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American Journal of Public Health Research
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The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
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American Journal of Medical and Biological Research
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The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
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Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
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The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

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Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
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Dear Label_Name,

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I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
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Nitrogen-fixing Potential of Bacillus Strains Isolated from Soils and Roots Across Different Levels of Anthropization in Côte d’Ivoire

N’DRI Ahou Roseline*, ALUI Konan Alphonse, KOKORA Aya Philomène, ANGORATCHI Marius Ebaley Yves-Magloire, ANGOUA Amanahan Mauricette Prisca, TOURE Kakoumani Lama Ruth, COULIBALY Yele Fatoumata, KARAMOKO Detto, MOROH Jean-Luc Aboya

Laboratory of Biotechnology and Agro-food Valorization (Labo BiVA), Faculty of Biological Sciences, Peleforo Gon Coulibaly University, Korhogo, Côte d’Ivoire

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.

Keywords: Plant growth–promoting rhizobacteria, biological nitrogen fixation, ammonia, Bacillus, anthropization gradient

Received January 14, 2026; Revised February 16, 2026; Accepted February 23, 2026

Citation: N’DRI Ahou Roseline, ALUI Konan Alphonse, KOKORA Aya Philomène, ANGORATCHI Marius Ebaley Yves-Magloire, ANGOUA Amanahan Mauricette Prisca, TOURE Kakoumani Lama Ruth, COULIBALY Yele Fatoumata, KARAMOKO Detto, MOROH Jean-Luc Aboya, Nitrogen-fixing Potential of Bacillus Strains Isolated from Soils and Roots Across Different Levels of Anthropization in Côte d’Ivoire. Journal of Applied & Environmental Microbiology, 14 (1). 1-8. January 2026. doi:10.12691/jaem-14-1-1

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

2.1. Materials

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.

2.2. Methods

2.2.1. Sample Collection

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].

2.2.2. Isolation and Enumeration of Cultivable Bacteria

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.

2.2.3. Screening of Nitrogen-fixing Bacteria

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.

2.2.4. Selection and Identification of Spore-forming PGPR Isolates

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.

2.2.5. Specific Nitrogen-fixation Efficiency of Bacterial Isolates

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.

2.2.6. Statistical Analysis

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

3.1. Enumeration and Isolation

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.

Figure 1. Macroscopic appearance of bacterial colonies observed on different culture media after incubation. Morphological diversity is reflected in colony pigmentation, size, shape, and texture, representing the first step in their phenotypic characterization

3.2. Nitrogen FIxation

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.

3.3. Selection and Identification of Spore-forming Isolates

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.

3.4. Specific Efficiency of the Selected Bacteria

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.

Table 1. Results of the biochemical and physiological tests performed on the selected isolates. The isolates were characterized based on NH₃ production (mg mL⁻¹), Gram staining, catalase and oxidase activities, and analysis using the API 50 CHB system. Percentages indicate the associated similarity levels

Figure 2. Ammonia (NH₃) production by the bacterial isolates investigated. Each bar represents the mean ammonia production per isolate, expressed in mg mL⁻¹, and error bars indicate the standard deviation. The x-axis represents the bacterial isolates, while the y-axis indicates the ammonia concentration produced

Figure 3. Comparative dynamics of bacterial biomass and ammonia production in three Bacillus isolates

Figure 4. Comparative plot of the mean slopes of NH₃ assimilation in three bacterial strains. The slopes represent the amount of NH₃ fixed per unit of log CFU, directly reflecting the specific efficiency of each strain

Figure 5. Comparison of the specific NH₃ assimilation efficiency (mg NH₃ per log CFU) among three bacterial strains (B. cereus, B. coagulans, Brevibacillus laterosporus). Bars represent the mean slopes. Different letters indicate significant differences between isolates (p < 0.05)

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.

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