed that leucine is present in all bean samples. Lysine could not be identified, most likely due to the low Rf in the mobile phase used. Tryptophan was detected only in two samples (Fs1-a and Fs2-a) (see Figure S3 in the Supplementary Materials). Moreover, cysteine appears to be present in some samples. It is well known that cysteine is important due to its role in plant health, its synergy with other bioactive molecules, and its implication in post-collection storage [79].

The samples with the highest leucine content are Fs1-b and Fs3-b, as shown in Figure 5A (track 5 and track 7, respectively), and Fs2-c and Fs4-c, as shown in Figure 5B (track 2 and track 4). The extracts obtained from leaves collected from samples watered with the fertigation mixture containing water + whey + GPHAE (c) are distinguished by a uniform accumulation of amino acids regardless of the soil supplementation; in addition to leucine, significant amounts of valine are also observed.

The Fs2-c sample stands out with the highest amino acid content (Figure 6), even though it did not have high biometric parameter values. From this result, it can be observed that all biofertilizers used in this study acted synergistically with respect to amino acid accumulation.

3.5. Principal Component Analysis and Hierarchical Cluster Analysis for the Measured Biometric and Phytochemical Parameters

From the PCA presented in Figure 7, it can be observed that all measured biometric and phytochemical parameters are located on the right side of the quadrant.

In the case of biometric parameters, the first two principal components explained 92.67% of the total variance, with PC1 and PC2 accounting for 77.72% and 14.96%, respectively. It can be seen that all variables point strongly toward the positive side of dimension 1. Arrows corresponding to fresh biomass, aerial part height, and root average length are located in proximity, showing that plants with greater shoot biomass also tend to have longer roots and taller aerial parts. This strong positive correlation reflects a well-known functional equilibrium in plant development; in particular, plants with greater shoot biomass inherently require taller aerial parts for light interception, which must be supported by a proportional elongation of the root system for physical anchorage and deep-soil supply acquisition. Only root weight points strongly upward on dimension 2. PC2 separates root biomass from shoot or length traits, which means that some treatments may specifically increase root mass, and others may increase shoot growth or length more than root weight.

Biologically, this implies that while root length and shoot expansion are heavily synchronized (driven by PC1), root mass accumulation can operate independently (driven by PC2). As a result, certain experimental treatments may trigger a morphological shift—favoring resource allocation toward root thickening and lateral branching (increased weight) rather than vertical elongation (length and height).

PCA for phytochemical parameters showed that the first two principal components explained 95.39% of the variability, with the first principal component accounting for 51.98%. Total chlorophyll and carotenoid contents found on the upper right side of the quadrant, with the arrows close to the quadrant, were strongly positively correlated, indicating coordinated accumulation of photosynthetic pigments. This correlation highlights that maintaining primary photoprotective mechanisms is vital, as carotenoids’ role is to dissipate excess light energy that could otherwise damage chlorophyll molecules.

Similarly, TPC and DPPH scavenging activity were closely associated, suggesting that phenolic compounds contributed substantially to antioxidant activity. The second principal component (43.14%) separated pigment-related variables from antioxidant-related parameters, indicating partial independence between them. This suggests that pigment synthesis (associated with primary metabolism and light-harvesting) and phenolic compound accumulation (associated with secondary metabolism and stress mitigation) operate via independent physiological pathways. Thus, experimental treatments may enhance or suppress a plant’s antioxidant defense system without necessarily compromising or altering its primary photosynthetic framework.

These aspects are consistent with the Pearson correlation coefficients presented in Tables S3 and S5 of the Supplementary Materials.

The PCA score plots (factor map) and HCA dendrograms show how samples behave and cluster together depending on biometric characteristics (Figure 8) and phytochemical parameters, respectively (Figure 9).

It can be observed that samples with similar growth responses are in the same cluster (Figure 8); thus, there are three clusters related to the biometric parameters. Considering that PC1 explains 77.71% (the most important axis), samples on the right side (e.g., Fs1-a, Fs2-a, Fs2-b, Fs4-a) represent the most effective treatments regarding growth performance. On the contrary, samples on the left side (e.g., Fs1-c, Fs3-c, F4-c, Fs4-b) have weaker growth, lower biomass, or shorter plants. Among these, Fs2-b appears to be particularly distinct from the point of view of PC2, which reflects root biomass accumulation.

The green cluster, containing the Fs1-a, Fs2-a, Fs2-b, and Fs4-a samples, appears to be the best-performing group with higher biometric characteristics. It becomes evident that the combinations used for the treatments found in the green cluster (water in soil s1, s2, and s4, and whey in soil s2) optimize both aerial and subterranean plant physiology, highlighting their practical potential as biofertilizers. Within this cluster, sample Fs2-b exhibits a distinct separation; its unique positioning is biologically significant. This shows that, while Fs2-b achieves robust overall vegetative growth along Dimension 1, it simultaneously induces an independent, substantial stimulation of root biomass, as captured by Dimension 2.

Occupying a transitional zone centered near the vertical origin, the red cluster contains six samples, with the treatments yielding an intermediate growth performance, demonstrating moderate shoot expansion and balanced, stable root elongation without outstanding tissue accumulation. Located predominantly on the negative quadrants of Dimension 1, the black cluster indicates that these treatment combinations resulted in noticeably weaker growth characteristics. It is noted that all samples in which solution c was used as a fertigation agent have the lowest biometric parameters; this is probably an inhibitory effect caused by the combination of bioactive compounds from whey and GPHAE.

The clustering pattern demonstrated clear differences among treatments and confirmed the strong association between growth parameters and the response of bean plants to the use of food by-products as potential biofertilizers.

By performing hierarchical cluster analysis (HCA) of the dataset for the bean leaf extracts (16 samples and four variables: chlorophyll, carotenoid, and polyphenolic compound contents and DPPH scavenging activity), similarities and differences were observed between the samples (Figure 9). It can be perceived that the 16 samples of bean leaf extracts were split into 3 clusters, and their hierarchical classification is in excellent agreement with the PCA results.

The black cluster represents the largest group with nine samples, followed by the red and green clusters comprising four and three samples, respectively.

The red cluster exhibits the best results in terms of chlorophyll and carotenoid content (Fs1-a and Fs1-d; and Fs4-b and Fs2-c, respectively), forming two groups based on their similarities in the hierarchical cluster analysis dendrogram (Figure 9B). These treatments strongly stimulate primary metabolism and light-harvesting capacities. Samples from the green cluster—Fs1-b, Fs3-b, and Fs1-c—are distinguished by the highest values with respect to total phenolic content and DPPH scavenging activity. The three combinations between soil and fertigation solution act as potent metabolic activators of plants’ secondary metabolites, without prioritizing pigment production.

Representing the largest cluster, the black cluster is characterized by the lowest overall values across all parameters. A minimal metabolic response—maintaining the plants at a baseline level of both primary and secondary biochemical synthesis—was associated with specific treatment combinations or application rates (samples Fs3-a, Fs4-a, Fs3-c, and Fs2-d found in the inferior left quadrant of the score plot).

PCA and HCA demonstrate that the food by-product treatments do not affect plant biochemistry uniformly. Some treatments can result in the enhancement of photosynthetic pigments, and others can contribute to secondary metabolite accumulation, confirming their targeted potential as biofertilizers.

4. Conclusions

The present work reveals the specific response of a nutritionally important crop, Phaseolus vulgaris L. var. communis Auria Bacăului, to the use of some local food by-products and waste as potential biofertilizers in the vegetative growth phase under controlled growth conditions in greenhouses.

The results obtained showed that bean plants grown on soil amended with EGP and sprayed with water present high biometric values with respect to average plant height (Fs2-a/55.50 cm) and fresh biomass (Fs2-a/24.55 g). It was also observed that bean plants grown in Fs2 soil developed better than those grown in the other two amended soils (Fs3 and Fs4), regardless of the fertigation solution applied (b, c, or d). This behavior validates the efficiency of applying the mixed treatment—namely, amending the soil with by-products rich in inorganic nutrients (EGP) and using products with high bioactive organic compound content (whey, SBPP, and GPHAE) as a fertigation solution.

The UV–Vis phytochemical screening that preceded the quantitative analysis of some representative metabolites emphasized, through the spectral profiles generated, both the importance of the treatment applied to stimulate their biosynthesis process in the plant and the methodology used to obtain the analyzed extracts.

Beyond the complexity of metabolite accumulation processes during the vegetative development phase of the studied plant, positive effects from the employed food-waste fertilizers were observed on the content of chlorophyll, amino acids, and polyphenols and implicitly on antioxidant activity:

• The use of mixture d (water + GPHAE) or b (water + whey) as a fertigation solution applied on non-amended soil (s1) or amended soils s3 (SBPP) and s4 (EGP + SBPP) resulted in a positive effect on the accumulation of chlorophyll (Fs1-d, Fs3-d, and Fs4-d) and on the total phenolic content of bean plants (Fs1-b and Fs3-b), when compared to the control sprayed with water (Fs1-a).
• PCA and HCA provided important information regarding the effects of food by-products on the biometric performance and phytochemical characteristics of bean plants. The use of certain food by-products enhanced overall plant vigor and biomass production, while others preferentially stimulated the accumulation of photosynthetic pigments and antioxidant metabolism.
• In addition, the HPTLC analysis carried out to screen amino acids underlined the effect of soil formulation and fertigation solutions. Among all samples, the highest synergetic effect in terms of amino acid accumulation was observed for bean plants grown on soil amended with EGP and sprayed with the c mixture (Fs2-c), even though this sample did not exhibit high biometric parameter values and ranked third in chlorophyll levels.

Overall, the positive effects observed during this exploratory study on the vegetative growth phase of Phaseolus vulgaris L. var. communis Auria Bacăului—in terms of vegetative development and phytochemical profile—underline the importance of continuing investigations on the benefits of using regional food by-products and waste for soil vitality, the possible synergistic interactions between active principles, and their limitations as biofertilizers.

For future perspectives, we aim to extend our research towards evaluating the influence of these food by-products—which are rich in organic and inorganic nutrients—by refining their application in accordance with sustainable agricultural practices and circular economy principles. This will include assessments throughout the reproduction stage of the studied crop, as well as investigations involving other legume and vegetable species.

Moreover, economic implications are essential for a large-scale implementation of these regional agri-food by-products as potential biofertilizers. They could serve as a cost-effective substitute for conventional fertilizers, providing an accessible solution for farmers who desire cleaner crops and enhancing the financial viability of the farming enterprise.