The Role of Bacillus sp. in Reducing Chemical Inputs for Sustainable Crop Production

Abstract

Bacillus species have emerged as promising agents for reducing chemical input in sustainable crop production. These bacteria possess diverse abilities, including nutrient cycling, phytohormone production, and stress tolerance enhancement in plants. Recent advances in omics technologies have revolutionized the understanding of Bacillus sp. biology and expanded their potential applications. Bacillus-based products have demonstrated effectiveness in reducing nitrogen and phosphorus fertilizer requirements while maintaining or improving crop yields. However, their field performance may be inconsistent, highlighting the need for further research to optimize formulations and delivery methods. The compatibility with other agricultural inputs varies depending on the specific chemicals and conditions involved. The introduction of Bacillus sp. can significantly affect the soil microbiome, potentially promoting plant growth and microbial diversity. Strain specificity and host compatibility play crucial roles in determining the success of host–parasite interactions. The regulatory and safety aspects require further investigation to ensure the safe and sustainable use of Bacillus species in various applications. The integration of Bacillus-based products into existing crop management systems, following the principles of Integrated Pest Management and Integrated Crop Management, is essential for their successful implementation. This review provides a comprehensive overview of the current knowledge on Bacillus sp. for reducing chemical inputs for sustainable crop production, highlighting the challenges and opportunities for future research and application.

1. Introduction

Agricultural inputs encompass a wide range of resources and materials that are used in farming systems. Traditional inputs include labor, water, arable land, and other resources such as energy and fertilizers [1]. Chemical fertilizers, pesticides, genetically modified organisms, antibiotics, and growth hormones are commonly used [2]. However, sustainable and organic farming practices emphasize the use of alternative inputs. Organic farming systems avoid synthetic chemical inputs and maximize the use of crop rotations, animal manure, organic waste, and biological systems for nutrient mobilization and plant protection [2]. Conservation agriculture (CA) integrates the management of available natural resources such as soil, water, flora, and fauna, with minimal external inputs [3]. Similarly, low external input sustainable agriculture (LEISA) focuses on farm-derived inputs and productivity based on ecological processes and functions [4].

Chemical fertilizers are synthetic substances used in agriculture to provide essential nutrients to plants, primarily nitrogen, phosphorus, and K, to enhance crop productivity and yield [5]. Fertilizers play a pivotal role in modern agricultural systems by maintaining the balance of nutrients in the soil and boosting plant growth [5]. However, the use of chemical fertilizers has been a subject of debate. Although they have contributed to increased food production, their excessive and imbalanced use has led to significant environmental and health concerns. The indiscriminate application of chemical fertilizers, particularly urea, has resulted in reduced soil health and increased cultivation costs [6]. Chemical fertilizers, which are designed to enhance crop growth and productivity, have been associated with numerous problems that affect human health, the environment, and agricultural sustainability. Excessive and uncontrolled use of chemical fertilizers has led to significant environmental issues, including water pollution, soil degradation, and climate change [7]. These fertilizers can contaminate groundwater, potentially causing health hazards, such as hormone disruption, reproductive abnormalities, and cancer when consumed over extended periods [8]. Additionally, nitrogenous fertilizers have been linked to human and animal diseases through groundwater pollution [9].

Interestingly, while chemical fertilizers were initially considered a boon for humankind, scientific advancements have revealed their detrimental effects on soil health and groundwater quality [8]. The persistent nature of these agrochemicals negatively affects soil microbial communities, which are crucial for sustaining various nutrient cycles [8]. Moreover, the inability to determine the appropriate amounts, types, and application periods of fertilizers contributes to global warming and climate change, further affecting agricultural productivity [10]. The problems caused by chemical fertilizers extend beyond environmental concerns, including serious human health risks and long-term agricultural sustainability issues. These findings highlight the urgent need for alternative approaches, such as biofertilizers and sustainable agricultural practices, to mitigate the negative effects of chemical fertilizers while meeting global food demands [10].

The pesticides used in agriculture include herbicides, insecticides, fungicides, nematicides, and rodenticides. Herbicides account for the largest market share (around 40%), followed by insecticides (17%) and fungicides (10%) [11]. These chemicals are used to control weeds, insects, diseases, and other pests that threaten crop production. However, the widespread use of pesticides has resulted in significant environmental and health problems. They pose risks to human health and the natural environment, affecting beneficial insect groups, aquatic species, and soil microorganisms. Pesticides can accumulate in soil and water, causing harm to ecosystems and loss. They have been linked to genotoxicity, including mutagenicity, carcinogenicity, and teratogenicity [12]. Acute pesticide poisoning is a significant cause of morbidity and mortality worldwide, with an estimated three million severe cases and 220,000 deaths annually [13]. Although pesticides play a crucial role in increasing agricultural productivity and feeding the growing human population, their use comes at a considerable cost to ecosystems and human health [14]. The negative impacts of pesticides highlight the need for more sustainable agricultural practices, such as integrated pest management and biopesticide development, to protect both the environment and human well-being [15].

Bacillus is a diverse genus of rod-shaped spore-forming bacteria belonging to the Firmicutes phylum [16]. These bacteria are ubiquitous in nature, predominantly found in soil, and have been reported in various ecological niches [17]. This genus is known for its remarkable genetic and metabolic diversity, which enables Bacillus species to serve multiple ecological functions and adapt to a wide range of environments [17]. Bacillus species possess numerous abilities that make them useful in various fields. In agriculture, they play crucial roles in nutrient cycling, conferring stress tolerance to plants and improving plant growth through phytohormone production [17]. Some Bacillus species, particularly those in the B. cereus group, are opportunistic pathogens that cause food poisoning [18]. Many Bacillus species are beneficial and have been extensively exploited in the agro-biotechnology industry [17]. They produce antimicrobial substances, making them valuable for controlling microbial infections [19]. Additionally, Bacillus spp. have industrial applications in enzyme production, vitamin synthesis, and plant-growth-promoting formulations [20].

The Bacillus genus encompasses a wide range of species with diverse abilities, ranging from beneficial soil bacteria to potential pathogens. Their capacity to form resistant endospores, produce antimicrobial compounds, and adapt to various environments makes them significant in ecological, agricultural, industrial, and medical contexts [17]. The versatility of Bacillus species continues to drive research and application in multiple fields, highlighting their importance in both natural ecosystems and human activities. Bacillus sp. is a promising genus for food production owing to its versatile characteristics and numerous beneficial applications.

Bacillus spp. are widely recognized for their ability to produce a variety of useful substances and extracellular enzymes, making them valuable in the food, pharmaceutical, agricultural, and environmental industries [21]. This genus is particularly attractive for industrial use because of its short growth time, capacity to secrete proteins, and safety in humans [22]. Bacillus species can grow efficiently using inexpensive carbon sources, making them cost-effective for industrial production [21,23]

Interestingly, although Bacillus sp. offers numerous benefits in food production, it also presents challenges. Some Bacillus species, particularly Bacillus cereus, have been implicated in foodborne gastroenteritis [24]. However, certain Bacillus strains exhibit antimicrobial properties against foodborne pathogens, potentially contributing to food safety [25]. This dual nature highlights the importance of careful strain selection and control measures for food applications. Bacillus sp. hold great promise for food production because of its versatility, efficiency, and potential for producing valuable enzymes and antimicrobial substances. Their ability to form spores allows them to survive various food-processing operations, making them both a challenge and an opportunity for food production [26]. The adaptability of this genus to different environmental conditions and its potential as a probiotic further enhance its value in the food industry [27].

Recent research has highlighted their benefits, including nitrogen fixation, phosphate solubilization, siderophore production, and growth hormone production. Bacillus species also act as biocontrol agents, mitigating phytopathogens, and protecting plants. Advances in molecular biology have helped elucidate the genetic and metabolic interactions between Bacillus species and plants. Additionally, biotechnological innovations have led to the development of Bacillus-based biofertilizers and biopesticides, offering sustainable alternatives to chemical inputs. The integration of biochar and Bacillus species is crucial for improving soil fertility and promoting sustainable agriculture through better nutrient retention and plant growth [23,28]

1.1. The Change in Classification of Bacillus sp.

Recent advances in sequencing technology have led to significant changes in the nomenclature of Bacillus species, particularly within the Bacillus subtilis group, which includes many commercially important strains that are used as biocontrol agents and plant growth promoters [29]. These changes have resulted in taxonomic revisions and the identification of strains that were previously registered under different species names. Interestingly, a study revealed that most of the 14 Bacillus strains registered as plant pathogen crop protection products in the United States were initially classified under the species nomenclature, which is inconsistent with their current taxonomy [29]. This discrepancy highlights the importance of keeping the nomenclature up to date with the latest scientific findings. To address this issue, researchers have developed selective primers to identify Bacillus velezensis, which was found to be the most commonly misidentified strain in the study. These changes in Bacillus nomenclature over the past decade have significant implications for research, regulatory actions, and product labeling. The findings of the study emphasize the need to establish a clear link between research literature and product labels, which should lead to improved knowledge and more accurate identification of Bacillus species [29]. This ongoing refinement of bacterial nomenclature reflects the dynamic nature of microbial taxonomy and the importance of adapting it to new scientific discoveries. One of the main issues is the difficulty in interpreting molecular data from environmental sequencing projects within a bacterial ecological framework. In particular, the genus Bacillus demonstrates ecological diversity that extends beyond what was previously imaginable, complicating our understanding of Bacillus ecology and evolution [30]. For instance, ecologically distinct clusters of B. simplex have been identified in evolutionary canyons, and species demarcation in the industrially and medically important B. cereus group has proven to be challenging.

1.2. The Importance of Omics Technology to Bacillus sp.

Omics technology is a comprehensive analytical approach that enables the study of biological systems at various molecular levels. These technologies include genomics, transcriptomics, proteomics, metabolomics, epigenomics, and lipidomics [31]. High-throughput techniques have been used to generate large-scale datasets that provide insights into complex interactions and functions within living organisms [32]. These technologies have revolutionized biomedical research by allowing scientists to examine biological processes in their entirety, rather than focusing on individual components. For instance, single-cell multi-omics technologies can simultaneously analyze RNA expression, single-nucleotide polymorphisms, epigenetic modifications, and protein abundance, thereby providing a more comprehensive understanding of cellular mechanisms [33]. Additionally, omics technologies have found applications in diverse fields such as crop improvement, human health, environmental studies, and industrial sectors [31,34]

Omics technologies represent a paradigm shift in life science research, moving from reductionist approaches to global-integrative analytical methods. They offer unprecedented opportunities to unravel the complexities of biological systems and have the potential to advance precision medicine, drug discovery, and our understanding of disease mechanisms [35]. However, the vast amount of data generated by these technologies presents challenges in terms of data processing, integration, and interpretation, necessitating the development of sophisticated bioinformatics tools and computational approaches [36].

Omics technologies have significantly contributed to studies using Bacillus spp., providing valuable insights into their genomic and proteomic characteristics. These advancements have enhanced our understanding of Bacillus spp. and have expanded their potential applications. Bioinformatic tools have been instrumental in characterizing the genomic and proteomic sequences of Bacillus sp., enabling the prediction of genes and proteins [37]. The integration of genomics and proteomics, coupled with two-dimensional electrophoretic separation of proteins and mass spectrometry, has become a high-throughput technique for analyzing gene and protein expression, discovering new genes or protein products, and understanding gene and protein functions in post-genomic studies [37].

Interestingly, the application of omics technologies to Bacillus sp. has led to the development of numerous databases, facilitating rapid information retrieval for both genomic and proteomic research. These databases have made it possible to identify sites for post-translational modifications based on specific protein sequence motifs, which play crucial roles in protein structure, activity, and compartmentalization [37]. Moreover, the study of secreted proteins from Bacillus sp. has gained attention because of their potential therapeutic value and applications in biomedicine [37].

Omics technologies have revolutionized Bacillus sp. research by providing comprehensive molecular profiling. These advancements have not only deepened our understanding of Bacillus sp. biology but have also opened up new avenues for their application in various fields, particularly in biomedical research and therapeutic development.

1.3. Mode of Action

Bacillus species, particularly B. subtilis, employ multiple mechanisms to promote plant growth and enhance stress tolerance. These plant-growth-promoting bacteria (PGPB) act through various direct and indirect modes of action. Bacillus strains facilitate plant nutrient acquisition by fixing nitrogen and solubilizing phosphorus, making these essential nutrients more accessible to plants [38]. They also produce phytohormones such as indole acetic acid (IAA), gibberellins, and polyamines, which stimulate plant growth and development [39]. Additionally, Bacillus species secrete stress-mitigating enzymes like 1-aminocyclopropane-1-carboxylate deaminase, which helps plants cope with various environmental stresses [40]. Bacillus strains act as biocontrol agents by suppressing soil-borne diseases through the production of antifungal compounds and hydrolytic enzymes [41]. They also induce systemic resistance in plants and enhance defense mechanisms against pathogens [42]. Some Bacillus species can inhibit quorum sensing in pathogenic bacteria, further contributing to plant protection [40].

Bacillus species promote plant growth through multiple mechanisms, including nutrient provision, phytohormone production, stress alleviation, and pathogen suppression. These diverse modes of action make Bacillus an effective PGPB for improving crop productivity and reducing the need for chemical inputs in agriculture [40]. However, further research is needed to fully understand and utilize the potential of Bacillus spp. in horticulture and sustainable agriculture [43].

1.4. Consistency in Field Performance

Laboratory experiments and field conditions often yield discrepancies in the results for Bacillus-based products, as observed across various scientific disciplines. This phenomenon is not unique to Bacillus studies but extends to multiple areas of research. In ecological studies, combining field observations with controlled laboratory experiments can provide more robust insights [44]. While field observations may reveal correlations between plant species composition and soil bacterial communities, laboratory microcosm experiments can help establish causal relationships by isolating specific factors, such as plant community composition. However, it is important to note that such controlled experiments may not fully capture the complexity of real-world conditions. Interestingly, similar discrepancies were observed in other studies. For instance, in hydraulic fracturing experiments, significant differences were observed between the calculated minimum stresses from the fracture closure pressure and the actual values obtained under the experimental conditions [45]. In weathering studies of silicate minerals, laboratory rates can be up to five orders of magnitude higher than those inferred from field studies [46]. These examples highlight the challenges of extrapolating laboratory results to real-world scenarios.

Novel approaches have been developed to address these discrepancies. For example, the use of Si isotope ratios to measure weathering rates under close-to-natural conditions [46] or conducting experiments that simulate both laboratory and actual field conditions, as observed in friction and wear behavior studies of alloys [47]. These efforts aim to bridge the gap between controlled experiments and real-world applications, providing more accurate and relevant data for the practical use of Bacillus-based products and other scientific applications.

1.5. Reduction in Nitrogen Fertilization

Several studies have demonstrated the effectiveness of Bacillus spp. in reducing nitrogen fertilizer requirements while maintaining or improving crop yield. In peanut cultivation, a combination of Bacillus spp. NTLG2-20 inoculant with a 50% reduction in nitrogen fertilizer (20 kg N ha⁻¹) resulted in a 17.6% higher yield compared with no nitrogen application and showed no significant difference from the recommended 100% nitrogen application [48]. Similarly, during potato cultivation, inoculation with Bacillus sp. OSU-142 increased tuber yield and yield components at all nitrogen fertilizer levels, with the most beneficial effect observed at 120 kg ha⁻¹ nitrogen [49]. Interestingly, the use of Bacillus sp. not only reduces the need for nitrogen fertilizers but also mitigates the environmental risks associated with excessive nitrogen use. For instance, biofertilizers made from Bacillus sp. T28 combined with sea buckthorn pomace reduced NO₃⁻-N content by 33.1–43.8% and decreased N₂O release rates by 8–26 times in paddy soil [50]. This demonstrates the potential of Bacillus spp. to improve soil health and reduce environmental pollution. In potato cultivation, the beneficial effects of Bacillus spp. OSU-142 on tuber yield was observed at 120 kg N ha⁻¹, suggesting a more effective inoculation in low-N input agriculture [49]. This indicates the possibility of reducing nitrogen fertilizers while maintaining productivity.

Interestingly, in soybean production, organomineral fertilization using 50% mineral and 50% organic fertilizer with bovine biofertilizer was the most efficient with and without Bacillus sp. inoculation [51]. This suggests a potential 50% reduction in mineral nitrogen fertilizer use when combined with organic sources. Research has consistently shown that Bacillus sp. can effectively reduce nitrogen fertilizer requirements across various crops while maintaining or improving yields. This approach not only benefits crop production but also contributes to more sustainable agricultural practices by mitigating the environmental risks associated with excessive nitrogen use. These studies highlight the potential of Bacillus sp. as a promising biological agent for the development of more efficient and environmentally friendly fertilization strategies in agriculture [50].

Reduction in Phosphorous Fertilization

Bacillus sp. has shown potential in reducing phosphorus fertilization requirements, but its effectiveness varies across studies. In soybean cultivation, the application of Bacillus subtilis and Bacillus megaterium, either alone or in combination with phosphate fertilization, did not significantly influence the nutritional, biometric, and productive parameters of the crop in soil with naturally low P content. This suggests that, in some cases, Bacillus sp. may not contribute to reducing the phosphorus fertilization needs. In a study of sugarcane, co-inoculation with Bacillus sp. BACBR04, Bacillus sp. BACBR06, and Rhizobium sp. RIZBR01, combined with compost as a P source, increased the P content in shoots compared with uninoculated treatments with compost or triple superphosphate [52]. This suggests that Bacillus sp. can enhance P uptake and potentially reduce the need for chemical P fertilizers.

Organic fertilizers, when combined with phosphorus-solubilizing bacteria, such as Bacillus megaterium var. phosphaticum, can potentially improve phosphorus availability and uptake in plants. However, its effectiveness may vary depending on the specific conditions and application methods. In a study of a natural meadow, the application of phosphorus-solubilizing bacteria (Bacillus megaterium var. phosphaticum) did not significantly affect dry matter yield or botanical composition, whereas phosphorus fertilization alone affected these parameters [53]. Interestingly, some studies have shown that a combination of organic fertilizers and phosphorus can have positive effects on crop yield and quality. For instance, in medicinal pumpkin seeds, the combination of vermicompost and phosphorus fertilizer application resulted in the highest seed number and oil percentage [54]. Similarly, the combined application of organic, NPK, and phosphorus fertilizers (OCPF) in a teak plantation improved soil quality and optimized bacterial community structure [55].

While the specific combination of organic fertilizers and Bacillus spp. for reducing phosphorus fertilization shows limited direct evidence of effectiveness, the overall approach of integrating organic and inorganic fertilizers appears promising for improving soil fertility, crop yield, and microbial community structure. Further research is required to determine the optimal combinations and application methods for different crops and soil types. Several studies have demonstrated the positive effects of Bacillus spp. on phosphorus availability and crop yield. For instance, inoculation with Bacillus atrophaeus combined with mycorrhizal fungi enhanced maize yield by 4.2% compared with standard fertilizer treatments [56]. Similarly, Bacillus subtilis inoculation improved some plant parameters in maize, although the effects were complex when combined with rock phosphate fertilization [57]. However, the effectiveness of Bacillus-based products has not always been consistent. In a study of natural meadows, phosphorus-solubilizing bacteria (Bacillus megaterium var. phosphaticum) did not show any significant effect on dry matter yield or botanical composition, whereas phosphorus fertilization had an impact [58]. This suggests that the effectiveness of Bacillus-based products may depend on specific environmental conditions and plant species. Figure 1 shows the aspects related to the effects of Bacillus sp. inoculation on plant production.

1.6. Compatibility with Other Inputs

Mixing operations in agriculture and food processing are crucial for blending ingredients to create uniform mixtures that are essential for producing acceptable food products [59]. This process can also be used to alter the properties of dough, indicating its versatility in food production. Blending agricultural products can enhance product attributes, increase product safety by replacing harmful chemicals with safer alternatives, reduce the consumption of non-sustainable materials, and decrease pollutant release into the atmosphere [60]. This demonstrates the potential of mixing to improve both product quality and environmental sustainability. In the context of sustainable agriculture, mixing or blending can contribute to minimizing inputs, waste, and pollution [61]. This approach can help address concerns related to the increasing world population, human diet, and overexploitation of natural resources. Interestingly, while mixing can offer benefits, it is important to note that existing cropping patterns may not always align with comparative advantages in agriculture. For instance, a study in Ilam Province showed that the current cropping pattern was not allocated according to comparative advantage but was related to the effective protection coefficient [62]. Mixing bacteria-based products with chemical products can be compatible in certain cases but depends on the specific components and their intended use. Some studies have demonstrated the successful combination of biological and chemical approaches. For example, researchers have combined chemical catalysis with bioconversion to valorize polyethylene deconstruction products [63]. They used chemical catalysis to break down polyethylene into n-alkanes, which were then utilized by the microbial consortia. This demonstrates that the carefully selected bacterial strains are compatible with chemically processed materials.

However, the compatibility is not universal. Many chemical products, especially those with antimicrobial properties, can inhibit or kill beneficial bacteria. For instance, a study on Z-Mix, a zinc-oxide-based medicament containing antibiotics, demonstrated its antimicrobial effects against various bacteria [64]. However, these products are not compatible with probiotic bacteria. Although some innovative approaches successfully combine bacterial and chemical products, compatibility must be assessed on a case-by-case basis. Factors such as the specific bacterial strains, chemical components, and intended applications need to be carefully considered to ensure that the mixture remains effective and the bacteria are viable.

Bacillus sp. products are compatible with certain chemical products, but their compatibility varies depending on the specific chemicals and conditions involved.

Some studies have shown promising results for combining Bacillus spp. with chemical products. For instance, a protease produced by the thermophilic Bacillus sp. demonstrated good stability and compatibility with commercial laundry detergents, retaining over 80% of its activity after 30 min of incubation at 60 °C with Tide^® detergent, USA Cheshire, CT [65]. Similarly, cutinase from Bacillus sp. KY0701 exhibits high stability and compatibility with commercial detergents, making it a potential candidate for application in the detergent industry [66].

However, compatibility is not universal. Some chemical agents can inhibit or deactivate enzymes in Bacillus sp. For example, while the protease from thermophilic Bacillus sp. was stable in the presence of EDTA and some detergents, it was inhibited by Triton X-100 and unstable in a 5% peroxide solution [65]. In addition, a study of cellulases from Bacillus sp. SMIA-2 showed that the compatibility of the enzyme varied with different laundry detergents, being more stable with Ultra Biz^® and less stable with Ariel^® USA, Cheshire, CT [67]. Although Bacillus sp. products can be compatible with certain chemical products, especially in detergent applications, their compatibility should be evaluated on a case-by-case basis. Factors such as pH, temperature, and specific chemical agents can significantly affect the stability and activity of Bacillus spp. Therefore, careful testing and optimization are necessary when considering mixtures of Bacillus sp. products and chemical agents for industrial or commercial applications.

1.7. Strain Specificity and Host Compatibility

Strain specificity and host compatibility play crucial roles in determining the success of host–parasite interactions across various biological systems. Studies have shown that the outcome of these interactions depends on both host and pathogen genetic factors as well as their specific combinations [68]. This specificity can influence infection rates, transcriptional responses, and the overall compatibility between hosts and pathogens. In the case of Mycobacterium tuberculosis, research has revealed that host–strain compatibility significantly affects infection outcomes. Compatible host–strain combinations exhibit higher infection rates and distinct transcriptional patterns than low-compatibility infections [69]. Similarly, in the Citrus tristeza virus (CTV) system, strain-specific interactions can overcome tissue tropism limitations in selective hosts, highlighting the importance of compatibility between gene products of different strains and the host [70].

Interestingly, contradictions and variations in host specificity have been observed in different systems. For instance, although bat flies have historically been viewed as non-specific, recent biodiversity surveys have demonstrated high host specificity for many species [71]. In contrast, studies on Frankia strains of Asian origin showed less diversity in host specificity compared with North American and European strains [72]. These findings underscore the complexity of host–parasite interactions and the need for careful consideration of genetic and environmental factors. Strain specificity and host compatibility are critical determinants of host–parasite interactions, influencing infection success, transcriptional responses, and evolutionary dynamics. Understanding these mechanisms is essential for developing effective strategies to manage diseases and predict the outcomes of host–parasite associations in various biological systems.

1.8. Impact on Soil Microbiome

Plant microbiomes can be manipulated to promote plant growth through various approaches. Microbiome engineering is an emerging biotechnological strategy to improve crop yield and resilience. This can be achieved through indirect methods, such as using soil amendments or selective substrates, and direct methods, such as inoculation with specific probiotic microbes, artificial microbial consortia, microbiome breeding, and transplantation [73]. High-efficiency top-down approaches based on high-throughput technology and synthetic communities (SynCom) can be used to identify and apply plant-growth-promoting rhizobacteria (PGPR) in the rhizosphere [74]. Interestingly, the composition of the microbial community is influenced by various factors, including soil type, plant genotype, plant exudates, and cultivation practices [75]. This complexity necessitates both reductionist and systems ecology approaches to identify biotic and abiotic factors involved in microbiome assembly [76]. In conclusion, several strategies can be employed to harness the rhizosphere microbiome and enhance crop productivity. These include the use of organic and inorganic amendments, microbial inoculants, synthetic microbial consortia, host-mediated microbiome engineering, prebiotics made from specific plant root exudates, and crop breeding to promote beneficial plant-microbiome interactions [77]. Additionally, bioengineering of the endophyte microbiome through methods such as host-mediated and multi-generation microbiome selection, inoculation into soil and rhizosphere, seed or seedling inoculation, tissue atomization, and direct injection into tissues or wounds can be effective [78].

The introduction of Bacillus sp. into plants can have a significant impact on the soil microbiome. Bacillus spp. can induce changes in the overall soil microbial community structure. For instance, it has been observed to alter the fungi/bacteria ratio and is negatively correlated with the relative abundance of phagotrophic protists [79]. This bacterial introduction can also increase the total number of microorganisms, amino heterotrophs, and Azotobacter spp. in the soil [80]. Interestingly, the impact of Bacillus sp. on plants and the soil microbiome can vary depending on environmental factors. For example, the effect of B. subtilis on plants decreases with increasing soil bulk density and fertilizer rate [81]. Additionally, Bacillus sp. can enhance the resistance of the plant–microbial system to the adverse effects of high nitrogen fertilizer rates by rearranging bacteria in the rhizosphere ecological niches [81].

The introduction of Bacillus sp. can lead to significant alterations in the soil microbiome, potentially promoting plant growth and enhancing the soil microbial diversity. However, its effects can be modulated by various environmental factors, highlighting complex interactions within the soil ecosystem. The ability of Bacillus sp. to influence the soil microbiome makes it a promising candidate for sustainable agricultural practices and bioremediation strategies [82].

1.9. Formulation and Delivery Methods

Bacillus-based bioinoculants face challenges related to stability, shelf life, and application methods, which require research in several key areas. Formulation additives play a crucial role in enhancing the survival and stability of liquid bio-inoculants. A previous study found that 2% polyvinylpyrrolidone, 0.1% carboxymethylcellulose, and 0.025% polysorbate 20 significantly improved the long-term survival of Bacillus megaterium var. phosphaticum, maintaining 5.6 × 10⁷ colony forming units (CFU) mL⁻¹ after 480 days when stored at 30 °C [83]. This highlights the importance of optimizing formulation components to extend the shelf life. Interestingly, although liquid formulations show promise, talc-based formulations have also demonstrated effectiveness as carriers for Streptomyces species, which are similar to Bacillus spp. as soil-dwelling bacteria [84]. This suggests that exploring various carrier materials could lead to improved stability and delivery methods of Bacillus-based bioinoculants.

To address these challenges, researchers should focus on novel encapsulation techniques and formulation strategies. Bioencapsulation using appropriate polymers and capsule sizes can enhance the survival, colonization efficiency, and storage period of bioinoculants [85]. Additionally, investigating the use of probiotics and their metabolites in edible packaging could provide innovative delivery methods, potentially improving both the shelf life and efficacy of Bacillus-based products in agricultural applications [86]. Root colonization by beneficial rhizobacteria is crucial for plant growth promotion and biocontrol. Effective delivery systems are essential to ensure the even distribution and sustained activity of Bacillus strains in the rhizosphere.

In-furrow spray application has been shown to be an effective method for inoculating plants with rhizosphere-competent bacteria. Studies have demonstrated that maximum root colonization can be achieved using cell suspensions of log 7–8 colony-forming units (cfu)/mL applied at a rate of 10 mL/m for crops, such as canola, soybean, and wheat [87]. This technique avoids interactions with formulation components and may be valuable for commercial delivery when compatible with agronomic practices. Interestingly, the efficacy of root colonization can be enhanced through genetic modifications. For instance, disrupting the global transcription regulator AbrB in Bacillus amyloliquefaciens SQR9 significantly improved its colonization and biocontrol activity [88]. Additionally, the type VII secretion system and its major secreted protein YukE were found to be critical for root colonization by promoting iron leakage from plant roots during the early stages of inoculation [89].

Although in-furrow spray application provides a practical delivery method, genetic modifications of Bacillus strains offer promising avenues to enhance their root colonization abilities and extend their activity periods. Further research into optimizing formulations and delivery systems, combined with genetic improvements, could lead to more effective and sustained colonization of beneficial Bacillus strains in agricultural applications.

1.10. Regulatory and Safety Aspects

Bacillus species are generally regarded as safe for use in various applications, including food production and biocontrol. However, as their large-scale use increases, the regulatory and safety aspects require further investigation. Further studies are needed to evaluate the long-term effects of Bacillus-based products on human health and the environment. Although B. subtilis and certain other species are classified as Generally Recognized As Safe (GRAS) by the Food Drug Administration (FDA), the safety of new strains or genetically modified Bacillus species used in industrial applications must be thoroughly evaluated [90]. The safety of novel strains or genetically modified Bacillus species used in industrial applications needs to be thoroughly assessed. This is particularly important when considering their use in food production or as biocontrol agents in agriculture [91].

Although Bacillus species show promise in mitigating heavy metal stress in plants and promoting plant growth [92], there are concerns regarding their potential to produce toxins or cause allergic reactions in some individuals [93]. Additionally, the use of Bacillus spp. in animal feed as probiotics raises questions about their colonization and dissemination in animal guts, necessitating the establishment of testing and production standards [94]. To ensure the safe and sustainable use of Bacillus species in various applications, more comprehensive studies are needed to address potential safety risks, establish clear regulatory frameworks, and develop standardized testing protocols. This includes assessing their environmental impact, potential allergenicity, and long-term effects on human and animal health [92]. Collaboration among researchers, industry, and regulatory bodies is crucial for developing science-based guidelines that balance technological progress with robust safety standards. Figure 2 shows the mode of action of Bacillus sp., demonstrating several abilities related to plant growth.

1.11. Integration into Crop Management Systems

Integrating Bacillus-based products into existing agricultural practices is crucial for their successful implementation in crop management systems. These biopesticides offer promising alternatives to conventional pest control methods based on integrated pest Management (IPM) and crop management (ICM) [95]. Bacillus thuringiensis (Bt) has gained widespread use in the global biopesticide market because of its effectiveness against target pests in economically important crops [95]. The integration of Bt-based products complements physical and cultural methods in pest and disease management, contributing to the reduction in pest populations below economic injury levels while minimizing impacts on the agro-ecosystem and environmental conditions [95]. Interestingly, the transition from conventional to organic farming systems presents both challenges and opportunities for the integration of Bacillus-based products. During this transition period, growers may face pest control difficulties and lower yields when abandoning conventional practices [96]. However, biopesticides, such as Bt, can play a crucial role in supporting this transition by providing effective pest control while aligning with organic farming principles.

The successful integration of Bacillus-based products into crop management systems requires a holistic approach that considers the principles of ICM and IPM. By incorporating these biopesticides into existing practices, farmers can enhance pest control efficacy, reduce environmental impact, and contribute to more sustainable agricultural systems. This integration aligns with the growing focus on crop quality, safety, and sustainability in modern agriculture [95].

Table 1. The impact of Bacillus sp. inoculation on plant growth through various improvements.

Bacillus spp.	Improvement	References
Bacillus sp.	50% reduction (20 kg N ha−1) 17.6% higher yield	[2]
Bacillus sp.	Effective at 120 kg N ha−1 Increased tuber yield	[5]
Bacillus sp.	Reduced NO3−-N by 33.1–43.8%, decreased N2O release rates by 8–26 times	[6]
B. subtilis B. megaterium	50% mineral and 50% organic fertilizer	[7]
Bacillus sp.	Increased P content in shoots	[8]
B. atrophaeus, B. subtilis	Enhanced yield by 4.2%, improved plant parameters	[9]
B. subtilis	Changed the microbiome and improved crop yield and resilience	[10]
Bacillus sp.	Increased microorganism heterotrophs	[29]
Bacillus sp.	Decreased nitrogen fertilization	[30]
Bacillus sp.	Promoted plant growth, enhance soil microbial diversity	[31]
Bacillus sp.	Changed the microbiome and promoted plant growth because of these changes	[13]
Bacillus sp.	Altered the relationships between the microbes and facilitated plant growth.	[15]

shows the impact of Bacillus sp. inoculation on plant growth through various improvements.

2. Conclusions

Although several studies have demonstrated the capacity of bacteria from the genus Bacillus to reduce the quantity of mineral fertilizers because of their ability to fix atmospheric nitrogen and decrease dependency on mineral phosphorus fertilizers through phosphorus solubilization, their application in food production remains limited. This limited usage can be attributed to several factors. Agricultural enterprises often lack access to scientific studies, and there are significant challenges in translating and applying the information contained in these studies to practical food production. Furthermore, the results obtained from controlled studies are not entirely replicable under farming conditions. In addition, in large-scale agricultural operations, aerial application of inputs is typically employed to enhance efficiency. To minimize application costs, multiple chemical and biological products are frequently combined. However, this practice does not ensure the viability of microorganisms present in the biological products. Substantial variations exist in the soil composition, climatic conditions, and production management practices. Consequently, the outcomes observed in scientific studies may not be precisely replicated in agricultural settings. There is significant pressure on farms to utilize mineral fertilizers, driven by numerous companies that market these products and their ease of use. Additionally, farms have more experience employing mineral fertilizers than products based on microorganisms. In addition, it is challenging to adopt measures to reduce the use of mineral fertilizers in food production and accept the risk of decreased productivity. Such a decision is likely to incur high costs and result in numerous problems. Numerous studies have demonstrated the efficacy of Bacillus sp. in reducing various agricultural inputs, particularly nitrogen and phosphorus fertilizers. As previously mentioned, this effect is a result of the combined ability of bacteria to fix atmospheric nitrogen, solubilize unavailable phosphorus in the soil, and enhance the efficiency of plants to explore the soil and uptake nutrients. This practice has the potential to reduce production costs and environmental impact without compromising productivity. However, the consumption of mineral fertilizers continues to increase annually. This discrepancy represents the gap between theoretical knowledge and practical applications.

3. Future and Perspectives

With a comprehensive understanding of how microorganism-based products function, as well as an in-depth knowledge of plant–microorganism interactions in various environments, soil types, and conditions, the outcomes generated from the utilization of microorganism-based products will demonstrate increased consistency. This technology has been widely recognized and disseminated. Consequently, an increasing number of farms will adopt it and be incentivized to reduce their reliance on mineral fertilizers. These farms are likely to benefit from reduced production costs, decreased environmental impacts, and more sustainable food production practices.