Improving properties of sandy soil and production of Phaseolus vulgaris cultivation using Azolla pinnata, eggshells, and biochar

Abstract

Sandy soils are characterized by low organic matter content, weak nutrient retention, and limited water-holding capacity, which restrict crop productivity in arid and semi-arid regions. This study evaluated the individual and combined effects of biochar, Azolla pinnata, and eggshell powder on soil properties and the productivity of common bean (Phaseolus vulgaris L.) grown in sandy soil. Field experiments were conducted during two consecutive growing seasons (2021–2022) at the Ismailia Agricultural Research Station, Egypt, using a randomized complete block design with eight treatments and three replicates. Soil chemical properties, photosynthetic pigments, vegetative growth, biomass accumulation, and yield parameters were evaluated. The combined application of biochar, Azolla, and eggshell powder significantly improved soil fertility compared with the control treatment. Soil organic matter increased to 0.70%, while available nitrogen, phosphorus, and potassium reached 51.10, 6.67, and 200 mg kg⁻¹, respectively. The integrated treatment also enhanced chlorophyll content, plant growth, biomass production, and seed yield across both seasons. Correlation analysis indicated strong positive relationships between soil organic matter, nutrient availability, and crop productivity. These results demonstrate that the integrated application of biochar, Azolla pinnata, and eggshell powder can effectively improve soil fertility and enhance the productivity of Phaseolus vulgaris in sandy soil systems.

Introduction

Sandy soils are widely distributed in arid and semi-arid regions and are generally characterized by low organic matter content, weak structural stability, poor nutrient-retention capacity, and limited water-holding ability¹. These constraints often lead to low nutrient-use efficiency and reduced crop productivity, particularly for leguminous crops that require balanced soil fertility to sustain growth and biological nitrogen fixation^2,3. Therefore, improving the fertility and functionality of sandy soils represents a major challenge for sustainable agricultural production. In recent years, the use of environmentally friendly soil amendments derived from natural or agricultural wastes has gained considerable attention as an effective strategy for enhancing soil quality and crop performance⁴. Among these materials, biochar has been extensively studied due to its ability to improve soil physical and chemical properties, increase cation-exchange capacity, enhance water retention, and reduce nutrient leaching in coarse-textured soils^5,6. Additionally, biochar provides a stable carbon source that can influence microbial activity and contribute to long-term soil fertility⁷. Azolla pinnata, a small floating aquatic fern associated with nitrogen-fixing cyanobacteria, has also been recognized as a valuable biofertilizer in sustainable agriculture^8,9. Through its symbiotic relationship with Anabaena azollae, Azolla can fix atmospheric nitrogen and enrich the soil with organic matter and nutrients after decomposition¹⁰. Previous studies have reported that Azolla incorporation into soil can enhance nutrient availability, improve soil biological activity, and promote plant growth^11,12. On the other hand, eggshell waste represents an abundant agricultural by-product that is primarily composed of calcium carbonate¹³. Recycling eggshell powder as a soil amendment can provide calcium, improve soil buffering capacity, and potentially influence nutrient availability and soil chemical balance¹⁴. The utilization of such waste materials not only contributes to soil improvement but also supports sustainable waste management practices¹⁵. Although the beneficial effects of biochar, Azolla, and calcium-rich organic residues have been widely documented individually, limited information is available regarding their combined application in sandy soils^16,17. The integration of these amendments may result in synergistic interactions within the soil–plant system. Biochar may act as a stable matrix capable of retaining nutrients released from decomposing Azolla, while eggshell-derived calcium may contribute to soil buffering, nutrient balance, and root-zone chemical stability.

Recent studies have highlighted the potential of sustainable soil amendments, such as biochar, biological fertilizers, and recycled agricultural residues, to improve soil fertility and crop productivity in degraded soils^18,19. Biochar has been reported to enhance soil structure, water retention, and nutrient-holding capacity^20,21, while Azolla pinnata serves as a natural biofertilizer capable of supplying biologically fixed nitrogen and organic matter to the soil^22,23. Likewise, eggshell waste, which is primarily composed of calcium carbonate, has attracted attention as an environmentally friendly amendment that can improve soil buffering capacity and supply calcium to plants^24,25. However, most previous studies have mainly focused on the individual effects of these amendments. Limited information is available regarding their combined application and possible synergistic interactions, particularly in sandy soils where low nutrient retention and poor biological activity restrict crop productivity^25–27. The integration of biochar as a stable carbon matrix, Azolla as a biologically active nitrogen source, and eggshell powder as a recycled calcium amendment may promote complementary processes that improve soil physicochemical properties, enhance nutrient cycling, and support plant growth. Therefore, the novelty of the present study lies in evaluating the integrated effects of biochar, Azolla pinnata, and eggshell powder on soil quality, plant physiological responses, and yield performance of Phaseolus vulgaris L. grown in sandy soil under field conditions. This approach provides new insights into sustainable strategies for improving soil fertility and legume productivity in low-fertility sandy soils.

Results

Soil chemical properties

Soil pH, Electrical Conductivity (EC), and Organic Matter (OM)

The application of biochar, Azolla pinnata, and eggshell powder significantly influenced the chemical properties of the sandy soil in both growing seasons (Table 1). A slight but consistent reduction in soil pH was observed across all amended treatments compared with the control (7.83–7.84). The most pronounced decrease occurred in the combined treatment of eggshell powder, biochar, and Azolla pinnata (ESP + B + A), where soil pH declined to 7.62 and 7.58 during the first and second seasons, respectively. Soil pH showed a slight decrease in treatments receiving organic amendments. Similarly, soil electrical conductivity (EC) decreased in all treatments receiving soil amendments. The highest EC values were recorded in the control treatment (1.35 and 1.33 dS m⁻¹), whereas the ESP + B + A treatment resulted in the lowest EC values, reaching 1.10 dS m⁻¹ in the second season. In contrast, soil organic matter content increased significantly following amendment application. The triple-amendment treatment (ESP + B + A) produced the highest organic matter levels, reaching 0.65% and 0.70% in the first and second seasons, respectively. These results indicate a strong synergistic effect among the three amendments in enhancing soil carbon accumulation and improving soil fertility.

Table 1.

Effect of biochar, Azolla pinnata, and eggshell powder on soil pH, electrical conductivity (EC), and organic matter (OM) during two growing seasons.

Treatment	pH (Season 1)	pH (Season 2)	EC (dS m⁻¹) Season 1	EC (dS m⁻¹) Season 2	OM (%) Season 1	OM (%) Season 2
Control	7.83 a	7.84 a	1.35 a	1.33 a	0.32 d	0.34 d
Eggshell powder (ESP)	7.78 ab	7.76 ab	1.30 b	1.28 b	0.41 c	0.43 c
Biochar (B)	7.70 bc	7.68 bc	1.22 c	1.20 c	0.52 b	0.55 b
Azolla pinnata (A)	7.72 bc	7.69 bc	1.25 c	1.23 c	0.50 b	0.53 b
ESP + B	7.66 cd	7.63 cd	1.18 d	1.15 d	0.58 ab	0.61 ab
ESP + A	7.67 cd	7.64 cd	1.20 cd	1.18 cd	0.56 b	0.59 b
B + A	7.65 cd	7.60 d	1.15 d	1.12 d	0.60 ab	0.63 ab
ESP + B + A	7.62 d	7.58 d	1.12 d	1.10 d	0.65 a	0.70 a
LSD (0.05)	0.12	0.11	0.07	0.06	0.05	0.05

Values represent means of three replicates (n = 3). Means followed by the same letter within each column are not significantly different at P ≤ 0.05 according to Tukey’s HSD test.

Statistical analysis revealed that the effects of soil amendments were significant (P ≤ 0.05) for most measured parameters.

Available macronutrients (N, P, and K)

The availability of essential macronutrients (N, P, and K) was significantly enhanced by the application of soil amendments (Table 2). The ESP + B + A treatment recorded the highest concentrations of available nitrogen (49.10 and 51.10 mg kg⁻¹), phosphorus (5.65 and 6.67 mg kg⁻¹), and potassium (192 and 200 mg kg⁻¹) across both seasons. Biochar applied alone also produced a noticeable increase in nutrient availability compared with the control treatment. Meanwhile, single applications of eggshell powder or Azolla pinnata produced moderate improvements. In general, combined treatments exhibited stronger effects than individual amendments, indicating complementary interactions among the tested soil conditioners. Overall, the results demonstrate that the integrated application of biochar, Azolla pinnata, and eggshell powder was the most effective strategy for improving soil nutrient status in sandy soils.

Table 2.

Effect of biochar, Azolla pinnata, and eggshell powder on available nitrogen (N), phosphorus (P), and potassium (K) in sandy soil during two growing seasons.

Treatment	N (mg kg⁻¹) Season 1	N (mg kg⁻¹) Season 2	P (mg kg⁻¹) Season 1	P (mg kg⁻¹) Season 2	K (mg kg⁻¹) Season 1	K (mg kg⁻¹) Season 2
Control	32.40 d	34.10 d	3.10 d	3.25 d	145 d	148 d
Eggshell powder (ESP)	36.50 cd	38.20 cd	3.80 cd	4.05 cd	158 cd	162 cd
Biochar (B)	40.80 bc	42.50 bc	4.50 bc	4.80 bc	170 bc	175 bc
Azolla pinnata (A)	42.20 bc	44.00 bc	4.70 bc	5.00 bc	172 bc	178 bc
ESP + B	44.60 b	46.30 b	5.10 b	5.40 b	182 b	188 b
ESP + A	45.10 b	47.20 b	5.20 b	5.55 b	185 b	190 b
B + A	47.80 ab	49.60 ab	5.50 ab	6.00 ab	190 ab	195 ab
ESP + B + A	49.10 a	51.10 a	5.65 a	6.67 a	192 a	200 a
LSD (0.05)	3.10	3.25	0.45	0.50	8.5	9.0

Values represent means of three replicates (n = 3). Means followed by the same letter within each column are not significantly different at P ≤ 0.05 according to Tukey’s HSD test.

Photosynthetic pigments

Significant increases in photosynthetic pigments were observed in response to soil amendment treatments (Fig. 1). Both chlorophyll and carotenoid concentrations in Phaseolus vulgaris leaves were higher in all amended plots compared with the control.

Fig. 1.

Effect of biochar, Azolla pinnata, and eggshell powder on carotenoid content (A) and chlorophyll content (B) in Phaseolus vulgaris plants during two growing seasons. Values represent the mean of three replicates ± standard error (SE). Different letters indicate significant differences among treatments according to Tukey’s HSD test at P ≤ 0.05.

The combined treatment (ESP + B + A) consistently produced the highest pigment concentrations during both growing seasons. These increases suggest enhanced photosynthetic capacity and improved plant physiological performance under the integrated amendment system.The consistent improvement in pigment concentrations indicates that the applied amendments enhanced nutrient uptake and promoted more efficient physiological functioning, contributing to improved plant growth and productivity.

Vegetative growth

All soil amendment treatments significantly improved vegetative growth parameters compared with the control (Fig. 2). Plants grown under amended treatments exhibited greater plant height, number of branches, and number of leaves. The highest values were consistently recorded under the ESP + B + A treatment, highlighting the synergistic effect of combining biochar, Azolla pinnata, and eggshell powder. These improvements in vegetative growth are likely associated with enhanced soil fertility, improved nutrient availability, and better soil physical conditions resulting from the combined amendments.

Fig. 2.

Effect of biochar, Azolla pinnata, and eggshell powder on plant height (A), number of branches (B), and number of leaves (C) of Phaseolus vulgaris during two growing seasons. Values represent the mean of three replicates ± standard error (SE). Different letters indicate significant differences among treatments according to Tukey’s HSD test at P ≤ 0.05.

Biomass production

Fresh and dry biomass were significantly affected by soil amendment treatments (Fig. 3). All amended plots showed higher biomass accumulation compared with the control. The ESP + B + A treatment produced the highest fresh and dry biomass values across both growing seasons. This improvement indicates that the combined amendments effectively stimulated plant growth by improving soil nutrient availability and root development.

Fig. 3.

Effect of biochar, Azolla pinnata, and eggshell powder on fresh weight (A) and dry weight (B) of Phaseolus vulgaris plants during two growing seasons. Values represent the mean of three replicates ± standard error (SE). Different letters indicate significant differences among treatments according to Tukey’s HSD test at P ≤ 0.05.

Seed yield and quality

Soil amendment treatments significantly enhanced seed yield and seed quality parameters (Fig. 4). The ESP + B + A treatment resulted in the highest seed yield, as well as the greatest seed protein and oil contents. The increase in seed yield is likely related to enhanced photosynthetic efficiency and improved nutrient acquisition. Higher seed protein content may be attributed to the additional nitrogen supplied by Azolla pinnata and the improved nitrogen retention promoted by biochar. Meanwhile, the increased oil percentage may be associated with improved potassium and calcium availability, which play important roles in lipid metabolism and seed development. The consistency of these responses across both growing seasons indicates the reliability of the integrated amendment strategy for improving crop productivity and seed nutritional quality in sandy soils.

Fig. 4.

Effect of biochar, Azolla pinnata, and eggshell powder on seed yield (A), seed protein content (%) (B), and seed oil content (%) (C) of Phaseolus vulgaris plants during two growing seasons. Values represent the mean of three replicates ± standard error (SE). Different letters indicate significant differences among treatments according to Tukey’s HSD test at P ≤ 0.05.

Relationship between soil fertility and yield components

Correlation analysis

The Pearson correlation analysis (Table 3) demonstrates a highly significant positive relationship between soil chemical properties and plant productivity. Available N and Organic Matter (OM) showed the strongest correlations with seed yield (r = 0.91** and r = 0.88**, respectively). These results confirm that the enhancement of soil fertility, particularly nitrogen availability through Azolla and carbon accumulation from biochar, was the primary driver of crop yield. Furthermore, the strong association between chlorophyll content and seed yield (r = 0.83**) suggests that the improved nutrient status boosted photosynthetic efficiency, leading to higher biomass and productivity in the sandy soil system.

Table 3.

Pearson correlation coefficients between soil properties, physiological traits, and yield parameters of Phaseolus vulgaris.

Variables	OM	N	P	K	Chlorophyll	Carotenoids	Fresh weight	Dry weight	Seed yield
Organic matter (OM)	1
Available N	0.82**	1
Available P	0.71**	0.76**	1
Available K	0.65*	0.69*	0.73**	1
Chlorophyll	0.78**	0.81**	0.66*	0.62*	1
Carotenoids	0.69*	0.74**	0.61*	0.59*	0.83**	1
Fresh weight	0.86**	0.88**	0.72**	0.70**	0.79**	0.73**	1
Dry weight	0.84**	0.86**	0.70**	0.67*	0.76**	0.71**	0.92**	1
Seed yield	0.88**	0.91**	0.75**	0.72**	0.83**	0.77**	0.94**	0.92**	1

Overall interpretation

Collectively, the results demonstrate that combining organic and mineral soil amendments can substantially improve soil fertility and crop productivity in sandy soils. The synergistic effects of biochar, Azolla pinnata, and eggshell powder appear to enhance nutrient cycling, soil organic matter accumulation, and plant physiological performance, ultimately leading to improved quantitative and qualitative yield traits in Phaseolus vulgaris. These findings highlight the potential of integrated soil amendment strategies as a sustainable approach for improving crop production in nutrient-poor sandy soils.

Discussion

The present study demonstrates that the integrated application of biochar, Azolla pinnata, and eggshell powder significantly improved soil fertility and enhanced the growth and productivity of Phaseolus vulgaris cultivated in sandy soil. The improvements observed in soil chemical properties, plant physiological characteristics, biomass accumulation, and yield performance indicate that the combined use of organic and mineral amendments can effectively overcome the inherent limitations of sandy soils. Such soils are typically characterized by low organic matter content, poor nutrient retention, weak structural stability, and high susceptibility to nutrient leaching, all of which limit crop productivity and nutrient-use efficiency.

The significant increase in soil organic matter observed in the amended treatments can primarily be attributed to the combined addition of stable carbon from biochar and organic residues derived from Azolla pinnata²⁸ Biochar is widely recognized as a carbon-rich material with high structural stability and resistance to microbial decomposition, allowing it to persist in soil for extended periods²⁹. This stability contributes to long-term carbon sequestration while improving soil physicochemical properties such as cation exchange capacity, nutrient retention, and soil aggregation. In addition³⁰, biochar provides a porous structure that serves as a habitat for soil microorganisms, thereby promoting microbial activity and nutrient cycling³¹. These characteristics are particularly beneficial in sandy soils where organic carbon is typically scarce and microbial activity is often limited³².

The improvement in soil nutrient availability (N, P, and K) observed in the present study further highlights the synergistic interaction between biochar and organic amendments³³. Biochar possesses a large surface area and high adsorption capacity, which allows it to retain nutrients released from organic matter decomposition and reduce their loss through leaching³⁴. Previous studies have demonstrated that biochar application can significantly enhance nutrient retention and microbial-mediated nutrient transformations in soil systems^35,36. The improved nutrient retention observed in the combined treatment likely resulted from the interaction between biochar surfaces and nutrients released during the mineralization of Azolla biomass.

The improvement in soil nutrient status and plant performance under the integrated amendment treatment is consistent with recent evidence on biochar-based and mineral-associated soil amendments. A recent global meta-analysis showed that biochar application can improve several soil chemical properties, including pH regulation, cation exchange capacity, organic carbon status, and nutrient availability³⁷. Moreover, calcium carbonate-modified biochar has been reported to ameliorate soil chemical constraints, enhance root development, improve microbial diversity, increase nitrogen accumulation, and promote plant growth³⁸. In the present study, the combined application of biochar, Azolla pinnata, and eggshell powder may have created complementary effects, where biochar acted as a porous nutrient-retention matrix, Azolla supplied biologically derived organic matter and nitrogen, and eggshell powder contributed a calcium carbonate-rich component that supported soil chemical balance. The observed increase in available potassium and plant nutrient uptake may also be explained by recent findings showing that potassium-enriched biochar-based fertilizers can improve K uptake, while biochar- and silica-mediated interactions can regulate K fractionation and release dynamics in calcareous soil^39,40. Furthermore, the improvement in plant physiological performance may be partly associated with enhanced rhizosphere functioning, as flavonoids and related secondary metabolites are known to play important roles in plant–rhizomicrobiome communication and stress-related belowground interactions⁴¹. Therefore, the superior performance of the combined amendment can be attributed not only to direct nutrient inputs, but also to improved nutrient retention, mineral-associated biochar interactions, potassium availability, and rhizosphere-mediated plant responses.

The role of Azolla pinnata as a biofertilizer also played a crucial role in improving soil fertility and plant growth in the present study. Azolla is well known for its symbiotic association with the nitrogen-fixing cyanobacterium Anabaena azollae, which enables it to fix atmospheric nitrogen and enrich soil nitrogen content⁴². The decomposition of Azolla biomass releases organic nitrogen compounds that gradually mineralize and become available to plants⁴³. This biological nitrogen supply is particularly valuable in sandy soils, where nitrogen is often lost through leaching or volatilization. Previous studies have reported that the incorporation of Azolla biomass can significantly enhance crop productivity, nitrogen-use efficiency, and soil fertility in agricultural systems^44,45. Similarly⁴⁶,demonstrated that the addition of fresh Azolla biomass improved growth and yield performance of rice under reduced nitrogen fertilizer application, highlighting its potential as a sustainable nitrogen source in crop production systems. In addition to improving nitrogen availability, the decomposition of Azolla biomass contributes to the accumulation of organic acids and bioactive compounds that may influence soil biochemical processes and microbial activity. These compounds can stimulate microbial mineralization processes, enhance nutrient cycling, and improve nutrient availability to plants. The slight decrease in soil pH observed in the amended treatments in the present study may be explained by the production of organic acids during microbial decomposition of organic residues. Similar trends have been reported in studies investigating the incorporation of organic amendments into soil, where microbial mineralization processes temporarily reduce soil pH due to the formation of organic acids and carbon dioxide^47,48.

Eggshell powder also played an important role in improving soil chemical balance through the supply of calcium carbonate. Eggshells are composed primarily of calcium carbonate (CaCO₃), which can act as a liming material and influence soil buffering capacity.

Calcium is an essential nutrient for plant growth and plays a key role in maintaining cell wall stability, root development, and nutrient uptake. The addition of calcium-rich materials such as eggshell powder may improve soil aggregation and structural stability, which are important factors for root growth and water retention in sandy soils. The potential use of eggshell-derived materials as soil stabilizers has been widely reported in geotechnical and environmental studies. For example⁴⁹,demonstrated that eggshell-derived lime could effectively improve the mechanical properties and environmental performance of stabilized soils. Similarly⁵⁰,reported that eggshell ash could partially replace lime in expansive soil stabilization while enhancing soil strength and durability. The beneficial effects of eggshell-based amendments have also been reported in other fields such as construction and materials engineering. Several studies have shown that eggshell ash can be used as a sustainable calcium-rich material for partial replacement of cement in concrete production, improving mechanical strength while reducing environmental impacts associated with cement manufacturing^51–53. These findings emphasize the broader potential of eggshell waste recycling as a sustainable strategy for resource recovery and environmental management.

The improvements in plant physiological traits observed in the present study further support the positive impact of integrated soil amendments on crop performance. The significant increase in chlorophyll and carotenoid contents in Phaseolus vulgaris leaves suggests that the amendments improved plant nutritional status and photosynthetic efficiency. Chlorophyll content is closely associated with nitrogen availability, as nitrogen is a key component of chlorophyll molecules and photosynthetic enzymes. Therefore, the higher chlorophyll concentrations observed in the amended treatments likely reflect improved nitrogen supply from Azolla decomposition and enhanced nutrient retention by biochar^54,55.

Enhanced photosynthetic capacity often leads to increased carbohydrate production and biomass accumulation, which ultimately contributes to improved yield formation. The observed improvements in vegetative growth parameters, including plant height, number of branches, and leaf number, further indicate that the integrated soil amendments created a more favorable root environment for plant development⁵⁶. Biochar has been reported to improve soil physical properties such as porosity, aeration, and water-holding capacity, which are particularly important in sandy soils that typically suffer from poor moisture retention⁵⁷.

The significant increase in fresh and dry biomass observed in the combined treatment reflects improved soil–plant interactions and enhanced nutrient-use efficiency. The synergistic effect of biochar, Azolla, and eggshell powder likely created a more balanced nutrient supply and improved soil conditions for plant growth. Similar synergistic effects have been reported in previous studies evaluating the combined use of organic amendments and biochar in degraded soils, where integrated soil management practices resulted in higher biomass production and improved soil fertility^58,59. Seed yield and seed quality parameters were also significantly improved by the integrated soil amendments. The highest seed yield, seed protein content, and seed oil percentage were consistently recorded in the combined treatment across both growing seasons. Increased seed yield may be attributed to improved nutrient availability, enhanced photosynthetic activity, and increased biomass production during the vegetative growth stage. The higher seed protein content observed in the amended treatments is likely related to improved nitrogen availability, as nitrogen is a fundamental component of amino acids and proteins in legume seeds^60,61.

Correlation analysis further confirmed the strong relationships between soil fertility indicators and crop productivity. Soil organic matter and available nitrogen showed strong positive correlations with biomass accumulation and seed yield, indicating that improvements in soil carbon and nutrient availability play a central role in enhancing crop productivity in sandy soils. The strong association between chlorophyll content and yield parameters also highlights the importance of plant physiological status in determining yield performance. Despite the promising results obtained in this study, several limitations should be acknowledged. The experiment was conducted over two growing seasons at a single experimental site, which may limit the extrapolation of the findings to other soil types and climatic conditions. Long-term field experiments under different agroecological environments are needed to better understand the sustainability and long-term impacts of these amendments. In addition, future studies should investigate the dynamics of soil microbial communities and nitrogen transformation processes in greater detail to better elucidate the mechanisms underlying the observed improvements in soil fertility and crop productivity. Overall, the results of the present study demonstrate that the integrated application of biochar, Azolla pinnata, and eggshell powder represents a promising strategy for improving soil fertility and crop productivity in sandy soils. The use of recycled organic resources such as biochar and agricultural wastes not only enhances soil health but also contributes to sustainable agricultural practices by reducing dependence on synthetic fertilizers and promoting circular resource utilization.

Materials and methods

Study area and experimental design

The field experiment was carried out at the Ismailia Agricultural Research Station, Ismailia Governorate, Egypt (30°35′41.9″ N, 32°16′45.8″ E), during two consecutive growing seasons (2021 and 2022). The region is characterized by sandy soils with inherently low fertility, poor water-holding capacity, and weak structural aggregation. These conditions present major challenges for crop productivity and make the site suitable for evaluating soil amendments aimed at improving soil quality. The common bean (Phaseolus vulgaris L.) cultivar ‘Dokki 126’ was selected as the test crop due to its economic importance and sensitivity to soil nutrient status and water availability.

A randomized complete block design (RCBD) was adopted in each season with three replicates. The experimental layout consisted of eight treatments representing single, dual, and combined applications of biochar (B), eggshell powder (ESP), and Azolla pinnata (A). Each experimental plot measured.

The experiment was analyzed as a two-factor factorial design, where:

• Factor A: Treatment (8 levels).

• Factor B: Season (2 levels).

Replicates (blocks) were considered random effects nested within each season.

Soil characterization before planting

Prior to sowing, composite soil samples were collected from the 0–30 cm depth to assess the initial soil physical and chemical characteristics. Samples were air-dried, sieved (2 mm), and analyzed according to standard procedures described by^62,63. The soil was classified as sandy, with low organic matter, low cation exchange capacity, and limited availability of key macronutrients—conditions typical of the arid and semi-arid soils of northeastern Egypt. The baseline soil properties are presented in Table 4.

Table 4.

Initial physicochemical properties of the experimental soil (0–30 cm depth). (*Determined in soil paste; **Determined in soil paste extract).

Soil properties			Values
Physical properties
Particle size distribution		Sand (%)	63.69
		Silt (%)	9.00
		Clay (%)	6.48
		Soil texture	Sandy
Available water %		5.40
Wilting point (%)		1.52
Field capacity (%)		9.13
Organic matter (%)		0.45
Bulk density (g cm–3)		1.62
Total porosity %		47.5
Saturation percentage (%)		44.1
Calcium carbonate (%)		5.95
*pH (Soil paste)		7.83
**EC (dS m–1)		1.35
Chemical properties
Soluble cations (meq L–1)		Ca++	5.51
		Mg++	2.75
		Na+	10.69
		K+	1.03
Soluble anions (meq L–1)		CO3−−	7.70
		HCO3	3.20
		CI−	14.80
		SO4 2	2.91
Available nutrients (mg kg–1)		Nitrogen	38.30
		Phosphorus	4.45
		Potassium	152

Description of soil amendments

Biochar

Biochar was produced from sugarcane bagasse through pyrolysis at 450 °C. The material was ground and sieved to < 0.5 mm particle size and applied at a rate of 7.35 m³ per feddan^64,65,. Table 5 summarizes its main properties. This rate was selected based on previous studies demonstrating that moderate biochar application improves soil structure, water retention, and nutrient availability in sandy soils without causing excessive alkalinity or nutrient imbalance.

Table 5.

Physical and chemical properties of biochar.

Biochar properties	Values
Physical properties
Moisture content %	25.72
Organic matter (%)	46.39
Bulk density (g cm–3)	0.16
Chemical properties
Total N	2.55%
Total P	0.65%
Total K	1.18%
*pH (Sus.1:10dSm-)1:2.5 : water suspension	6.87
*EC (dSm− 1) (1:10 bio char water suspension)	0.85

Azolla pinnata

Fresh Azolla pinnata biomass was obtained from the Agricultural Microbiology Research Department, SWERI, ARC, Egypt. It was applied at 180 kg per feddan as a single dose before planting. Its chemical composition^30,31 is shown in Table 6. This rate was chosen based on studies showing that Azolla incorporation at similar levels significantly enhances nitrogen availability, microbial activity, and crop productivity^65,66.

Table 6.

Some characteristics of Azolla pinnata.

Total nitrogen	3.00	C/N: carbon/nitrogen	15/1
Organic matter	78.20	TP: total phosphorus,	0.7
Organic carbon	45.35	TK: total potassium	3.5
Ash	21.8

Eggshell powder

Eggshell waste was collected from domestic sources, washed, dried, and ground into fine powder. The material is rich in calcium carbonate and serves as a natural soil amendment³². Table 7 summarizes its chemical composition. Eggshell powder was applied at a rate of 3-ton feddan⁻¹ (≈ 7.2-ton ha⁻¹). This rate was selected based on previous studies indicating that moderate applications of calcium carbonate-rich materials improve soil structure, nutrient availability, and buffering capacity without inducing excessive alkalinity^53,54. Similar application ranges have been reported to enhance soil physicochemical properties and plant performance under sandy soil conditions.

Table 7.

Chemical composition of eggshell powder.

Chemical composition	Weight (%)
C	21.1286
Na2O	0.1046
MgO	0.9261
P2O3	0.4149
SO3	0.3264
K2O	0.0542
C3O	76.9922
Fe2O3	0.0132
SrO	0.0396

Experimental design and treatments

The experiment employed a Randomized Complete Block Design (RCBD) with three replications. The eight treatments, comprising single, dual, and triple combinations of the amendments, were as follows:

Control: No soil amendments (only recommended chemical fertilizers).

• Biochar (B)

• Eggshell Powder (ESP)

• Azolla pinnata (A)

• A + ESP

• A + B

• ESP+ B

• ESP + B + A

All treatments, including the control, received the same recommended doses of chemical fertilizers. The use of fertilized control reflects standard agricultural practices; however, it may limit the ability to isolate the sole effects of organic amendments. This aspect was considered in data interpretation.

Agronomic practices and fertilization

The common bean cultivar ‘Dokki 126’ was used. All soil amendments were applied and incorporated into the soil before sowing. The recommended fertilization for all plots was as follows:

• Phosphorus: as superphosphate (15% P₂O₅) at 200 kg feddan⁻¹, applied entirely before cultivation.

• Potassium: as potassium sulfate (48% K₂O) at 50 kg feddan⁻¹, split into two equal doses applied at sowing and 30 days after sowing.

• Nitrogen: as ammonium nitrate (33.5% N) at 150 kg feddan⁻¹, split into four equal doses applied at 15, 30, 45, and 60 days after sowing.

Soil and plant measurements

Soil analysis

After harvest, soil samples (0–30 cm) were collected and analyzed for:

• pH (1:2.5 soil: water)

• EC (1:5 extract)

• Organic matter

• Available N, P, and K

Plant measurements

• Growth parameters: plant height, number of branches and leaves.

• Biomass: fresh and dry weight.

• Yield: seed yield per plot.

• Photosynthetic pigments: chlorophyll and carotenoids (spectrophotometric method)⁶⁷.

• Seed quality: protein and oil content.

Statistical analysis

Data were analyzed using a two-way ANOVA⁶⁸ to evaluate the effects of:

• Treatment (T)

• Season (S)

• Interaction (T × S)

The statistical model used was:

Where:

• µ = overall mean

• Ti = treatment effect

• Sj = season effect

• Bk(j) = block nested within season

• εijk = experimental error

Before analysis, data were tested for:

• Normality (Shapiro–Wilk test)

• Homogeneity of variance (Levene’s test)

Mean comparisons were performed using Tukey’s HSD test (p ≤ 0.05) instead of Duncan’s MRT to reduce Type I error.

Data presentation

Results are presented as mean ± standard deviation (SD).

All figures include error bars and sample size (n = 3).

Different letters indicate significant differences among treatments within each season.

Limitations and recommendations

Despite the robust increases observed in pigment concentrations, growth, and yield, further research is recommended to:

• Quantify gas exchange parameters and chlorophyll fluorescence to directly link pigment changes to photosynthetic performance.

• Investigate root architecture and microbial community dynamics to better understand nutrient mineralization pathways.

• Optimize amendment application rates and timing to avoid potential localized pH variations from eggshell powder or biochar ash.

• Characterize biochar feedstock properties, Azolla incorporation timing, and eggshell particle size to improve reproducibility and strengthen field-level recommendations^27,29,32.

Author contributions

D.M.K.: Conceptualization, Data curation; D.M.K. and A.A.A.: Formal analysis; M.N.M.A. and A.M.A.: Funding acquisition; A.M.A. and D.M.K.: Investigation; A.M.A., D.M.K. and E.R.M.: Methodology; D.M.K.: Resources; D.M.K. and H.G.I.: Software; D.M.K.: Writing – original draft preparation; A.M.A., A.A.A. and A.M.A., and H.G.I.: Writing – review and editing. All authors have read and agreed to the published version of the manuscript.

Data availability

The data supporting the findings of this study are included within the article.

Declarations

Competing interests

The authors declare no competing interests.

Consent to Participate

All authors provided consent to participate in this submission.