Abstract

In the context of declining soil fertility associated with agricultural intensification in Côte d’Ivoire, the use of plant growth promoting rhizobacteria (PGPR) represents a sustainable alternative to nitrogen-based inputs. This study assessed the atmospheric nitrogen-fixing potential of cultivable bacteria isolated from rhizospheric soils and roots (endophytes) collected along a gradient of anthropization. A total of sixty-five (65) samples were collected from anthropized areas (maize and cassava fields) and low-anthropized areas (Banco Forest, Haut Bandama Reserve, and Lataha). Following isolation on nutrient agar and Pseudomonas Agar, 234 isolates were purified (78.63% rhizospheric; 21.37% endophytic). Nitrogen-fixing capacity was screened in vitro using nitrogen-free peptone water, with colorimetric quantification of ammonia production. Cultivable bacterial densities were generally higher in soils than in roots and tended to increase in low-anthropized sites. Among the isolates, 96.15% produced ammonia, with 14 isolates showing high performance after 72 h of incubation. The concentrations produced by these bacteria ranged from 18.60 x 10^-3 to 11.05 x 10^-3 mg/ml. Three sporulating isolates were selected and identified using the API 50 CHB system as Bacillus cereus (88.6%), Bacillus coagulans (70.5%), and Brevibacillus laterosporus (83%). Ammonia assimilation increased linearly with bacterial abundance, and strain-specific efficiency differed significantly, with Brevibacillus laterosporus exhibiting the highest performance. These results highlight the potential of sporulating bacilli as promising biofertilizer candidates.

1. Introduction

In Côte d’Ivoire, agriculture plays a central role in the national economy, accounting for approximately 22% of the gross domestic product (GDP) and providing employment for nearly two-thirds of the population ¹. With an estimated population of nearly 32 million in 2024 and sustained demographic growth projected to reach 36 million by 2030, increasing agricultural productivity has become a critical priority ². Accordingly, the government promotes the valorization of several staple and cash crops to strengthen national food security. However, despite their recognized socio-economic importance, the average yield of food crops is declining. This has been attributed to several factors such as unpredictable weather conditions, that is, prolonged droughts and sometimes heavy rainfall and the lack of crop maintenance. Another cause is biotic stress caused by phytopathogenic microorganisms that lead to crop diseases ³.

Consequently, enhancing agricultural productivity is essential to achieve food self-sufficiency in the context of rapid population growth and increasing nutritional demands ².

To mitigate soil fertility, decline and stress-related constraints, the application of chemical inputs has been widely adopted and has shown short-term effectiveness. However, their intensive use raises serious environmental and public health concerns ^{4 5}. As a result, alternative and more sustainable agricultural practices have been increasingly explored. These include the use of organic fertilizers, fallow systems, the development of improved crop varieties by research institutions such as the Centre National de Recherche Agronomique (CNRA), as well as agroecological approaches ^{6 7}.

Among these alternatives, microbial based technologies particularly plant bacteria interactions have gained considerable attention in sustainable agriculture. Although bacteria are often perceived as pathogenic organisms, only a small proportion are harmful to plants ^{8 9}. Numerous studies have demonstrated that certain soil bacteria possess beneficial traits that enhance plant growth and health, either through direct growth stimulation or biological control of pathogens. These microorganisms are collectively referred to as plant growth promoting rhizobacteria (PGPR). Due to their high agronomic potential, PGPR have been extensively investigated worldwide, with documented benefits in a wide range of crops, including tomato ¹⁰, wheat ¹¹, rice ¹², cotton ¹³, cassava, and maize ^{14 15 16}. PGPR enhance plant performance through multiple mechanisms, including phytostimulatory activities (production of indole-3-acetic acid and other phytohormones), phytoprotective effects (increased resistance to pathogens), and biofertilization processes such as mineral solubilization (e.g., phosphorus and potassium) and biological fixation of atmospheric nitrogen (N₂) ^{14 17}.

In agricultural systems, crop productivity is frequently limited by the availability of essential nutrients, particularly nitrogen, which is a major determinant of plant growth and yield ¹⁸. Although plants cannot directly assimilate atmospheric nitrogen (N₂), this element can be converted into plant-available forms, such as ammonia and nitrate, through biological nitrogen fixation (BNF) carried out by specific microorganisms ¹⁹. Consequently, BNF has attracted increasing interest as a sustainable strategy to improve soil fertility and reduce dependence on synthetic nitrogen fertilizers, as it enables the transformation of atmospheric nitrogen into bioavailable forms via nitrogen-fixing microorganisms ^{20 21}.

In this context, the present study aimed to identify bacteria capable of fixing atmospheric nitrogen in order to increase the likelihood of discovering novel PGPR with biofertilizer potential. Specifically, the objectives were to: (i) perform microbiological analyses of soil and root samples collected along a gradient of anthropization, and (ii) select and characterize spore-forming bacteria exhibiting the ability to fix atmospheric nitrogen.

2. Materials and Methods

The materials used in this study consisted of soil and root samples collected from different ecosystems. The targeted areas were represented in the North and South of the country due to the high production of cassava and maize in South and North respectively. Samples from anthropized areas included soils and roots from maize and cassava fields located in the departments of Agboville, Abidjan, Korhogo, and Tafiré. Samples from low-anthropized areas included soil samples collected in Abidjan (Banco National Park), Katiola–Niakara (Haut Bandama Reserve), and Korhogo (Lataha). In addition, a total of 234 bacterial isolates obtained from soil and root samples were used. These isolates were screened to determine their capacity to fix atmospheric nitrogen.

Soil and root sampling was conducted under aseptic conditions along a toposequence defined for each site, taking into account the upper, middle, and lower slope positions, with sampling intervals of 100 m for low-anthropized sites and 50 m for anthropized sites. After removing the surface layer with an alcohol-sterilized knife, soil samples were collected from the humic horizon at a depth of approximately 10 cm. Samples were placed in sterile plastic containers. For maize and cassava root sampling, plants were carefully uprooted, and adhering soil was removed from the roots. Root samples were then placed in sterile plastic containers. All 65 samples were transported to the laboratory in coolers at 4 °C for subsequent analysis ²².

For the isolation of soil bacteria, soil samples were cleared of stones, plant debris, and other solid materials. One gram of each soil sample was suspended in 10 mL of sterile distilled water and shaken for 30 min. For the isolation of endophytic bacteria from maize and cassava roots, rhizospheric soil was removed from the roots. Surface sterilization of roots was performed by initial washing, followed by immersion in 50 mL of 70% ethanol and subsequently in a 1% sodium hypochlorite solution, then rinsed three times with sterile distilled water ²³. Serial tenfold dilutions were prepared from 1 g of homogenized soil and crushed root samples. Aliquots of 100 µL from dilutions 10⁻⁴ to 10⁻⁶ for roots and 10⁻⁴ to 10⁻⁷ for soils were spread onto Pseudomonas Agar (PA) and Nutrient Agar (NA) supplemented with the fungicide amphotericin B (100 mg mL⁻¹). Colony counts were performed after incubation at 30 °C for 24–48 h. Results were expressed as colony-forming units per gram (CFU g⁻¹) of soil or root. Representative colonies with distinct morphologies were selected, purified, and preserved.

A total of 234 purified cultivable bacterial isolates were screened for plant growth–promoting traits.Atmospheric nitrogen fixation (reduction of N₂ to NH₃) was assessed according to the method described by ²⁴. Briefly, 10 mL of nitrogen-free peptone water was inoculated with 100 µL of 18–24 h bacterial suspension and incubated at room temperature for 72 h. The concentration of ammonia produced was determined using a colorimetric assay by adding 500 µL of Nessler’s reagent, followed by absorbance measurement at 430 nm using a spectrophotometer ²⁵. The standard calibration curve for ammonia is established using diluted standards treated with Nessler’s reagent. Their absorbance reading is taken at a wavelength of 430 nm using a spectrophotometer and the obtained values are used to plot a trend curve, with the resulting line equation being y= 147.21x and coefficient R²= 0.9993 (x= Ammonia concentration and y= sample absorbance). [Ammonia] in mg/ml: 0.0156; 0.0078125; 0.00390625; 0.00195313, 0.00097656, 0.00048828, 0.00024414. Optical density (430nm): 2.5865; 1.1935; 0.5535; 0.2680; 0.1605; 0.0410; 0.0150.

Based on their high PGPR activity, three sporulating isolates were selected for further analyses.

Among the isolates exhibiting the highest nitrogen-fixing activity, spore-forming bacilli were identified using Gram staining, heat resistance testing at 80 °C, and biochemical characterization with the API 50 CHB system.

Specific nitrogen-fixation efficiency was determined by estimating the amount of nitrogen fixed per colony-forming unit as a function of incubation time. Growth kinetics and nitrogen-fixation activity were monitored for each isolate over a 10 h incubation period. Inocula were prepared from the three selected isolates and cultured in 50 mL of peptone water. At hourly intervals, optical density was measured at 600 nm to estimate microbial abundance, followed by absorbance measurement at 430 nm after the addition of Nessler’s reagent to quantify fixed nitrogen. Prior to these analyses, a correlation was established between optical density at 600 nm and microbial abundance (CFU) for each isolate.

All data collected in this study were entered into Microsoft Excel 2021. Statistical analyses were performed using XLSTAT software (version 2019). One-way analysis of variance (ANOVA) followed by the Newman–Keuls post hoc test was conducted at a 5% significance level to compare ammonia concentrations produced by the bacterial isolates.

3. Results

Microbiological analysis of soil and root samples revealed varying bacterial counts depending on the sample. Analysis of the means showed significant differences among all tested samples. Overall, microbial densities in anthropized soils were higher (1.12 × 10¹¹ and 5.72 × 10¹⁰ CFU/g on Nutrient Agar (NA) and Pseudomonas Agar (PA), respectively) than in roots (3.90 × 10⁸ and 7.39 × 10⁷ CFU/g on NA and PA, respectively). However, low-anthropized areas generally exhibited the highest microbial densities in most cases (6.9 × 10¹⁰ CFU/g; 1.12 × 10¹¹ CFU/g; and 1.55 × 10¹⁰ CFU/g). Additionally, bacterial abundance in maize fields was higher than that observed in cassava fields overall. Colonies were selected based on distinct morphological characteristics, including shape, color, size, regularity, and margin curvature (Figure 1). Isolation yielded a total of 234 bacterial strains based on macroscopic criteria, of which 78.63% were rhizospheric and 21.37% endophytic.

The nitrogen-fixing ability of the isolates was demonstrated by their growth on a medium lacking any assimilable nitrogen source. Following the addition of Nessler’s reagent, a yellow-orange color appeared, indicating ammonia production. Results showed that 96.15% of the 234 tested isolates were capable of producing ammonia (Figure 2). Ammonia production after 72 hours varied among isolates, with 14 strains exhibiting high nitrogen-fixing capacity. Of these 14, only 3 were spore-forming bacteria.

Based on the highest nitrogen-fixing activity, three spore forming isolates were selected using Gram staining and heat resistance at 80 °C. The selected isolates and their corresponding ammonia production levels are presented in Table 1. Biochemical tests were subsequently performed using the API 50 CH system for identification. The results of the identification are also shown in Table 1. The isolates were subsequently identified using biochemical tests and were found to belong to the same genus group. Isolates AN1G1, KR1G2, and LT4G3 were identified as Bacillus coagulans (similarity score of 70.5%), Brevibacillus laterosporus (83%), and Bacillus cereus (88.6%), respectively.

Figure 3 illustrates the bacterial abundance of AN1G1, LT4G3, and KR1G2 expressed as log CFU mL⁻¹, together with ammonia production over time expressed in mg mL⁻¹. The figure shows a linear relationship between bacterial abundance and the amount of NH₃ produced. Ammonia concentration increases proportionally with bacterial abundance.

Figure 4 presents the specific efficiency of the bacteria, defined as the correlation between the amount of nitrogen fixed and the number of colony-forming units for each strain. Strain LT4G3 (Bacillus cereus) exhibited stable nitrogen-fixing activity that was less homogeneous than that of isolate AN1G1 (B. coagulans). Finally, strain KR1G2 (Brevibacillus laterosporus) showed an increase in NH₃ mass proportional to bacterial abundance, similar to the other strains, but at a lower microbial density (log CFU). KR1G2 exhibited the highest apparent NH₃ assimilation efficiency per log CFU.

Figure 5 highlights three distinct levels of specific efficiency, with a clear statistical separation. Strains LT4G3 and AN1G1 did not differ statistically from each other, whereas KR1G2 showed a significantly higher efficiency than the other two strains.

4. Discussion

Overall, samples from the different zones contained rhizobacteria as part of their microflora. Indeed, samples were collected from the humiferous horizon at a depth of 0–20 cm and from the roots of maize and cassava plants. This soil depth is known to host intense biological activity ²⁶.

The plate count method on solid media was adopted to provide an approximate estimation of the cultivable bacterial load, allowing the isolation of both rhizospheric and endophytic bacteria from the different samples. Previous studies, both earlier ¹⁰ and more recent ²⁵, have also reported the presence of rhizospheric and endophytic bacteria. A microorganism is considered endophytic when it is isolated from a plant organ whose surface has been previously sterilized ²⁷.

In addition, microbial density in soil samples was very high. In contrast, the internal root tissues harbored fewer bacteria than the surrounding soil. This difference is likely due to greater nutrient availability in the rhizospheric zone compared to the interior of plant tissues such as roots ²⁸. Moreover, colonization sites within plant tissues are less favorable than the external root environment.

The bacterial density observed in our rhizospheric soil can be considered approximately comparable, in some cases, to reported values of cultivable bacterial communities in 1 g of rhizospheric soil, which may reach up to 10⁹ cells ²⁹. However, such comparisons should be interpreted with caution, as several factors can influence bacterial density, including soil microbial community composition ³⁰, plant species ³¹, soil characteristics, and regional climate ³². Furthermore, the bacterial abundance observed in maize samples compared with cassava samples contrasts with the findings reported by ²⁵. This discrepancy may be explained by differences in sampling regions, maize and cassava varieties, or isolation media.

The ability of our isolates to fix atmospheric nitrogen was demonstrated by their capacity to grow on a medium lacking an assimilable nitrogen source. Following the addition of Nessler’s reagent, nitrogen fixing bacteria were identified by their ability to induce a yellow–orange color change in the medium, indicating ammonia production. Nitrogen is widely available in nature (over 78%) in the form of inert N₂ gas; however, it is not directly accessible to plants ³³. Plants can nevertheless utilize this gas to meet their nitrogen requirements through interactions with microorganisms ³⁴. Out of the 234 isolates tested, 225 were able to fix atmospheric nitrogen, corresponding to 96.15%. This result is consistent with previous studies by. ²⁰ and ²¹. In addition, ³⁴ demonstrated that nine isolates obtained from cassava plants were capable of fixing N₂. Atmospheric nitrogen is converted into plant-available forms through biological nitrogen fixation, during which it is reduced to ammonia by microorganisms ³⁵. Ammonia production by rhizospheric microorganisms is therefore commonly used as a selection criterion for plant growth-promoting bacteria ³⁶.

The present study highlights marked differences in ammoniacal nitrogen utilization efficiency among the three bacterial isolates tested, a trait frequently associated with plant growth-promoting rhizobacteria (PGPR). The three selected bacillary isolates, mainly identified as belonging to the genus Bacillus, exhibited variable levels of NH₃ production, suggesting metabolic heterogeneity among strains. These findings are comparable to those of ³⁷, who reported that several Bacillus spp. strains are capable of producing significant amounts of NH₃ under in vitro conditions. Several Bacillus species offer substantial advantages over other bacterial genera due to their rapid growth and their ability to form spores resistant to variations in pH, temperature, agrochemicals, fertilizers, and storage duration. These characteristics enable their use in the formulation of stable bioproducts and in biological control programs ³⁸.

Phenotypic identification using the API 50 CHB system allowed the assignment of the isolates to the species Bacillus cereus, Bacillus coagulans, and Brevibacillus laterosporus, with high similarity percentages. These species have previously been described in the literature as soil bacteria associated with cassava ³⁹ and maize ⁴⁰, and as exhibiting significant agronomic and biotechnological potential ⁴¹.

For the three strains studied (Bacillus cereus LT4G3, Bacillus coagulans AN1G1, and Brevibacillus laterosporus KR1G2), a strong linear correlation was observed between bacterial abundance (log CFU) and the amount of NH₃ assimilated, indicating that nitrogen assimilation was directly proportional to bacterial biomass. When comparing the isolates based on their specific NH₃ assimilation efficiency, Brevibacillus laterosporus KR1G2 exhibited a significantly higher efficiency than Bacillus cereus LT4G3 and Bacillus coagulans AN1G1. This high performance was evident even at low bacterial densities, which may represent an important functional advantage under low-biomass conditions.

In contrast, LT4G3 and AN1G1 displayed intermediate efficiencies that were statistically comparable. Overall, these results indicate that all three strains possess a biomass-dependent capacity for ammoniacal nitrogen utilization, but with distinct specific efficiencies. The observed differences suggest that the bacterial strains mobilize ammoniacal nitrogen through distinct metabolic, enzymatic, or physiological strategies. These differences may have practical implications for the selection of candidate bacteria in agronomic applications where nitrogen is a key limiting factor. In particular, Brevibacillus laterosporus is well known for its phytostimulatory properties ⁴², while Bacillus cereus has been reported to exhibit plant growth–promoting and biocontrol activities ⁴³, as well as the ability to produce ammonia in nitrogen-free culture conditions ⁴⁴.

5. Conclusion

The present study enabled the isolation and characterization of bacteria capable of producing ammonia, an important mechanism associated with plant growth promotion. Biochemical analyses showed that the selected isolates belong to the genera Bacillus and Brevibacillus. The three strains studied exhibited NH₃ assimilation proportional to their bacterial abundance, with distinct specific efficiencies. Among them, Brevibacillus laterosporus KR1G2 stood out for its highest apparent capacity to utilize ammoniacal nitrogen, whereas Bacillus coagulans AN1G1 showed high and particularly reproducible performance. These results confirm the relevance of Bacillus and related genera as promising candidates for the development of biofertilizers and biostimulants. However, complementary analyses, such as the detection of nif genes, are required to confirm potential diazotrophic activity. In addition, molecular analyses, particularly 16S rRNA gene sequencing, as well as assays under controlled conditions, will be necessary to validate their effectiveness.

ACKNOWLEDGEMENTS

The authors acknowledge the “Strategic Support Program for Scientific Research” (PASRES) for financial support through the PASRES Research Fellowship.

Declaration of Conflict of Interest

The authors declare no conflicts of interest.

References