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Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Sustainable Improvement of Soil Fertility and Phaseolus vulgaris Yield using Biochar, Azolla pinnata and Plant Growth-Promoting Rhizobacteria

M
Marwa Yousry A. Mohamed1,*
https://orcid.org/0000-0002-8411-7067
D
Doaa M. Khalifa2
1Department of Biology, College of Sciences, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11623, Kingdom of Saudi Arabia.
2Department of Soil Chemistry and Physics Res., Soil, Water and Environment Research Institute (SWERI), Agricultural Research Center (ARC), Giza 12619, Egypt.

  • Submitted24-11-2025|

  • Accepted27-03-2026|

  • First Online 08-04-2026|

  • doi 10.18805/LRF-916

ABSTRACT

Background: Enhancing crop productivity while maintaining soil fertility remains a major challenge in arid and semi-arid regions, where sandy soils are low in organic matter and nutrient retention. Sustainable alternatives to chemical fertilizers are therefore essential. This study evaluated the effectiveness of integrating plant growth-promoting rhizobacteria (Bacillus circulans), Azolla pinnata and biochar as soil amendments to improve soil health and the productivity of Phaseolus vulgaris (common bean).

Methods: A field experiment was conducted over two consecutive growing seasons (2022-2023) at the Ismailia Agricultural Research Station, Egypt. Treatments included individual and combined applications of B. circulans, Azolla and biochar. Soil physicochemical properties, macronutrient availability (N, P and K), microbial activity, including dehydrogenase enzyme activity and plant growth and yield parameters were evaluated.

Result: The integrated treatment significantly improved soil properties, increasing organic matter (0.66-0.71%) while slightly reducing pH (7.61-7.59) and electrical conductivity (1.11-1.09 dS m-1). Nutrient availability was enhanced, with nitrogen reaching 48.10-49.10 mg kg-1, phosphorus 5.62-6.62 mg kg-1 and potassium 185-187 mg kg-1. Microbial biomass and enzymatic activity were markedly stimulated. Plant growth traits, including biomass, leaf number, plant height and photosynthetic pigments, were improved, resulting in higher seed yield. These findings demonstrate that the synergistic use of biochar, Azolla and B. circulans effectively enhances soil fertility, microbial activity, plant growth and yield, providing a practical and eco-friendly strategy for sustainable agriculture in nutrient-poor, semi-arid regions.

INTRODUCTION

The global land cover is diminishing due to the rapid growth of the human population. The use of chemical fertilizers in agriculture, the destruction of large ecological systems throughout the biosphere, such as plants that cannot absorb CO2 and the production of energy fuels that both increase the demand for fossil fuels and offer low-energy fuels at high energy costs are examples of how human activity also affects the environment (Korsa et al., 2024).
       
The continuous decline in fertile agricultural land has intensified the need for sustainable strategies to improve soil productivity, particularly in newly reclaimed areas. Land reclamation has become a common practice to expand agricultural production; however, reclaimed soils often suffer from low fertility, poor organic matter content and limited water-holding capacity, which restrict crop productivity and increase reliance on chemical fertilizers and irrigation (Hou et al., 2024; Wang et al., 2024). Sandy loam soils, frequently found in reclaimed lands, are characterized by high permeability but weak nutrient retention and low microbial activity, making them highly susceptible to nutrient loss and soil degradation (Musei et al., 2024). Therefore, developing sustainable soil management strategies that enhance soil fertility and resource-use efficiency is essential for maintaining agricultural productivity in such environments.
       
Biological and organic soil amendments have emerged as promising approaches to improve soil health while reducing environmental impacts associated with intensive chemical fertilizer use. Among these, the aquatic fern Azolla has attracted considerable attention due to its capacity to fix atmospheric nitrogen through its symbiotic association with the cyanobacterium Anabaena azollae. This natural biofertilization process enhances soil nutrient availability and reduces dependence on synthetic nitrogen fertilizers. In addition to its role as a biofertilizer, Azolla contributes to carbon sequestration, environmental remediation and sustainable biomass production and can be used as livestock and aquaculture feed (Korsa et al., 2024; Al-Sayed et al., 2022).
       
Several studies have demonstrated that the incorporation of Azolla into cropping systems improves nutrient availability and crop yield. For example, Azolla co-cultivation in rice systems significantly increased grain yield due to nutrient release during decomposition (Bazihizina et al., 2025), while its application in wheat cultivation enhanced nitrogen uptake and productivity (Prabakaran et al., 2022).
       
Another promising soil amendment is biochar, a carbon-rich material produced through the pyrolysis of organic biomass. Biochar has been widely recognized for its ability to improve soil structure, increase water-holding capacity and enhance nutrient retention, particularly in sandy soils with low fertility (Sharma, 2024). Its highly porous structure facilitates the adsorption and gradual release of nutrients, thereby reducing nutrient leaching and improving plant nutrient uptake (Das and Ghosh, 2023). Furthermore, biochar amendments have been shown to stimulate soil microbial activity and enhance biological processes involved in nutrient cycling (Wang et al., 2022). These improvements in soil physicochemical and biological properties can enhance chlorophyll synthesis, photosynthetic activity and ultimately crop productivity (Zhang et al., 2024).
       
Plant growth-promoting rhizobacteria (PGPR) represent another key component of sustainable soil management. These beneficial microorganisms stimulate plant growth through multiple mechanisms, including phytohormone production, nitrogen fixation and phosphate solubilization (Kunal et al., 2023; Singh and Srivastava, 2025). Their application as biofertilizers has gained increasing attention due to their ability to improve nutrient availability, enhance root development and increase crop productivity (Ling et al., 2022; Singh and Singh, 2018). For instance, inoculation with Azospirillum brasilense has been reported to increase wheat yield while reducing nitrogen fertilizer requirements (Galindo et al., 2022). In addition, phosphate-solubilizing bacteria such as Pseudomonas and Bacillus improve phosphorus availability and may also suppress soil-borne pathogens, thereby enhancing plant health and productivity (Sun et al., 2023).
       
The common bean (Phaseolus vulgaris) is an important leguminous crop widely cultivated in temperate, subtropical and tropical regions. The crop is valued for its high nutritional content, providing significant amounts of protein, dietary fiber, vitamins and essential minerals (Lisciani et al., 2024). In Egypt, common bean cultivation plays an important role in both human nutrition and agricultural economies, particularly for smallholder farmers (Karavidas et al., 2022; Draz et al., 2022). As a leguminous species capable of biological nitrogen fixation, P. vulgaris also contributes to improving soil fertility and supports sustainable cropping systems (Amer et al., 2025; Singh et al., 2016). Despite its importance, the productivity of common bean in reclaimed sandy soils remains limited due to poor soil fertility and unfavorable physicochemical properties.
       
Although previous studies have separately demonstrated the beneficial effects of Azolla, biochar and PGPR on soil fertility and crop performance, research investigating their combined and synergistic effects in reclaimed sandy soils remains limited. Integrated soil amendment strategies may provide a more effective approach to improving soil health, enhancing nutrient availability and increasing crop productivity under nutrient-poor conditions.
       
Therefore, the objective of this study was to evaluate the integrated effects of biochar, Azolla and plant growth-promoting rhizobacteria inoculation (Bacillus circulans) on the physicochemical properties and fertility of sandy soil, as well as their combined influence on the growth and productivity of Phaseolus vulgaris cultivated in reclaimed agricultural land.

MATERIALS AND METHODS

Site description and soil characterization
 
The field experiments were conducted during the two successive summer seasons of 2022 and 2023 at the Ismailia Agriculture Research Station farm, Ismailia Governorate, Egypt (30°35'41.9"N, 32°16'45.8"E). To determine the initial physical and chemical properties, representative soil samples were collected from the surface layer (0-30 cm depth) before planting in each season. The analyses were performed following the standard methodologies described by (Page et al., 1982) and (Bottomley et al., 2020), and the results are presented in Table 1. Preliminary analysis confirmed the soil was sandy in texture and exhibited low fertility status with respect to available macronutrients.

Table 1: Chemical and physical characteristics of the investigated surface layer of soil (0-30 cm).


 
Experimental design and treatments
 
The experiment was laid out in a randomized complete block design (RCBD) with three replications. Each experimental plot measured 12 m2 (4 m length × 3 m width). Eight treatments were evaluated to assess the individual and combined effects of three soil amendments: Azolla pinnata, biochar and the plant growth-promoting rhizobacterium (PGPR) Bacillus circulans. All treatments, including the control, received the full recommended dose of NPK chemical fertilizers as described in Section 2.3. The eight treatments were as follows:
 
1. Control: No organic or bio-fertilizer amendments applied.
 
2. Bacillus circulans (B.c.): Soil inoculated with B. circulans.
 
3. Azolla pinnata (A): Soil amended with Azolla pinnata.
 
4. Biochar (B): Soil amended with biochar.
 
5. A + B.c.: Soil amended with Azolla pinnata and inoculated with B. circulans.
 
6. A + B: Soil amended with both Azolla pinnata and biochar.
 
7. B.c. + B: Soil inoculated with B. circulans and amended with biochar.
 
8. B.c. + B + A: Soil inoculated with B. circulans and amended with both biochar and Azolla pinnata.
 
Crop management and fertilizer application
 
The common bean (Phaseolus vulgaris L.) cultivar ‘Dokki 126’ was used as the test crop. A uniform basal dose of phosphorus fertilizer as calcium superphosphate (15% P2O5) was applied to all plots at a rate of 200 kg fed-1 just before sowing. Potassium fertilizer, in the form of potassium sulfate (48% K2O), was applied at a rate of 50 kg fed-1 in two equal splits: at sowing and 30 days after sowing (DAS). Nitrogen fertilizer, as ammonium nitrate (33.5% N), was applied at a rate of 350 kg N fed-1 in four equal splits at 15, 30, 45 and 60 DAS.
 
Amendments and their characterization
 
• Biochar (B): Biochar was produced from sugarcane bagasse via slow pyrolysis at 450°C. The produced biochar was ground and sieved to a particle size of <0.5mm. Its physicochemical properties, including pH, EC and nutrient content (N, P, K), were determined according to (Page et al., 1982) and are presented in Table 2. Biochar was applied to the soil at a rate of 17. 5 m³ fed-¹) before sowing.

Table 2: Chemical and physical characteristics and available nutrients of biochar.


 
• Azolla (A): Fresh Azolla pinnata was obtained from the Agricultural Microbiology Research Department, SWERI, ARC, Giza, Egypt. The primary chemical composition (e.g., N, P, K, organic matter) was analyzed following the procedures of (El-Shahat, 1988) and is presented in Table 3. Azolla was applied into the soil as a dose of 430 kg ha-1 (180 kg fed-1) at the time of sowing.

Table 3: Some characteristics of Azolla pinnata.


 
•  Bacillus circulans (B.c.): The PGPR strain Bacillus circulans was used in this study. The bacterial culture was maintained on nutrient broth with 15% glycerol at -80°C (Salem et al., 2024). For the field application, the bacterium was cultivated aerobically in nutrient broth on a rotary shaker (150 rpm) at 28°C for 48 hours. The final concentration of the inoculum was adjusted to 109 CFU mL-1. This bacterial suspension was diluted with irrigation water and applied to the soil at a rate of 10 L fed-1 immediately after sowing and again 45 days later.
 
Soil sampling and post-harvest analysis
 
At the end of the growing season (harvest), soil samples were collected from a depth of 0-30 cm from each experimental plot. The samples were air-dried, crushed, homogenized and passed through a 2 mm sieve before analysis for the following parameters according to standard methods (Cottenie et al., 1982).
 
• Soil pH was measured in a 1:2.5 (soil: water) suspension.
• Electrical conductivity (EC) was measured in a 1:5 (soil: water) extract.
 
• EC of biochar was determined in a 1:10 biochar-water suspension.
 
• Organic matter (OM) content was determined.
 
• Available nitrogen (N), phosphorus (P) and potassium (K) were extracted and quantified.
 
Soil biological activity
 
• Dehydrogenase activity (DHA): Soil dehydrogenase activity was assessed at 30 and 60 DAS following the method described by (Thalmann, 1967).
 
• Total bacterial count: The total bacterial population in the soil was enumerated at 30 and 60 DAS using the method of (Holm and Jensen, 1972).
 
Plant analysis
 
At harvest, plants from each plot were collected to determine the yield and its components. Plant samples (straw and seeds) were oven-dried at 70°C to constant weight, then ground. The dried plant material was digested using a H2SO4-H2O mixture (Page et al., 1982). The digests were analyzed for total N, P and K content following the methodologies described by (Cottenie et al., 1982).
 
Photosynthetic pigment determination
 
Fresh leaf samples were collected at the flowering stage to determine photosynthetic pigments. Chlorophyll *a*, chlorophyll *b* and carotenoids were extracted and their concentrations were measured spectrophotometrically at wavelengths of 663, 647 and 470 nm, respectively. The pigment concentrations were calculated using the formulas provided by (Moran, 1982) and expressed as mg 100 g-1 fresh weight (F.W.).
 
Statistical analysis
 
All data collected were subjected to a two-way analysis of variance (ANOVA) appropriate for a Randomized Complete Block Design (RCBD) using SPSS software (Version 20). When a significant F-test was observed (P≤0.05), treatment means were compared using Duncan’s Multiple Range Test. All figures were generated using Origin Pro software (Version 2024).

RESULTS AND DISCUSSION

The integration of biochar, Azolla pinnata and Bacillus circulans markedly enhanced soil properties, nutrient availability, microbial activity and Phaseolus vulgaris growth and yield over two seasons.
 
Soil physicochemical properties
 
The integrated application of biochar, Azolla pinnata and Bacillus circulans significantly improved soil physicochemical properties over two seasons. Organic matter increased to 0.66-0.71%, pH decreased slightly to 7.61-7.59 and electrical conductivity (EC) was reduced to 1.11-1.09 dS m-1 (Table 4). Biochar and Azolla applied individually also improved these parameters moderately, whereas the Azolla + B. circulans combination showed less pronounced effects. These results align with Kabir et al., (2023), Fakhar et al., (2025) and Kozioł et al. (2024), who reported biochar amendments enhance soil aggregation, organic carbon and pH stabilization (Kabir et al., 2023; Fakhar et al., 2025; Kozioł et al., 2024). Similarly, Marzouk et al., (2024) observed Azolla incorporation reduces pH in calcareous soils (Marzouk et al., 2024). Contrastingly, some studies report excessive Azolla can over-acidify soils (Thapa and Poudel, 2021), highlighting the need for balanced application. Biochar improved soil structure, moisture retention and carbon stabilization; Azolla decomposition released organic acids enhancing phosphorus solubility (Marzouk et al., 2024; Kumar et al., 2024) and B. circulans contributed to phosphate solubilization (Setiawati et al., 2022). Individually, biochar or Azolla moderately improved soil properties, while Azolla + B. circulans was less effective. Biochar and microbial inoculants also stabilized EC by adsorbing Na+ and Cl- ions (Wang et al., 2024).

Table 4: Effect of biochar, Azolla pinnata and Bacillus circulans (B.c.) on soil pH, electrical conductivity (EC) and organic matter (OM) during two growing seasons.


 
Soil nutrient availability
 
Soil NPK levels were highest in the integrated treatment (N: 48.10-49.10 mg/kg, P: 5.62-6.62 mg/kg, K: 185-187 mg/kg; Table 5, whereas individual treatments showed moderate improvements. These findings are consistent with Al Tawaha et al. (2025) and Marzouk et al., (2025), who reported combined organic amendments enhance nutrient cycling (Al Tawaha et al., 2025; Marzouk et al., 2025). Biochar’s high cation exchange capacity (CEC) and Azolla’s rapid N release explain the observed effects (Ngambia et al., 2024; Ebrahim et al., 2024). Bacillus circulans further mobilizes phosphorus through enzyme activity, agreeing with (Mehta et al., 2010). Some studies, however, suggest biochar alone may not significantly improve P availability in very alkaline soils (Dai et al., 2017), contrasting with the strong synergistic effect seen here (Dai et al., 2017). Biochar enhances nutrient retention by increasing CEC and reducing nutrient leaching, particularly in sandy soils (Ngambia et al., 2024; Wang et al., 2024; Bekchanova et al., 2024).

Table 5: Effect of biochar, Azolla pinnata and Bacillus circulans on available soil nitrogen (N), phosphorus (P) and potassium (K) during two growing seasons.


 
Soil microbial activity
 
Microbial counts and dehydrogenase activity increased markedly under integrated amendments (Fig 1), reflecting improved biological soil health. This agrees with (Abd-Elhameed et al., 2022) and (Chhetri et al., 2022), demonstrating biochar provides microbial habitats, while Azolla and B. circulans supply nutrients and promote enzymatic activity. Seasonal variation influenced microbial dynamics, consistent with environmental effects reported by (Wu et al., 2024).

Fig 1: Effect of biochar, Azolla pinnata and Bacillus circulans on soil microbial count (A) and dehydrogenase activity (B) during two growing seasons.


 
Plant growth, photosynthetic pigments and yield
 
Plant height, branch and leaf numbers increased (Fig 3) indicating enhanced nutrient uptake, root development and overall plant vigor. Fresh and dry biomass improved illustrated in Fig 4, higher fresh weight indicates improved water retention and nutrient absorption, while increased dry weight reflects enhanced carbon assimilation.
       
Chlorophyll and carotenoid content rose as shown in Fig 2, indicating enhanced photosynthesis (Al-Sayed et al., 2022). Seed yield reached 3.18 t ha-1 (~75% higher than control), with 31.8 pods per plant, 100-seed weight 41.2 g, biological yield 7.23 t ha-1 and harvest index 44%. Seed and biological yields, as well as pods per plant and 100-seed weight, were significantly enhanced by all soil amendments compared to the control, with the combined treatment of Azolla + Bacillus circulans + Biochar showing the highest improvement in both seasons, reflecting synergistic effects on plant growth and productivity (Table 6) reflecting more efficient nutrient allocation and higher productivity. These results align with Mayly and Hidayat, (2024), Nadeem et al., (2017) and Zhang et al., (2024), confirming synergistic biochar, Azolla and PGPR application improves growth and yield  (Mayly and Hidayat, 2024; Nadeem et al., 2017; Zhang et al., 2024). Contrastingly, some single amendments show less pronounced effects, highlighting the importance of combined application (Sadegh et al., 2019).

Fig 2: Effect of biochar, Azolla pinnata and Bacillus circulans on carotenoid pigment content (A) and chlorophyll pigment content (B) of Phaseolus vulgaris during two growing seasons.



Fig 3: Effect of biochar, Azolla pinnata and Bacillus circulans on plant length (A), number of branches (B) and number of leaves (C) of Phaseolus vulgaris during two growing seasons.



Fig 4: Effect of biochar, Azolla pinnata and Bacillus circulans on fresh weight (A) and dry weight (B) of Phaseolus vulgaris during two growing seasons.



Table 6: Yield parameters of Phaseolus vulgaris as influenced by biochar, Azolla pinnata and Bacillus circulans during two growing seasons.


 
Implications and future perspectives
 
This study highlights the potential of combining biochar, Azolla and Bacillus circulans to improve soil health, nutrient availability and crop productivity in nutrient-poor soils. Their synergistic interaction provides a sustainable strategy for enhancing agricultural productivity.
       
Future research should evaluate the long-term effectiveness of this integrated approach across different agro-climatic regions and cropping systems. Advanced molecular techniques could also be used to investigate changes in soil microbial communities and functional diversity. In addition, economic analyses and optimization of application rates and timing are needed to ensure practical scalability and maximize the benefits of these amendments under field conditions (Zhang et al., 2024).

CONCLUSION

This study highlights the benefits of integrating plant growth-promoting rhizobacteria (PGPR), Azolla pinnata and biochar to improve soil fertility and the productivity of Phaseolus vulgaris. The combined application of Bacillus circulans, Azolla and biochar showed a synergistic effect, improving soil properties by increasing organic matter while reducing soil pH and electrical conductivity, creating more favorable conditions for plant growth.
       
These amendments also enhanced nutrient availability, resulting in better plant performance. The integrated treatment significantly improved growth parameters, including plant height, number of branches and leaves and fresh and dry biomass. In addition, higher levels of photosynthetic pigments such as chlorophyll and carotenoids indicated improved photosynthetic efficiency and yield potential.
       
Overall, the results demonstrate that combining PGPR, Azolla and biochar is an effective and sustainable strategy for improving soil health and crop productivity. This integrated approach can help reduce dependence on chemical fertilizers while supporting resilient agricultural systems in Egypt and similar agro-ecological regions.

ACKNOWLEDGEMENT

The authors deeply thank the Soils, Water and Environment Research Institute (SWERI), Agricultural Research Center, Giza, Egypt for providing all facilities and consulting to conduct this research work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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    Full Research Article

    Sustainable Improvement of Soil Fertility and Phaseolus vulgaris Yield using Biochar, Azolla pinnata and Plant Growth-Promoting Rhizobacteria

    M
    Marwa Yousry A. Mohamed1,*
    https://orcid.org/0000-0002-8411-7067
    D
    Doaa M. Khalifa2
    1Department of Biology, College of Sciences, Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh 11623, Kingdom of Saudi Arabia.
    2Department of Soil Chemistry and Physics Res., Soil, Water and Environment Research Institute (SWERI), Agricultural Research Center (ARC), Giza 12619, Egypt.

    • Submitted24-11-2025|

    • Accepted27-03-2026|

    • First Online 08-04-2026|

    • doi 10.18805/LRF-916

    ABSTRACT

    Background: Enhancing crop productivity while maintaining soil fertility remains a major challenge in arid and semi-arid regions, where sandy soils are low in organic matter and nutrient retention. Sustainable alternatives to chemical fertilizers are therefore essential. This study evaluated the effectiveness of integrating plant growth-promoting rhizobacteria (Bacillus circulans), Azolla pinnata and biochar as soil amendments to improve soil health and the productivity of Phaseolus vulgaris (common bean).

    Methods: A field experiment was conducted over two consecutive growing seasons (2022-2023) at the Ismailia Agricultural Research Station, Egypt. Treatments included individual and combined applications of B. circulans, Azolla and biochar. Soil physicochemical properties, macronutrient availability (N, P and K), microbial activity, including dehydrogenase enzyme activity and plant growth and yield parameters were evaluated.

    Result: The integrated treatment significantly improved soil properties, increasing organic matter (0.66-0.71%) while slightly reducing pH (7.61-7.59) and electrical conductivity (1.11-1.09 dS m-1). Nutrient availability was enhanced, with nitrogen reaching 48.10-49.10 mg kg-1, phosphorus 5.62-6.62 mg kg-1 and potassium 185-187 mg kg-1. Microbial biomass and enzymatic activity were markedly stimulated. Plant growth traits, including biomass, leaf number, plant height and photosynthetic pigments, were improved, resulting in higher seed yield. These findings demonstrate that the synergistic use of biochar, Azolla and B. circulans effectively enhances soil fertility, microbial activity, plant growth and yield, providing a practical and eco-friendly strategy for sustainable agriculture in nutrient-poor, semi-arid regions.

    INTRODUCTION

    The global land cover is diminishing due to the rapid growth of the human population. The use of chemical fertilizers in agriculture, the destruction of large ecological systems throughout the biosphere, such as plants that cannot absorb CO2 and the production of energy fuels that both increase the demand for fossil fuels and offer low-energy fuels at high energy costs are examples of how human activity also affects the environment (Korsa et al., 2024).
           
    The continuous decline in fertile agricultural land has intensified the need for sustainable strategies to improve soil productivity, particularly in newly reclaimed areas. Land reclamation has become a common practice to expand agricultural production; however, reclaimed soils often suffer from low fertility, poor organic matter content and limited water-holding capacity, which restrict crop productivity and increase reliance on chemical fertilizers and irrigation (Hou et al., 2024; Wang et al., 2024). Sandy loam soils, frequently found in reclaimed lands, are characterized by high permeability but weak nutrient retention and low microbial activity, making them highly susceptible to nutrient loss and soil degradation (Musei et al., 2024). Therefore, developing sustainable soil management strategies that enhance soil fertility and resource-use efficiency is essential for maintaining agricultural productivity in such environments.
           
    Biological and organic soil amendments have emerged as promising approaches to improve soil health while reducing environmental impacts associated with intensive chemical fertilizer use. Among these, the aquatic fern Azolla has attracted considerable attention due to its capacity to fix atmospheric nitrogen through its symbiotic association with the cyanobacterium Anabaena azollae. This natural biofertilization process enhances soil nutrient availability and reduces dependence on synthetic nitrogen fertilizers. In addition to its role as a biofertilizer, Azolla contributes to carbon sequestration, environmental remediation and sustainable biomass production and can be used as livestock and aquaculture feed (Korsa et al., 2024; Al-Sayed et al., 2022).
           
    Several studies have demonstrated that the incorporation of Azolla into cropping systems improves nutrient availability and crop yield. For example, Azolla co-cultivation in rice systems significantly increased grain yield due to nutrient release during decomposition (Bazihizina et al., 2025), while its application in wheat cultivation enhanced nitrogen uptake and productivity (Prabakaran et al., 2022).
           
    Another promising soil amendment is biochar, a carbon-rich material produced through the pyrolysis of organic biomass. Biochar has been widely recognized for its ability to improve soil structure, increase water-holding capacity and enhance nutrient retention, particularly in sandy soils with low fertility (Sharma, 2024). Its highly porous structure facilitates the adsorption and gradual release of nutrients, thereby reducing nutrient leaching and improving plant nutrient uptake (Das and Ghosh, 2023). Furthermore, biochar amendments have been shown to stimulate soil microbial activity and enhance biological processes involved in nutrient cycling (Wang et al., 2022). These improvements in soil physicochemical and biological properties can enhance chlorophyll synthesis, photosynthetic activity and ultimately crop productivity (Zhang et al., 2024).
           
    Plant growth-promoting rhizobacteria (PGPR) represent another key component of sustainable soil management. These beneficial microorganisms stimulate plant growth through multiple mechanisms, including phytohormone production, nitrogen fixation and phosphate solubilization (Kunal et al., 2023; Singh and Srivastava, 2025). Their application as biofertilizers has gained increasing attention due to their ability to improve nutrient availability, enhance root development and increase crop productivity (Ling et al., 2022; Singh and Singh, 2018). For instance, inoculation with Azospirillum brasilense has been reported to increase wheat yield while reducing nitrogen fertilizer requirements (Galindo et al., 2022). In addition, phosphate-solubilizing bacteria such as Pseudomonas and Bacillus improve phosphorus availability and may also suppress soil-borne pathogens, thereby enhancing plant health and productivity (Sun et al., 2023).
           
    The common bean (Phaseolus vulgaris) is an important leguminous crop widely cultivated in temperate, subtropical and tropical regions. The crop is valued for its high nutritional content, providing significant amounts of protein, dietary fiber, vitamins and essential minerals (Lisciani et al., 2024). In Egypt, common bean cultivation plays an important role in both human nutrition and agricultural economies, particularly for smallholder farmers (Karavidas et al., 2022; Draz et al., 2022). As a leguminous species capable of biological nitrogen fixation, P. vulgaris also contributes to improving soil fertility and supports sustainable cropping systems (Amer et al., 2025; Singh et al., 2016). Despite its importance, the productivity of common bean in reclaimed sandy soils remains limited due to poor soil fertility and unfavorable physicochemical properties.
           
    Although previous studies have separately demonstrated the beneficial effects of Azolla, biochar and PGPR on soil fertility and crop performance, research investigating their combined and synergistic effects in reclaimed sandy soils remains limited. Integrated soil amendment strategies may provide a more effective approach to improving soil health, enhancing nutrient availability and increasing crop productivity under nutrient-poor conditions.
           
    Therefore, the objective of this study was to evaluate the integrated effects of biochar, Azolla and plant growth-promoting rhizobacteria inoculation (Bacillus circulans) on the physicochemical properties and fertility of sandy soil, as well as their combined influence on the growth and productivity of Phaseolus vulgaris cultivated in reclaimed agricultural land.

    MATERIALS AND METHODS

    Site description and soil characterization
     
    The field experiments were conducted during the two successive summer seasons of 2022 and 2023 at the Ismailia Agriculture Research Station farm, Ismailia Governorate, Egypt (30°35'41.9"N, 32°16'45.8"E). To determine the initial physical and chemical properties, representative soil samples were collected from the surface layer (0-30 cm depth) before planting in each season. The analyses were performed following the standard methodologies described by (Page et al., 1982) and (Bottomley et al., 2020), and the results are presented in Table 1. Preliminary analysis confirmed the soil was sandy in texture and exhibited low fertility status with respect to available macronutrients.

    Table 1: Chemical and physical characteristics of the investigated surface layer of soil (0-30 cm).


     
    Experimental design and treatments
     
    The experiment was laid out in a randomized complete block design (RCBD) with three replications. Each experimental plot measured 12 m2 (4 m length × 3 m width). Eight treatments were evaluated to assess the individual and combined effects of three soil amendments: Azolla pinnata, biochar and the plant growth-promoting rhizobacterium (PGPR) Bacillus circulans. All treatments, including the control, received the full recommended dose of NPK chemical fertilizers as described in Section 2.3. The eight treatments were as follows:
     
    1. Control: No organic or bio-fertilizer amendments applied.
     
    2. Bacillus circulans (B.c.): Soil inoculated with B. circulans.
     
    3. Azolla pinnata (A): Soil amended with Azolla pinnata.
     
    4. Biochar (B): Soil amended with biochar.
     
    5. A + B.c.: Soil amended with Azolla pinnata and inoculated with B. circulans.
     
    6. A + B: Soil amended with both Azolla pinnata and biochar.
     
    7. B.c. + B: Soil inoculated with B. circulans and amended with biochar.
     
    8. B.c. + B + A: Soil inoculated with B. circulans and amended with both biochar and Azolla pinnata.
     
    Crop management and fertilizer application
     
    The common bean (Phaseolus vulgaris L.) cultivar ‘Dokki 126’ was used as the test crop. A uniform basal dose of phosphorus fertilizer as calcium superphosphate (15% P2O5) was applied to all plots at a rate of 200 kg fed-1 just before sowing. Potassium fertilizer, in the form of potassium sulfate (48% K2O), was applied at a rate of 50 kg fed-1 in two equal splits: at sowing and 30 days after sowing (DAS). Nitrogen fertilizer, as ammonium nitrate (33.5% N), was applied at a rate of 350 kg N fed-1 in four equal splits at 15, 30, 45 and 60 DAS.
     
    Amendments and their characterization
     
    • Biochar (B): Biochar was produced from sugarcane bagasse via slow pyrolysis at 450°C. The produced biochar was ground and sieved to a particle size of <0.5mm. Its physicochemical properties, including pH, EC and nutrient content (N, P, K), were determined according to (Page et al., 1982) and are presented in Table 2. Biochar was applied to the soil at a rate of 17. 5 m³ fed-¹) before sowing.

    Table 2: Chemical and physical characteristics and available nutrients of biochar.


     
    • Azolla (A): Fresh Azolla pinnata was obtained from the Agricultural Microbiology Research Department, SWERI, ARC, Giza, Egypt. The primary chemical composition (e.g., N, P, K, organic matter) was analyzed following the procedures of (El-Shahat, 1988) and is presented in Table 3. Azolla was applied into the soil as a dose of 430 kg ha-1 (180 kg fed-1) at the time of sowing.

    Table 3: Some characteristics of Azolla pinnata.


     
    •  Bacillus circulans (B.c.): The PGPR strain Bacillus circulans was used in this study. The bacterial culture was maintained on nutrient broth with 15% glycerol at -80°C (Salem et al., 2024). For the field application, the bacterium was cultivated aerobically in nutrient broth on a rotary shaker (150 rpm) at 28°C for 48 hours. The final concentration of the inoculum was adjusted to 109 CFU mL-1. This bacterial suspension was diluted with irrigation water and applied to the soil at a rate of 10 L fed-1 immediately after sowing and again 45 days later.
     
    Soil sampling and post-harvest analysis
     
    At the end of the growing season (harvest), soil samples were collected from a depth of 0-30 cm from each experimental plot. The samples were air-dried, crushed, homogenized and passed through a 2 mm sieve before analysis for the following parameters according to standard methods (Cottenie et al., 1982).
     
    • Soil pH was measured in a 1:2.5 (soil: water) suspension.
    • Electrical conductivity (EC) was measured in a 1:5 (soil: water) extract.
     
    • EC of biochar was determined in a 1:10 biochar-water suspension.
     
    • Organic matter (OM) content was determined.
     
    • Available nitrogen (N), phosphorus (P) and potassium (K) were extracted and quantified.
     
    Soil biological activity
     
    • Dehydrogenase activity (DHA): Soil dehydrogenase activity was assessed at 30 and 60 DAS following the method described by (Thalmann, 1967).
     
    • Total bacterial count: The total bacterial population in the soil was enumerated at 30 and 60 DAS using the method of (Holm and Jensen, 1972).
     
    Plant analysis
     
    At harvest, plants from each plot were collected to determine the yield and its components. Plant samples (straw and seeds) were oven-dried at 70°C to constant weight, then ground. The dried plant material was digested using a H2SO4-H2O mixture (Page et al., 1982). The digests were analyzed for total N, P and K content following the methodologies described by (Cottenie et al., 1982).
     
    Photosynthetic pigment determination
     
    Fresh leaf samples were collected at the flowering stage to determine photosynthetic pigments. Chlorophyll *a*, chlorophyll *b* and carotenoids were extracted and their concentrations were measured spectrophotometrically at wavelengths of 663, 647 and 470 nm, respectively. The pigment concentrations were calculated using the formulas provided by (Moran, 1982) and expressed as mg 100 g-1 fresh weight (F.W.).
     
    Statistical analysis
     
    All data collected were subjected to a two-way analysis of variance (ANOVA) appropriate for a Randomized Complete Block Design (RCBD) using SPSS software (Version 20). When a significant F-test was observed (P≤0.05), treatment means were compared using Duncan’s Multiple Range Test. All figures were generated using Origin Pro software (Version 2024).

    RESULTS AND DISCUSSION

    The integration of biochar, Azolla pinnata and Bacillus circulans markedly enhanced soil properties, nutrient availability, microbial activity and Phaseolus vulgaris growth and yield over two seasons.
     
    Soil physicochemical properties
     
    The integrated application of biochar, Azolla pinnata and Bacillus circulans significantly improved soil physicochemical properties over two seasons. Organic matter increased to 0.66-0.71%, pH decreased slightly to 7.61-7.59 and electrical conductivity (EC) was reduced to 1.11-1.09 dS m-1 (Table 4). Biochar and Azolla applied individually also improved these parameters moderately, whereas the Azolla + B. circulans combination showed less pronounced effects. These results align with Kabir et al., (2023), Fakhar et al., (2025) and Kozioł et al. (2024), who reported biochar amendments enhance soil aggregation, organic carbon and pH stabilization (Kabir et al., 2023; Fakhar et al., 2025; Kozioł et al., 2024). Similarly, Marzouk et al., (2024) observed Azolla incorporation reduces pH in calcareous soils (Marzouk et al., 2024). Contrastingly, some studies report excessive Azolla can over-acidify soils (Thapa and Poudel, 2021), highlighting the need for balanced application. Biochar improved soil structure, moisture retention and carbon stabilization; Azolla decomposition released organic acids enhancing phosphorus solubility (Marzouk et al., 2024; Kumar et al., 2024) and B. circulans contributed to phosphate solubilization (Setiawati et al., 2022). Individually, biochar or Azolla moderately improved soil properties, while Azolla + B. circulans was less effective. Biochar and microbial inoculants also stabilized EC by adsorbing Na+ and Cl- ions (Wang et al., 2024).

    Table 4: Effect of biochar, Azolla pinnata and Bacillus circulans (B.c.) on soil pH, electrical conductivity (EC) and organic matter (OM) during two growing seasons.


     
    Soil nutrient availability
     
    Soil NPK levels were highest in the integrated treatment (N: 48.10-49.10 mg/kg, P: 5.62-6.62 mg/kg, K: 185-187 mg/kg; Table 5, whereas individual treatments showed moderate improvements. These findings are consistent with Al Tawaha et al. (2025) and Marzouk et al., (2025), who reported combined organic amendments enhance nutrient cycling (Al Tawaha et al., 2025; Marzouk et al., 2025). Biochar’s high cation exchange capacity (CEC) and Azolla’s rapid N release explain the observed effects (Ngambia et al., 2024; Ebrahim et al., 2024). Bacillus circulans further mobilizes phosphorus through enzyme activity, agreeing with (Mehta et al., 2010). Some studies, however, suggest biochar alone may not significantly improve P availability in very alkaline soils (Dai et al., 2017), contrasting with the strong synergistic effect seen here (Dai et al., 2017). Biochar enhances nutrient retention by increasing CEC and reducing nutrient leaching, particularly in sandy soils (Ngambia et al., 2024; Wang et al., 2024; Bekchanova et al., 2024).

    Table 5: Effect of biochar, Azolla pinnata and Bacillus circulans on available soil nitrogen (N), phosphorus (P) and potassium (K) during two growing seasons.


     
    Soil microbial activity
     
    Microbial counts and dehydrogenase activity increased markedly under integrated amendments (Fig 1), reflecting improved biological soil health. This agrees with (Abd-Elhameed et al., 2022) and (Chhetri et al., 2022), demonstrating biochar provides microbial habitats, while Azolla and B. circulans supply nutrients and promote enzymatic activity. Seasonal variation influenced microbial dynamics, consistent with environmental effects reported by (Wu et al., 2024).

    Fig 1: Effect of biochar, Azolla pinnata and Bacillus circulans on soil microbial count (A) and dehydrogenase activity (B) during two growing seasons.


     
    Plant growth, photosynthetic pigments and yield
     
    Plant height, branch and leaf numbers increased (Fig 3) indicating enhanced nutrient uptake, root development and overall plant vigor. Fresh and dry biomass improved illustrated in Fig 4, higher fresh weight indicates improved water retention and nutrient absorption, while increased dry weight reflects enhanced carbon assimilation.
           
    Chlorophyll and carotenoid content rose as shown in Fig 2, indicating enhanced photosynthesis (Al-Sayed et al., 2022). Seed yield reached 3.18 t ha-1 (~75% higher than control), with 31.8 pods per plant, 100-seed weight 41.2 g, biological yield 7.23 t ha-1 and harvest index 44%. Seed and biological yields, as well as pods per plant and 100-seed weight, were significantly enhanced by all soil amendments compared to the control, with the combined treatment of Azolla + Bacillus circulans + Biochar showing the highest improvement in both seasons, reflecting synergistic effects on plant growth and productivity (Table 6) reflecting more efficient nutrient allocation and higher productivity. These results align with Mayly and Hidayat, (2024), Nadeem et al., (2017) and Zhang et al., (2024), confirming synergistic biochar, Azolla and PGPR application improves growth and yield  (Mayly and Hidayat, 2024; Nadeem et al., 2017; Zhang et al., 2024). Contrastingly, some single amendments show less pronounced effects, highlighting the importance of combined application (Sadegh et al., 2019).

    Fig 2: Effect of biochar, Azolla pinnata and Bacillus circulans on carotenoid pigment content (A) and chlorophyll pigment content (B) of Phaseolus vulgaris during two growing seasons.



    Fig 3: Effect of biochar, Azolla pinnata and Bacillus circulans on plant length (A), number of branches (B) and number of leaves (C) of Phaseolus vulgaris during two growing seasons.



    Fig 4: Effect of biochar, Azolla pinnata and Bacillus circulans on fresh weight (A) and dry weight (B) of Phaseolus vulgaris during two growing seasons.



    Table 6: Yield parameters of Phaseolus vulgaris as influenced by biochar, Azolla pinnata and Bacillus circulans during two growing seasons.


     
    Implications and future perspectives
     
    This study highlights the potential of combining biochar, Azolla and Bacillus circulans to improve soil health, nutrient availability and crop productivity in nutrient-poor soils. Their synergistic interaction provides a sustainable strategy for enhancing agricultural productivity.
           
    Future research should evaluate the long-term effectiveness of this integrated approach across different agro-climatic regions and cropping systems. Advanced molecular techniques could also be used to investigate changes in soil microbial communities and functional diversity. In addition, economic analyses and optimization of application rates and timing are needed to ensure practical scalability and maximize the benefits of these amendments under field conditions (Zhang et al., 2024).

    CONCLUSION

    This study highlights the benefits of integrating plant growth-promoting rhizobacteria (PGPR), Azolla pinnata and biochar to improve soil fertility and the productivity of Phaseolus vulgaris. The combined application of Bacillus circulans, Azolla and biochar showed a synergistic effect, improving soil properties by increasing organic matter while reducing soil pH and electrical conductivity, creating more favorable conditions for plant growth.
           
    These amendments also enhanced nutrient availability, resulting in better plant performance. The integrated treatment significantly improved growth parameters, including plant height, number of branches and leaves and fresh and dry biomass. In addition, higher levels of photosynthetic pigments such as chlorophyll and carotenoids indicated improved photosynthetic efficiency and yield potential.
           
    Overall, the results demonstrate that combining PGPR, Azolla and biochar is an effective and sustainable strategy for improving soil health and crop productivity. This integrated approach can help reduce dependence on chemical fertilizers while supporting resilient agricultural systems in Egypt and similar agro-ecological regions.

    ACKNOWLEDGEMENT

    The authors deeply thank the Soils, Water and Environment Research Institute (SWERI), Agricultural Research Center, Giza, Egypt for providing all facilities and consulting to conduct this research work.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article.

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