Comprehensive Review of Microbial Inoculants: Agricultural Applications, Technology Trends in Patents, and Regulatory Frameworks

Abstract

Agriculture is essential for nutrition and the global economy, becoming increasingly important due to population growth and higher food demand. This situation boosts interest in creating bioproducts that enhance productivity sustainably while reducing environmental issues and strain on natural resources. Bioinoculants are important innovations that use beneficial microorganisms to boost crop growth and resilience. They enhance the interaction between soil and plants by solubilizing essential nutrients and producing phytohormones. This not only boosts agricultural productivity but also promotes environmentally sustainable practices by decreasing reliance on chemical fertilizers. Considering the relevance of this subject to advances in agro-industrial biotechnology, this review analyzes recent studies and patent advances on the production and use of bioinoculants, as well as their integration into agricultural practices and plant development. It also explores the dynamics of production and downstream processes on an industrial scale, regulations in different countries, and growing market demands, which is an important feature of this review. Furthermore, future perspectives for the application of bioinoculants in agro-industrial biotechnology are discussed, emphasizing the critical role that these biological agents play in advancing agricultural sustainability.

1. Introduction

To address population growth and increasing food demand, sustainable agricultural practices are encouraged to lessen the strain on natural resources and farmland while reducing fertilizer use. In 2021, it is estimated that the agricultural sector utilized 109 million tons of nitrogen fertilizers and 46 million tons of phosphate fertilizers [1]. About 65% is lost to the environment due to processes like volatilization, denitrification, and reactions with soil’s organic components. This issue has led to a greater demand for chemical products that resulted in the excessive use of fertilizers, which can have negative impacts on the soil and cultivars, in addition to the environment, since synthetic fertilizers can cause harmful effects on several physiological processes of living beings and their biological interactions [2,3].

In this context, the potential of bioinoculants has been investigated as a means of enhancing the growth of plants in agricultural crops through the implementation of more sustainable practices. These are defined as products comprising living organisms that, when introduced into the environment, can facilitate the bioavailability of nutrients or act in the biocontrol of harmful biological activities [4,5]. A variety of microorganisms are employed as bioinoculants, including bacteria, fungi, and algae. These organisms facilitate the solubilization of nutrients (such as phosphorus and nitrogen), stimulate the synthesis of hormones, mitigate the adverse effects of continuous cultivation, reduce the prevalence of pathogens, and promote the growth of agricultural crops [6,7,8,9].

Introducing bioinoculants like mycorrhizal fungi, beneficial bacteria, and biocontrol agents can significantly improve soil and plant health. They fix nitrogen, solubilize nutrients like phosphorus, zinc, and potassium, and produce plant hormones, antibiotics, enzymes, and siderophores [10]. Several microorganisms, including Rhizobium, Bacillus, Azobacter, Aspergillus, Trichoderma, Pseudomonas, Paenibacillus, and Acinetobacter, have been identified as potential candidates to produce bioinoculants. These microorganisms have been selected based on their demonstrated capacity to enhance plant growth, promote greater accumulation of nutrients, and improve soil health [11,12,13,14,15]. These benefits of applying bioproducts to the soil are generally associated with the colonization of plant roots and are largely linked to the density of the bioinoculant in the rhizosphere environment [16].

Considering the growing number of innovations in the field, companies have been seeking enhanced performance and alternative systems in agriculture. This has led to a corresponding expansion of the bioinoculants market. Over the past five years, 822 patents have been filed within this field, 114 of which are related to bioinoculants. Moreover, advances in bioinoculant studies have prompted the assessment of diverse agricultural crops and facilitated the scaling up of these bioprocesses, such as industrial-scale production by piloting bioreactors that produce microbial biomass efficiently for demand with a minimum concentration of 1 × 10⁸ cell mL⁻¹ and downstream processes that facilitate the application of bioproducts, whether in solid formulations using vehicles (e.g., talc, vermiculite, or immobilization in waste) or liquid formulations, together with other nutrients, minerals, and bioactives [17,18].

Because of these significant developments and increased commercialization, regulatory bodies have established quality, safety, and efficacy standards. India, Brazil, Canada, Australia, and Uruguay have strict quality standards for microorganism concentration, identification, purity, and application conditions. Several challenges still exist in bioinoculant production, such as environmental interactions, downstream processes, half-life, large-scale applications, and field efficiency.

This review aims to study bioinoculants in agro-industrial biotechnology. It covers current lab and large-scale production, analyzes regulations by country, and reviews existing patents. This study enhances our understanding of biofertilizers in agriculture, focusing on their mechanisms, patent developments, and recent global regulations, and promotes discussion on sustainable biotechnology in crop production.

2. Synthetic Fertilizer and Biofertilizer Overview

Agricultural activities generate significant problems in relation to the fertility of soil, which is the main raw material for agriculture. The decline in organic carbon and nutrient levels in soil due to intensive agriculture necessitates the use of substances to restore and enhance soil fertility for continuous cropping [19].

These substances are known as fertilizers, chemical compounds of minerals (extracted from mines) or organic (extracted from animal and/or vegetable waste) sources, that can be natural or synthetic, combined or not. These chemicals are sources of nutrients and micronutrients that are essential for plant development, especially nitrogen, phosphorus, and potassium [19]. Thus, when used and managed correctly, fertilizers can result in improvements in the growth and development of agricultural crops, increasing the productivity and quality of cultivated plants [20].

Since the Green Revolution, which took place in the 1960s and aimed to encourage an increase in global food production to combat hunger, the use of fertilizers and other agricultural chemicals (such as pesticides) has been promoted [21]. According to recent data from the Food and Agriculture Organization [1], in 2021 the global agricultural sector used 109 million tons of nitrogen fertilizers, 46 million tons of phosphate fertilizers, and 40 million tons of potassium fertilizers, totaling 195 million tons of inorganic fertilizers. Figure 1 shows the percentage of inorganic fertilizers used across the globe in 2021, according to the FAO survey [22].

Fertilizers provide essential nutrients to the soil for agriculture, which are lost due to intense cultivation over the years. When used appropriately, rationally, and in a balanced manner, fertilizers can provide better and faster plant development, the maintenance of soil and crop quality parameters, and an increase in crop yields [23]. Also, they have advantages such as being easily absorbed by the soil as they are soluble in water and allow an application that guarantees a more unanimous plant absorption of nutrients [24].

A large portion of nutrients from synthetic and organic fertilizers is lost due to volatilization, leaching, denitrification, and reactions with soil components, with only 30–35% being absorbed by crops. This necessitates the use of greater quantities of fertilizers in agroecosystems to boost the effectiveness of these inputs in enhancing agricultural productivity [2].

The inappropriate and excessive application of fertilizers can significantly harm the natural physical, chemical, and biological characteristics of the soil, potentially resulting in effects that are contrary to their intended benefits [3]. Excessive fertilization can alter the content of nitrogen and carbon, important organic components of the soil, and influence soil aggregation, pH, moisture, density, and nutrient availability [25].

Soil structure changes as aggregate units break down, leading to smaller pore spaces and reduced soil volume and porosity. This increases soil density and compaction, decreasing air circulation and drainage. The soil pH is affected by nitrogen compounds such as ammonium ion, nitrate ion, and urea. The transformation of nitrogen from one form to another and its absorption by plants influences soil acidity. In turn, the hydrogen ions emitted by the acid lead to the loss of alkaline nutrients from the soil, and the free oxygen generated in these reactions can cause the oxidation of organic matter in the soil [26].

Furthermore, the production and use of synthetic fertilizers are sources of salt (which increases soil salinity) and many toxic metals (e.g., cadmium, arsenic, mercury, nickel, lead, and copper). The salt and metals contained in fertilizers can accumulate in the soil and/or be leached into surface waters close to the area of application and can cause toxicity to living organisms [27].

These changes can impact soil microbial populations, which are essential for soil fertility. Bacteria, fungi, protozoa, algae, and viruses in the soil, along with their enzymes, play a crucial role in maintaining soil balance and fertility in agriculture. They act on processes such as nutrient cycling and the degradation of inorganic and organic matter. Once disturbed, it can result in a major loss of soil fertility [28,29].

The atmosphere is also threatened by the excessive use and production of these compounds. Nitrogen fertilizers are a major source of nitrous oxide (N₂O) emissions, which are harmful to the ozone layer. They also contribute to greenhouse gas emissions, which significantly impact global warming and climate change [2,30].

Excessive application of fertilizers rich in nitrogen, whether synthetic or organic, can also lead to increased leaching and runoff of nitrates and cations into deeper soil layers and nearby bodies of water, causing contamination, pollution, and eutrophication of surface and groundwater. Eutrophication caused by the deposition of nitrates in surface waters can lead to hypoxia or anoxia of the aquatic environment and the proliferation of harmful microalgae, causing damage to human health, and loss of aquatic biodiversity and quality of fishing resources [31,32]. Examples of how fertilizer use can affect biota are shown in

Table 1. Effects of fertilizers on the physiology of different species.

Fertilizer	Type	Experimental Conditions	Reported Environmental Impacts	Reference
Urea	Nitrogen	Exposure of Oreochromis mossambicus to different concentrations of urea for 24, 48, 72, and 96 h	Urea concentrations of 22,000 and 38,000 mg L−1 were sublethal and lethal, respectively; decreased feeding rate and growth rate were observed	[33]
Urea	Nitrogen	Exposure of Danio rerio to different urea concentrations (0, 10, 50, and 100 mM) from 4 to 96 h post-fertilization (hpf)	Affected cell proliferation and the number of cells that express nos1, a gene involved in the formation of neuronal cells during embryonic development	[34]
NPK (3 different ratios)	Nitrogen, phosphorus, potassium	Exposure of Biomphalaria alexandrina to sublethal concentrations (1/10 LC50, ¼ LC50, and ½ LC50) of each fertilizer for 24 h	Decreased the growth rate of juvenile snails; activation of the antioxidant system	[35]
Ceselio	Phosphorus	Exposure of Lanistes carinatus to 200 and 600 µL L−1 of fertilizer for 0, 1, 3, and 7 days	Increase in lipid peroxidation; tissue necrosis; increase in lipofuscin pigment (related to aging); and bioaccumulation of metals in the shell and organs	[36]
Weatfert (NPK)	Nitrogen, phosphorus, and potassium (higher phosphorus content)	Exposure of Eobania vermiculata to the recommended agricultural dose (D1 = 500 mg/400 cm2) and the recommended agricultural dose × 2 (D2 = 1000 mg/400 cm2)	Histological and biochemical alterations in the digestive gland; negative impact on growth rate	[37]
Copper sulfate	Copper sulfate	Exposure of Friesella schrottkyi 5.0 g L−1 for 72 h	Compromised the survival of bees mainly via oral exposure	[38]
5% S, 5% Zn, 3% Mn, 0.6% Cu, 0.5% Be, 0.06% Mo	Micronutrient mixture	Exposure of Friesella schrottkyi 2.0 mL L−1 for 72 h	Reduction in respiratory rate	[38]
Urea	Nitrogen	Exposure of Drawida willsi to 100, 200, 300, 400, 500, 600, 700, and 800 mg of urea kg−1 of dry soil for 96 h	At a dose of 800 mg of urea kg−1 of dry soil, 100%, 76%, and 52% mortality were observed for juvenile, immature, and adult earthworms, respectively	[39]
Ammophos	Phosphorus	Exposure of Cyprinus carpio to 97.21 mL L−1 of fertilizer for 96 h	Behavioral and hematological changes	[40]
Kristalon (NPK)	Nitrogen, phosphorus, and potassium	Exposure of Cyprinus carpio to 265.18 mL L−1 of fertilizer for 96 h	Behavioral and hematological changes were observed	[40]

.

The environmental impacts of the unrestrained use of fertilizers in agriculture have given rise to a new need: the creation of more sustainable methods and processes that can replace nutrients in the soil to maintain quality agricultural activity and, at the same time, do not harm the environment and natural resources [41].

A sustainable agricultural practice that emerged is the use of biofertilizers. These eco-friendly compounds reduce environmental pollution and the negative effects of traditional fertilizers while providing essential nutrients to enhance soil fertility and boost agricultural production [4,5]. Biofertilizers are divided according to their mechanism of action and functions, with the most used being nitrogen-fixing, potassium-solubilizing, phosphate-solubilizing, and plant growth-promoting rhizobacteria (PGPR) biofertilizers [42,43,44].

Biofertilizers consist of living organisms that enhance the chemical and biological properties of soil by transferring nutrients and naturally regulating pH levels. These processes effectively stimulate the growth of crops [45]. The organisms that compose biofertilizers can be very diverse, such as nitrogen-fixing cyanobacteria, green microalgae, and cyanobacteria that stimulate plant growth, mycorrhizal bacteria and fungi, and even some species of seaweed that increase organic matter, balance pH, and reduce the carbon/nitrogen ratio in the soil [46].

Species of eukaryotic green algae and prokaryotic blue algae (cyanobacteria), both known as photosynthetic microalgae, have been extensively studied in biofertilizer development, where they have shown promising results [47,48]. They are the largest primary producers on Earth and help improve the fertility and quality of various soils by naturally producing plant growth hormones, polysaccharides, and antimicrobial compounds, as well as adding organic matter and fixing nitrogen [49,50].

Macroalgae are also quite interesting in this context. They adapt well to bright environments with limited nutrients and water, still carrying out photosynthesis. This produces amino acids, growth promoters, hormones, and fixes nitrogen, all essential for crop growth. These organisms provide bioactive compounds and organic matter that enhance germination, rooting, nutrient absorption, soil quality, and crop yield [32].

Some species of microorganisms, especially bacteria and mycorrhizal fungi, called phosphate solubilizers, can mineralize and solubilize phosphorus in the soil, making it available for consumption by cultivated plants [51]. These microorganisms offer several advantages when used as biofertilizers, including low cost and high efficiency in promoting plant growth, nitrogen fixation, and the synthesis of plant hormones [6,7].

Yeasts have also been the target of studies for possible applications as biofertilizers, as they can provide various nutrients, phytohormones, and enzymes to plants. They absorb carbon and nitrogen, can ferment carbohydrates, can adapt to different environments and soil types, and proliferate at high rates. Furthermore, they can absorb toxic substances, which can be used in the bioremediation of contaminated soils [52]. Table 2 presents different organisms that show promise in the composition of biofertilizers in different types of crops.

The advantages of using biofertilizers instead of traditional fertilizers are numerous. They can colonize the rhizosphere and the interior of the plant. Therefore, they can boost plant growth through their application to the seed, leaf surface, or soil. Furthermore, once applied continuously to the crop for approximately 4 years, there is no need for reapplication, as the parental inoculums can maintain their own growth and multiplication over time [53].

Table 2. Different organisms used as biofertilizers and their benefits.

Species Used as Biofertilizer	Biological Division	Culture in Which It Was Applied	Benefits	Reference
Chlorella sp.	Chlorophytes (green algae)	Tomato and cucumber	Increased seed growth and germination	[54]
Spirulina platensis	Cyanobacteria	Yellow-lupin	Improved pigment production and photosynthetic capacity	[8]
Palmaria palmata	Rhodophyta (red algae)	Pea	Improved concentrations of NH4+ and NO3− in the soil	[55]
Laminaria digitata	Pheophyceous (brown algae)	Pea	Improved concentrations of NH4+ and NO3− in the soil	[55]
Ascophyllum nodosum	Pheophyceous (brown algae)	Grapevine	Stimulatory effect on fertility, shoot length, shoot diameter, and leaf area	[56]
Rhizoglomus irregulare, Funneliformis mosseae, and F. caledonium	Arbuscular mycorrhizal fungi	American ginseng	Alleviated the negative effects of continuous cultivation; promoted the content of nutrients available in the rhizosphere; and decreased the abundance of pathogenic fungi	[9]
Macrocystis pyrifera with Azospirillum brasilense	Brown macroalga (kelp type) and plant growth-promoting bacteria, respectively	Lettuce	Increased root and plant growth	[57]
Ulva lactuca	Green macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Cystoseira sp.	Brown macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Gelidium crinale	Red macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Bradyrhizobium japonicum	Bacteria	Bambara groundnut	Positively influenced the growth characteristics, biomass yield, and yield traits	[59]
Bacillus amyloliquefaciens	Bacteria	Pakchoi	Reduced ammonia volatilization, increased crop yield and nitrogen recovery, inhibited urease activity, and enhanced the potential of ammonia oxidation	[60]

Table 2. Different organisms used as biofertilizers and their benefits.

Species Used as Biofertilizer	Biological Division	Culture in Which It Was Applied	Benefits	Reference
Chlorella sp.	Chlorophytes (green algae)	Tomato and cucumber	Increased seed growth and germination	[54]
Spirulina platensis	Cyanobacteria	Yellow-lupin	Improved pigment production and photosynthetic capacity	[8]
Palmaria palmata	Rhodophyta (red algae)	Pea	Improved concentrations of NH4+ and NO3− in the soil	[55]
Laminaria digitata	Pheophyceous (brown algae)	Pea	Improved concentrations of NH4+ and NO3− in the soil	[55]
Ascophyllum nodosum	Pheophyceous (brown algae)	Grapevine	Stimulatory effect on fertility, shoot length, shoot diameter, and leaf area	[56]
Rhizoglomus irregulare, Funneliformis mosseae, and F. caledonium	Arbuscular mycorrhizal fungi	American ginseng	Alleviated the negative effects of continuous cultivation; promoted the content of nutrients available in the rhizosphere; and decreased the abundance of pathogenic fungi	[9]
Macrocystis pyrifera with Azospirillum brasilense	Brown macroalga (kelp type) and plant growth-promoting bacteria, respectively	Lettuce	Increased root and plant growth	[57]
Ulva lactuca	Green macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Cystoseira sp.	Brown macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Gelidium crinale	Red macroalgae	Canola	Alleviated the harmful effects of salinity under a salinity stress condition and increased antioxidative compounds	[58]
Bradyrhizobium japonicum	Bacteria	Bambara groundnut	Positively influenced the growth characteristics, biomass yield, and yield traits	[59]
Bacillus amyloliquefaciens	Bacteria	Pakchoi	Reduced ammonia volatilization, increased crop yield and nitrogen recovery, inhibited urease activity, and enhanced the potential of ammonia oxidation	[60]

Types of Inoculant Formulations

When developing inoculants, different aspects must be considered. For example, the manufacturer wants to obtain the highest profit margin without compromising the quality of the product, while farmers seek maximum crop yield [61]. On the other hand, producers expect inoculants to be compatible with routine field activities, both in the planting process and in fertilization and spraying activities. Other desirable characteristics in inoculants are ease of use, long shelf life, compatibility with seeding equipment, and human, animal, and plant safety [61,62].

Microbial inoculants are formulated in sterile or non-sterile vehicles. The former is more advantageous since the microorganisms are supplied in a precise concentration, avoiding antagonistic processes between the autochthonous microorganisms. Thus, there is greater control over the potential of the inoculant; however, sterilization is an operation that increases the production costs of the inoculants. According to Bashan et al. and Rojas-Sánchez et al. [61,63], the formulation of the inoculant is a crucial process that will determine the effectiveness and shelf life of the biological agent.

Inoculants can be found in liquid (suspension), solid (powder, granular), and paste form. Liquid formulations offer marked advantages over solid formulations, such as longer shelf life, which can be retained throughout the cropping period. Furthermore, liquid formulations have higher survival rates and can withstand greater stress conditions due to external effects, such as temperature changes [64].

In the case of liquid inoculants, one of the vehicles generally used is polyvinylpyrrolidone (PVP). According to Chaudhary et al. and Maitra et al. [64,65], the synthetic polymer PVP acts as a preservative of the inoculant, providing a favorable environment for the growth of microorganisms. This polymer is used in concentrations between 1.0 and 1.8% and is sometimes mixed with other compounds to enhance its protective effect, such as sodium alginate, the latter in concentrations of 0.2% [65]. For solid inoculation, the microbial strain is mixed with solid carriers, mainly peat, rock phosphate, charcoal, coconut peat, etc. [61]. In the case of peat, particles with diameters between 0.35 and 1.2 mm are desired. In solid inoculants, the vehicle must be dried to a maximum of 10% moisture, the vehicle material is bagged, and the cell suspension is injected.

Table 3. Different types of inoculants and their properties.

Inoculant Type	Vehicle	Microorganism	Microbial Concentration	Plant Species	Positive Aspects of Inoculant	References
Liquid	Polyvinylpyrrolidone (1.8%) and sodium alginate (0.3%)	Bradyrhizobium japonicum	1.93 × 109 cells mL−1	Soybean	Increase in the number of nodules on plants, number of pods per plant, and number of seeds per pod	[65]
	Polyvinylpyrrolidone, Fe-EDTA	Several rhizobia, Bradyrhizobium japonicum and Bacillus megaterium	106–108 CFU/mL	Soybean	Increase in the number of nodules on plants and seeds produced per plant	[66]
	Arabic gum	Bradyrhizobium sp. J-81 and Bradyrhizobium sp. J-237	1010 CFU/mL	Peanut (Arachis hypogaea)	Increased yield in peanut pods by up to 44%	[67]
	Trehalose (2%), polyvinylpyrrolidone (10 mM), and glycerol (10 mM)	Pseudomonas fluorescens	3 × 1010 CFU/mL	Tomato	Increased yield in tomato	[68]
Solid	Vermiculite—carboxymethylcellulose	Bacillus subtilis	About 108 CFU/mL	Cucumber, lettuce, and American ginseng	Significant increase in weight and roots	[69]
	Alginate and starch	Raoultella terrigena, Azospirillum brasilense	Between 109 and 1011 CFU/mL	n.s.	n.s.	[70]
	Alginate and starch	Raoultella planticola Rs-2	Between 107 and 1013 CFU/mL	n.s.	n.s.	[71]

n.s., not specified; CFU, colony forming unit.

shows some inoculant formulations with their respective vehicles.

3. Beneficial Microbes and Their Multifaceted Agricultural Applications

3.1. Plant Growth Promoters

Rhizobacteria are microorganisms that colonize plant roots and promote growth through various mechanisms, such as nitrogen fixation, nutrient solubilization, and phytohormone production [72]. Species of the genus Bacillus are widely recognized for their growth-promoting and biocontrol properties, due to their ability to enhance nutrient availability in the soil, which is crucial for healthy plant development. For instance, inoculation with Bacillus megaterium, in combination with antioxidants, improved olive performance, fruit weight, and oil content in the pulp, particularly in calcareous soils [73].

Actinobacteria are a group of bacteria notable for their bioactive properties, predominantly found in soil. They are known for their ability to produce a variety of secondary metabolites, including enzymes, hormones, and antibiotics, which are not used during their developmental and reproductive phases. In an agricultural context, actinobacteria play a crucial role in promoting plant growth and improving soil fertility. They colonize the rhizosphere, the zone around the roots, where their high production of antimicrobial compounds provides a competitive advantage, aiding in plant protection against phytopathogens and resisting environmental stress [10].

The genus Rhizobium is widely recognized for its critical role in nitrogen fixation in leguminous plants but is also noted for its importance in the rhizosphere of other crops, such as wheat. Rhizobium bacteria are described as beneficial microorganisms that can promote plant growth through various positive interactions with roots [74].

3.1.1. Mechanisms of Action

Nitrogen-fixing microorganisms, such as Rhizobium species, convert atmospheric nitrogen (N₂) into plant-usable forms, such as ammonia (NH₃) [74]. The key enzyme in this process is nitrogenase, which catalyzes the reduction of N₂ to NH₃. Nitrogenase activity is sensitive to the presence of oxygen; thus, many nitrogen fixers develop mechanisms to protect it, such as forming anaerobic environments.

Nutrient-solubilizing rhizobacteria, such as those from the genera Bacillus and Pseudomonas, produce organic acids that dissolve insoluble phosphates in the soil, making them available to plants. They also secrete enzymes, such as phosphatases, which release phosphorus from organic and inorganic compounds, increasing their availability to plants [74].

Some microorganisms induce systemic resistance in plants through the modulation of phytohormones, such as salicylic acid and jasmonic acid, which are crucial for activating plant defenses against pathogens and biotic stresses. For example, Trichoderma sp. is noted for its ability to alter the phytohormone profile in plants, promoting resistance against nematodes and other pathogens. Additionally, interactions with arbuscular mycorrhizae are mentioned as factors that promote systemic resistance by activating defense-related genes in plant leaves [75].

Actinobacteria play a crucial role in plant protection through various mechanisms of action, including the production of secondary metabolites such as antibiotics and enzymes that inhibit pathogen growth. For instance, compounds such as actinobacillin and streptomycin have shown effectiveness against phytopathogenic fungi and bacteria, including Fusarium and Phytophthora. Furthermore, these microorganisms provide indirect protection by competing with pathogens for nutrients and space in the soil and plant roots, thus limiting the availability of essential resources for pathogens and reducing their ability to cause diseases [76].

3.1.2. Application in Agriculture and Its Benefits

Rhizobacteria improve soil quality through the decomposition of organic matter and the release of essential nutrients. They can solubilize phosphorus and other minerals, making them more available to plants. These bacteria produce phytohormones like auxin, cytokinins, and gibberellins, and they help solubilize essential nutrients such as phosphorus and potassium. This stimulates root and shoot growth, leading to healthier plants that absorb water and nutrients more effectively. Moreover, rhizobacteria can help plants tolerate biotic stresses (such as pathogens and pests) and abiotic stresses (such as drought and salinity). They can induce defense mechanisms in plants, increasing their resistance to diseases and adverse conditions [76].

Reducing the use of chemical fertilizers in agriculture is increasingly valued, particularly in the context of environmental and sustainability concerns. Rhizobacteria, like potassium-solubilizing bacteria (KSB), are important for nutrient solubilization and biofilm formation. They enhance soil structure and microbial health, contributing to a more balanced and sustainable environment [77].

Rhizobacteria can stimulate plant growth through the production of growth hormones, such as auxins, and by solubilizing essential nutrients like phosphorus and potassium. Healthy plants are more effective in absorbing soil contaminants and stabilizing degraded areas [74].

Rhizobacteria can be used as biopesticides through mechanisms that control plant pests and pathogens. They produce secondary metabolites, such as antibiotics, that inhibit the growth of harmful organisms and induce systemic resistance in plants, activating their natural defenses. For instance, species of Bacillus and Streptomyces are known to produce antibiotics that can suppress pathogenic fungi and bacteria in the soil and on plants [74].

3.2. Nitrogen Fixers

The nitrogen cycle involves a series of steps in which many biotic and abiotic factors are involved [78]. A key part of the nitrogen cycle is nitrogen fixation, where atmospheric nitrogen (N₂) is transformed into ammonia (NH₃), a form that plants and other organisms can use [79]. This process is mainly carried out by prokaryotic organisms such as symbiotic bacteria belonging to the genus Rhizobium, which form an association with plants and are found in root nodules of legumes [80]. The non-symbiotic bacteria such as those belonging to the genus Azotobacter can also be found in the same environment together with plants, but do not necessarily form a symbiotic relationship [4].

In the process of biological nitrogen fixation, the enzyme nitrogenase plays an important role since it is responsible for catabolizing the reduction reaction of N₂ to NH₃ (later to NH₄ due to protonation in the aqueous medium of the cell cytoplasm), which occurs in the absence of oxygen due to the high denaturation sensitivity of the enzyme [81]. The general reaction occurs using protons and electrons in the presence of ATP, which is broken down into ADP and inorganic phosphate:

N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16P_i

Protonation of ammonia to ammonium ion:

NH₃ + H⁺ → NH⁺₄

Many nitrogen-fixing microorganisms have been identified, particularly those that form direct symbiosis with plants by creating root nodules. This involves a complex interaction between the plants and the microorganisms [82]. These nodules, formed mainly in plants belonging to the orders Fabales, Cucurbitales, and Rosales, represent a survival strategy that is beneficial for both plants and symbiont microorganisms [83]. The formation of nodules creates a hypoxic environment, with a dissolved oxygen concentration between 10 and 30 mmol/L, which generates optimal conditions for the stability of the nitrogenase enzyme, expressed by a group of genes known as niF and fix [81,84]. This environment prevents nitrogenase degradation and facilitates nitrogen fixation.

Among the microbial groups capable of forming endosymbiosis with plants, creating root nodules to fix atmospheric nitrogen, we find mainly the Rhizobia group, among which the Rhizobium genus stands out as a symbiont of leguminous plants such as alfalfa (Medicago sativa), clover (Trifolium), and beans (Phaseolus vulgaris) [85,86,87]; Bradyrhizobium and Mesorhizobium associated with plants such as soybean (Glycine max) and peanut (Arachis hypogaea) [88]; Sinorhizobium that is commonly associated with alfalfa plants (Medicago sativa); and Azorhizobium that can form nodules at the stem and root level in aquatic legumes such as Sesbania virgata [89,90]. Likewise, Frankia is a genus of actinobacteria known to form a symbiotic association, creating nodules in non-leguminous plants, especially in plants of the orders Fagales, Rosales, and Cucurbitales, such as alder (Alnus glutinosa) and various species of casuarinas (Casuarina sp.) [91,92].

On the other hand, nitrogen-fixing microorganisms that do not generate a strict association with plants usually live freely in the soil at the rhizosphere level. Unlike the fixing microorganisms capable of forming root nodules on plants, free-living ones present other strategies to avoid degradation of the nitrogenase enzyme. Species of the genus Azotobacter, for example, can produce a mucilage layer around bacterial cells that reduces the diffusion of oxygen through the cell, and it has been found that they can accelerate their rate of cellular respiration to uptake oxygen more rapidly and allow a microaerophilic condition around the nitrogenase [93]. There are also other important free-living nitrogen-fixing microbial genera such as Klebsiella, Clostridium, and Azospirillum [94,95,96].

Recent studies have taken advantage of this property of nitrogen-fixing microorganisms to improve the growth-promoting efficiency of plants and consequently increase the production of crops of interest when used as microbial inoculants. Rhizobium mayense is a strain that was isolated from the rhizosphere soil of tea and peanut plantations, which produced up to 80.49 g/L in a nitrogen source-free medium in vitro. In addition, the influence of bacterial inoculum in finger millet (Eleusine coracana) and green gram (Vigna radiata) on total growth and dry weight was demonstrated in comparison to controls [97].

In another work by Ke et al. [98], it was found that a new strain identified as Pseudomonas stutzeri A1501 contributed between 0.30 and 0.82 g nitrogen/plant. It significantly altered the composition of the diazotrophic community at the rhizosphere level, indicating a great survival and stability mechanism that could allow its scaling up when used as a microbial inoculant. Finally, Azotobacter in consortium with other microorganisms significantly increased different growth attributes in wheat plantations such as total number of tillers m⁻², spike length, number of grain spikes⁻¹, and 1000-grain weight compared to the control [99], demonstrating that a microbial inoculum could increase its efficiency when composed of a varied consortium with particular properties and symbiotic with each other.

3.3. Phosphorus Solubilizers

Phosphorus is essential for crop growth, but not all forms are absorbable. Much of it exists as insoluble phosphates or complex organic compounds [100]. In view of this, phosphate-solubilizing microorganisms (PSMs) are a group of diverse species of bacteria, fungi, and actinomycetes, which have the capacity to convert insoluble forms of phosphorus into soluble forms that are then available to plants and can be absorbed more easily [101].

The mechanism of phosphorus solubilization by microorganisms involves the release of extracellular enzymes, the liberation of phosphorus during substrate degradation, and the secretion of compounds such as siderophores, protons, hydroxyl ions, and organic acids [102,103].

During the process of mineralization of organic phosphates, microorganisms produce enzymes such as phosphatases, phytases, phosphonatases, and lyases, which act on diverse organophosphorus substrates such as phytic acid, phosphonoacetaldehyde, phosphonoacetate, and other more complex ones such as phosphonates, whose decomposition reaction produces hydrocarbons and inorganic phosphates [104,105,106]. These last mentioned, as well as others in their free form, can still be insoluble for many organisms such as plants, and are also the most abundant in the soil, representing 70–80% of the total phosphorus compounds [107]. This is why another group of microorganisms have the capacity to produce compounds such as organic acids capable of lowering soil pH and producing the chelation of phosphorus-associated cations, finally releasing phosphorus in the form of soluble phosphate [108,109]. It has also been found that this process can be produced by enzymolysis, production of siderophores (which function as high-affinity iron chelators), high molecular weight exopolysaccharides (which have a high affinity for binding to metal ions), and production of hydrogen sulfide (H₂S), among others [110,111,112,113].

There is a great variety of microorganisms described in the literature that present this capacity, which includes bacterial genera such as Pseudomonas, Bacillus, Rhizobium, and Enterobacter; fungi genera Aspergillus, Penicillium, and Trichoderma; and some Actinomycetes, mainly of the genus Streptomyces [103]. Jastrzębska, Kostrzewska, and Saeid [114] used a biofertilizer made from renewable raw materials and activated by microbial fermentation with phosphorus solubilizing microorganisms (Bacillus megaterium and Acidithiobacillus ferrooxidans) to evaluate its effects on quality parameters of wheat grain, which were not affected when concentrations of up to 35.2 kg ha⁻¹ were used, indicating that it can be a viable alternative as a biofertilizer since it complies with environmental and consumer safety standards.

On the other hand, Bacillus subtilis and Bacillus pumilus have been used in field trials for their evaluation as microbial inoculants and their influence on productivity in corn plantations, obtaining an improvement in yield (6532 kg ha⁻¹) and in the accumulation of phosphorus in grains (15.95 kg ha⁻¹) [115]. Regarding fungi, their great efficiency for phosphorus solubilization has also been demonstrated, for example, Trichoderma harzianum presents different mechanisms for solubilizing in vitro soluble or poorly soluble minerals such as calcium phosphate, through acidification, production of chelating metabolites, and redox activity [116]. Finally, actinomycetes also play a relevant role in the solubilization of phosphorus, especially iron-bound phosphates. Cui et al. [117] evaluated the ability of Streptomyces sp. endophytic activity on Camellia oleifera to mobilize phosphorus in acidic and deficient soils, finding a release of 72.49 mg L⁻¹ for FePO₄, which was mediated by the production of siderophores.

3.4. Potassium and Zinc Solubilizers

Potassium and zinc are essential nutrients for plants, typically found in insoluble forms in the soil. Like phosphorus, the mechanisms for their solubilization and subsequent transport include the release of organic acids and the production of specific enzymes that help decompose the minerals containing potassium and zinc [118]. Potassium, a macronutrient crucial for plant growth and development, has 98% of its soil reserves in non-available forms [119].

Potassium-solubilizing microorganisms (KSMs) in the rhizosphere can solubilize insoluble potassium. This process has been documented in various bacteria, such as Enterobacter hormaechei, which can solubilize up to 82.012 μg mL⁻¹ of potassium in an aqueous medium in vitro [120], and Bacillus mojavensis is useful in saline agricultural soils, such as maize and rice crops [121]. Furthermore, plant growth can be enhanced not only by a single microorganism but also by incorporating a previously selected consortium, thus simulating the natural microsystem. Kour et al. [122] tested a consortium composed of a nitrogen-fixing bacterium, Acinetobacter guillouiae, and a potassium-solubilizing bacterium, Acinetobacter calcoaceticus. The combination of these strains significantly impacted shoot length, root length, and plant biomass, among other components of onion (Allium cepa).

In the case of zinc, it is a micronutrient whose bioavailability in the soil is limited due to its insoluble form, causing biochemical and physiological consequences for plants as it serves as a cofactor for various chemical reactions [123]. Zinc-solubilizing microorganisms (ZSMs) convert insoluble zinc into accessible forms, increasing its bioavailability in the soil and helping mitigate zinc deficiency in crops. The main microbial genera with this capability include Pseudomonas, Burkholderia, Gluconacetobacter, Thiobacillus, Acinetobacter, and Bacillus [124]. The solubilization mechanisms, similar to those of KSMs and PSMs, include the solubilization of metallic forms through proton extrusion, and the production of chelated ligands, phytohormones, vitamins, and redox systems at the membrane and cell surface level [125]. A study by Rahman et al. [11] demonstrated that the strain Acinetobacter pittii OR335753, used as an inoculant in tomato plants, promoted growth and yield, showing an increase in plant height, leaf number, and leaf area, as well as higher zinc accumulation (0.75 ppm) compared to controls (0.48 ppm).

4. Advances in Inoculants for Agricultural Production

The use of microbial inoculants in agriculture is one of the main strategies adopted to improve crop productivity and make agricultural practices more sustainable. According to O’Callaghan, Ballard, and Wright [62], agricultural inoculates are mainly made from bacteria and fungi, produced in bioreactors that create optimal conditions for microorganism growth. Therefore, variables such as pH, dissolved oxygen, temperature, type of culture, and amounts of inoculum are factors that must be evaluated for each type of microorganism.

The primary bioinoculants produced industrially are nitrogen fixers, then phosphorus solubilizers, and fewer mycorrhizal fungi. Most of the bioinoculants used in agriculture are Rhizobia (nitrogen-fixing microorganisms), accounting for approximately 79% of global production, followed by phosphorus or phosphate solubilizers and mycorrhizal, with a share of 15% and 7%, respectively [126].

Nitrogen deficiency in crops significantly limits their productivity, so the inoculant market continues to be dominated by nitrogen-fixing microbial species [62]. Among the nitrogen-fixing bacteria, the genera Azotobacter, Azospirillum, Acetobacter, Pseudomonas, and Rhizobia stand out [126]. These are characterized by being bacteria that grow under aerobic conditions, some are microaerobic or facultative. According to Prando et al. [127], inoculation of soybean plantations with Bradyrhizobium has the potential to fulfill the plant’s nitrogen requirements by eliminating fertilization processes with nitrogen sources, which represents an annual cost savings of approximately USD 13 billion [127].

Another group of microorganisms that are highly important in agriculture are phosphate solubilizers since phosphorus is a vital macronutrient for plant metabolism, which participates in key physiological processes, such as energy production, growth, enhancing plant resistance against diseases, and enzyme catalysts, among others. Currently, different bacterial and fungal species are known to be capable of solubilizing phosphate [128,129]. According to Suyal et al. [126], bacteria are more efficient since they can solubilize up to 50%, while fungi can only solubilize up to 0.5%. Phosphate-solubilizing species include bacterial genera such as Pseudomonas, Bacillus, Arthrobacter, Enterobacter, Klebsiella, Xanthomonas, Chryseobacterium, Azotobacter, Rhodococcus, Serratia, Vibrio, and fungal genera such as Aspergillus and Trichoderma [12,126].

Other organisms produced on an industrial scale as inoculants are arbuscular mycorrhizal fungi (phylum Glomeromycota), which can establish mutualistic symbiosis with different plants. Of the seven known groups of mycorrhizae, the most important, widespread, ecologically important, and commercially exploited are arbuscular and ectomycorrhizae [126].

For the manufacture of bioinoculants, microbial biomass must be produced in high concentrations under aseptic conditions, which must reach minimum concentrations of 1 × 10⁸ CFU/mL and no contaminants at a dilution of 10⁻⁵ [130]. For biomass production, stirred tank bioreactors designed with aeration systems and pH and temperature controls are mainly used. The operating modes used in these systems are generally simple batches due to their ease. Recent findings show that fed-batch processes can achieve higher cell concentrations because cells are not stressed or inhibited by high substrate levels early in the process. This allows for a better understanding of substrate consumption and product formation kinetics [131,132].

In the fed batch, the substrate is gradually supplied to the system as required. This extends the exponential phase and increases the efficiency of substrate conversion into biomass [133], reducing sporulation time, since this process occurs after the depletion of the main carbon source [134]. Furthermore, fed systems also prevent the formation of inhibitory metabolites that are toxic to the growth of microbial biomass. For example, batch cultivation of Bacillus subtilis generally produces acetate, which affects the growth of microbial biomass [132].

Since the deficiency or excess of oxygen can affect the growth of the microorganisms, one variable to consider in the scaling of these processes is the Volumetric Mass Transfer Coefficient (kLa), a parameter that can define the limits of the gas transference into and out of a liquid. Trujillo-Roldán et al. [135] found, in the scaling process of biomass production of Azospirillum brasiliense, that kLa can be used as the main scaling parameter of these processes. Thus, with a kLa of 31 h⁻¹ in the prototype and the pilot scale (1000 L), it is possible to achieve a biomass concentration between 5 and 8 × 10⁸ cells mL⁻¹.

Different carbon sources are used to produce microbial biomass for bioinoculants, but the most used are glucose and sucrose. Some nitrogen sources include yeast extract and micronutrients such as K₂HPO₄, MgSO₄.7H₂O, NaCl, CaCl₂, FeSO₄, Na₂MoO₄, MnSO₄, KOH, NH₄Cl, and H₃BO₄. The temperature conditions are similar for both nitrogen-fixing organisms and phosphorus or phosphate solubilizers (28–30 °C) [132]. A notable difference is that phosphate-solubilizing microorganisms such as Bacillus species generally require a greater amount of aeration, between 1.0 and 2.5 vvm. While nitrogen-fixing microorganisms such as Azospirillum species usually require maximum air flows of 0.5 vvm [135].

After biomass production, the inoculant is formulated (downstream). This can be liquid or solid, depending on the biological aspect, whether microbial cells or spores. Microorganisms can show marked efficiency under laboratory conditions. However, to formulate microorganisms/spores and apply them in the field, the appropriate carrier must be selected.

The products can be liquid, such as emulsions, oil, and even water, solid in the form of granules, powders, and wettable powders, and immobilized, such as encapsulation and microencapsulation. Solid formulations are the best options for organisms that can produce spores and are generally applied to seed furrows and even in the soil itself. While liquid formulations contain active microorganisms and can be applied directly to the seeds or through a fertigation process [61,136], immobilized products can use cells and the same spores and can be applied to the soil and the seeds themselves [132].

Liquid formulations generally contain fermentation broth because these are sources of nutrients and minerals that contain bioactive compounds useful for plants [17,18]. In addition, other nutrients and additives such as sorbitol, glycerol, and sugars such as glucose, sucrose, and trehalose, and some organic oils, minerals, or a mixture of these are added. Additives are used to improve stability, increase adhesion, and sometimes for surfactant capacity. To apply the bioinoculant, the seeds are dipped in the liquid inoculant before sowing, sprayed, or applied at sowing [17,18].

On the other hand, for solid formulations, the biological agent is mixed with solid vehicles such as vermiculite, talc, polysaccharides, clays, and agro-industrial waste, among others [17,137], in addition to adding binding, dispersing, and wetting agents. Solid formulations are applied directly to the soil, and compared to liquid products, solid formulations have a longer shelf life, in addition to being easier to handle, store, and transport [17,132].

Finally, there are the formulations obtained by encapsulation processes, which have marked advantages over solid and liquid formulations obtained by conventional processes, since conventional formulas can have their efficiency compromised by large losses during application, in addition to being more susceptible to deterioration by environmental conditions [61,138]. According to Florencio et al. [132], encapsulation can be used both to protect microorganisms and to facilitate application processes with gradual release. For the encapsulation of microorganisms or spores, polymeric materials such as gums (xanthan, gellan, and guar), starch, milk, and whey proteins are used; however, the most used are carrageenan and alginate. During encapsulation, large quantities of cells are affected by the high temperatures used in drying, in addition to being more costly processes [138]. The encapsulation process can be carried out using different techniques, among which the most common are extrusion, spray drying, fluidized bed, and molecular inclusion.

5. Patents: Innovations and Technological Perspectives

The market for inoculants in agricultural production has consistently been a hub of innovation, driven by the challenges of population growth and the pressing need to enhance global agricultural output. According to the Derwent Innovations Index search platform, in the last five years, 822 patents have been filed, 114 of which are related to inoculant microorganisms.

The patents aim to enhance plant growth, boost resistance to stress and pests, improve root nodule formation, enhance soil quality, and promote decontamination and use of environmental effluents. In most of the documents, the most common strains were from the genera Rhizobium sp., Azotobacter sp., Pseudomonas sp., Bacillus sp., and Lactobacillus sp.

Most of the results deposited were related to applications in agriculture, and biotechnology applied to microbiology, chemistry, and polymer science. This has generated products for the market such as bioinoculants, biofertilizers, biopesticides, extracts, and liquid compositions. However, as this article is about the agriculture field, it was necessary to refine the search algorithm.

Creating a search algorithm in the patent database was necessary to obtain better results. To help with the search, an International Patent Classification (IPC) code group A01N-063/00 was set up, where class A01 (agriculture, forestry, animal husbandry, hunting, trapping, and fishing), subclass A01N (preservation of bodies of humans or animals or plants or parts thereof), and group A01N-063/00 (biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals, or substances produced by, or obtained from, microorganisms, viruses, microbial fungi, or animals, e.g., enzymes or fermentates) were chosen. Along with this IPC, the names “microorganism*”, “microbe*”, “bacter*”, “fung*”, “yeast”, “inoculant*”, “inoculum*”, and “bioinoculant*” were added. This resulted in the final algorithm: (microorganism*)) OR TS = (microbe*)) OR TS = (bacter*)) OR TS = (fung*)) OR TS = (yeast)) AND TS = (inoculant*)) OR TS = (inoculum*)) OR TS = (bioinoculant*))) AND IP = (A01N-063/00)) OR IP = (A01N-063*).

With this algorithm, 16,180 patents were filed. Refining the search to include only patents in “Agriculture” and “Microbiology Applied to Biotechnology” with the IPC A01N-063/00, and excluding those filed in the last five years, resulted in 822 patents. After reading and analyzing the search results, patents referring to bioinoculants or biofertilizers that promote better plant growth and yield were selected. More specifically, the scope focused on patents containing nitrogen-fixing microorganisms, phosphorus, phosphate and zinc solubilizers, and potassium mobilizers. As a result, 114 patents were selected.

5.1. Trends in Patents Applied over Time

The first known patent was filed in the Netherlands in 1974 (NL7315471-A). The patent was assigned by Hershey Foods Company, an American company. The patent involves using Bacillus uniflagellatus spores on seeds or root systems of plants before cultivation, applicable to plants with thin, widespread roots like cereals, vegetables, and citrus fruits. This bioinoculant helps plants fix nitrogen better, leading to improved growth under stress and increased resistance to pests like weeds and insects [139].

The patents in this market field started to become more significant in the 1980s. Before the start of the 21st century, the origins of patents can be traced back to European countries, the United States, Japan, Australia, and New Zealand. It should be noted that these patents were largely developed in countries with limited territory for large-scale agricultural production.

In 1984, patent AU8317489-A, filed by the company Microlife Genetics Inc., developed the application of a siderophore microorganism. The active ingredient of interest is the ability of the bacterium Pseudomonas putida to inhibit the growth of other phytopathogenic microorganisms, and therefore prevent the inhibition of plant growth. According to the patent, the microorganism is cultivated on plates with ethylenediaminodi-(o-hydroxyphenylacetic) acid. As P. putida can solubilize and sequester ferric ions from the soil, this allows the prevention of the growth of other microorganisms that are phytotoxic to the plant. The treatment can be carried out using 10,000 cells/g of soil [140].

In the same decade, a year later, in 1985, another patent (US4551164-A) was filed by Bio Organics Inc., where a microbial consortium composed of bacteria and algae is applied to promote better growth of fruit plants, and their fruits reach commercial size more quickly. The consortium is a mixture of bacteria of the Bacillus subtilis species and algae of the Chlorella sacchorophila species. The cultures were prepared to have a concentration of 4 × 10⁶ to 6 × 10⁶ bacterial cells mL⁻¹ and 14 × 10⁶ to 16 × 10⁶ algae cells mL⁻¹. With these two cultures prepared, they are mixed and incubated to obtain a third nutrient medium. In the end, a liquid medium is obtained consisting of cells from the microbial consortium together with 0.5–2% vv⁻¹ of sorosepin, 0.5–2% vv⁻¹ of lipase, and 1.5–5 g gal⁻¹ of vitamin B-12. Improved plant growth is stimulated through the ability of microorganisms to fix nitrogen from the soil and atmosphere [141].

As the decade drew to a close in 1987, patent WO1987004182-A emerged, leading to the development of a groundbreaking bioinoculum. This innovative solution proved capable of infecting and modulating the roots of non-leguminous plants, such as wheat, barley, rice, and sorghum, while also facilitating nitrogen fixation. This is all possible through the production of transconjugated Rhizobia strains by means of techniques such as recombinant DNA, using phages and other vectors to infect Rhizobia with the appropriate sequences. As a result, this has generated plants with a higher protein, dry matter, and nitrogen content compared to uninfected and nodulated plants [142].

In the 1990s, the focus was on producing mutant or recombinant bacteria. Patent WO9015138-A, filed by the Wisconsin Alumni Research Foundation in 1990, features recombinant Rhizobium bacteria. This microorganism received a gene capable of producing trifolitoxin, an antibiotic that gives it resistance, as well as preventing root nodulation by bacterial strains sensitive to trifolitoxin. The patent does not specify to which plant seeds the bioinoculant can be applied, but it does accurately state the ability of this strain to generate root nodules capable of improving nitrogen fixation for “some plants”. As a result, this could improve plant growth and reduce the need to use fertilizers. The patent does not show a comparison of these improvements [143].

In 1993, an individual filed a patent for a new recombinant bacterium capable of improving nitrogen fixation in plants. This patent is considered applicable to any species of bacterium capable of fixing nitrogen by means of the nifL or nifL-like genes as part of its nitrogenase enzyme system, which exclusively control the transcription of nifA or nifA-like genes that regulate the transcription of other nif genes necessary for the structure and activity of nitrogenase itself. According to the patent, a preferable species of bacterium for this application would be Azotobacter vinelandii. The improved nifL gene is isolated by heterologous hybridization techniques and then reintroduced into a new strain of bacteria that will have its wild nifL gene replaced. The patent does not indicate specific plants for the transformed strain to be applied but recommends that it should ideally be used for plants that have bacteria naturally associated with their roots or stems by means of glycoproteins [144].

Moving towards the middle of the decade, in 1995, patent EP0634893-B1, filed by the Hungarian company Piacfejlesztesi Alapitvany, developed a method of fixing nitrogen in a more efficient way for plants, through the inoculation of plant protoplasts, cells, tissues, embryos, or plant organs grown in vitro by bacterial cells of the genera Azotobacter, Azomonas, Beijerinckia, or Derxia. This has led to plants with faster and more resistant growth. Another factor worth mentioning is that these genera of bacteria are not capable of colonizing and entering symbiosis with plants under in vivo conditions, according to the patent. Therefore, a way was created to establish a symbiosis between microorganisms and plants that would not occur naturally. According to the patent, this technique can be applied to any species of plant, as well as any part of the plant [145].

5.2. Current Prospects for Innovation

As we look to the 2000s, particularly the patents submitted in the past five years (2020–2024), it is evident that the trends and innovations in the development of bioinoculants, biofertilizers, and inoculates have shown remarkable consistency. However, a new trend has emerged, especially regarding the use of microorganisms applied as bioinsecticides, bioherbicides, antifungal, and antibacterial agents. This trend can be primarily attributed to the ongoing climate changes occurring globally, coupled with the rise in pest populations that have significantly harmed agricultural production on a worldwide scale. The goal is to develop biological pest control products to protect crops and ensure food security for people now and in the future [146].

Of the 822 patents that were found in the Derwent Innovations Index, 220 have the sole scope of acting as a bioinsecticide against bacteria, fungi, nematodes, insects, and weeds that can hinder the growth and yield of agricultural plants. An analysis of these patents shows that the main genera of microorganisms used for this protection function are Bacillus, Pseudomonas, Paecilomyces, and Pasteuria. The main goal of the inventions is to develop a natural, effective, and eco-friendly pest control method by using competition between microorganisms and plant pathogens.

With advances in research and technology, inventions now go beyond using microorganisms as bioinsecticides; they also utilize their biochemical products and genetic engineering [147]. These trends will become clearer in the examples cited below.

Patent EP4335866A1, filed by the Max Planck Gesellschaft zur Foerderung der Wissenschaften eV, in 2024, relates to an invention of a genetically modified microorganism. The bacterial species Yersinia entomophaga has been genetically modified to produce, in large quantities, a toxin capable of acting as an insecticide against various insect pests. The genetic modification of the bacterium takes place through a plasmid with the modified gene of interest inserted. In this way, plants with these bacteria present produce a toxin capable of protecting them against various (unspecified) insects [148].

Another example is patent WO2020142366-A1, filed by Locus Agriculture Ip Company LLC, in 2020, which deals with the use of a hydrolysate of one or more biochemicals produced by a microorganism. The composition is made from biosurfactants (surfactin, iturin, phenicin, artrofactin, and lichenisin) and enzymes (chitinase or exo-b-1, 3-glucanase). The invention uses yeasts and bacteria to produce these biocompounds, such as Bacillus subtilis and Pseudozyma aphidis. The patent recommends that the biocompost, in the form of powder or granules, be applied to the roots of a plant. This approach safeguards the plant from a wide range of threats, including nematodes, arthropods, bacteria, viruses, fungi, protozoa, and parasites, particularly targeting the diseases known as greening and canker. The active principle of biochemicals is to cause osmotic shock or thermal inactivation in invading microorganisms [149].

Moving on to biofertilizers, of the 114 patents mentioned above, we can start with the patent registered in 2020, where the company Pivot Bio Inc. filed patent WO2020014498-A1. The invention consists of producing a consortium of remodeled, non-intergeneric microorganisms. These bacteria can colonize the root surface of plants and provide atmospheric nitrogen in an optimal amount, dispensing with the use of exogenous microorganisms or fertilizers. The aim of the invention is to help plants such as corn, rice, wheat, barley, sorghum, millet, oats, or rye to produce better. The bacteria selected need to be epiphytic or rhizospheric, for example, Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, A. marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, K. sacchari, and combinations thereof. The non-intergeneric remodeled bacterium comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, through recombinant DNA technology. With this bioinoculant, it is hoped to achieve faster and more efficient cereal production [150].

Another patent, WO2020219932-A1, also filed by Pivot Bio Inc. in 2020, also used genetic engineering to generate microorganisms capable of helping agricultural production of cereals such as corn, soybeans, wheat, and rice. In this case, the species of bacteria that would have its gene modified was not specified, but the various genes that were modified using CRISPR-Cas technology were mentioned. The plant treatment method involves applying a liquid formulation containing the genetically modified bacteria to a plant seed. The application is made after planting and before the plant is harvested, preferably between two months and eight months after germination [151].

As time progressed, in 2021, patent WO2021086695-A1, filed by Novozymes Bioag A/S., showed a bioinoculant obtained in a simpler way. A pure culture of Microbacterium trichothecenolyticum was isolated, which was combined with another strain, Azospirillum brasilense, whose objective is to improve the growth and yield of unspecified plants. According to the document, the bacteria are capable of increasing the absorption and/or accumulation of nutrients, such as calcium, copper, iron, manganese, magnesium, nitrogen, potassium, and zinc in a plant or part of the plant, as well as increasing the solubilization of phosphate, revealing a bioinoculant with expanded activities. The application method is summarized as infecting the seed of the desired plant with an ideal amount of the microbial consortium before germinating it [152].

In addition to nitrogen fixers, there are also many patents relating to phosphate or phosphorus solubilizers. Patent IN202041019739-A, filed by individual inventors in 2021, refers to phosphorus and phosphate biofertilizer, which aims to increase phosphate solubilization and phosphorus bioavailability for the plants that are treated. The product is applied in the form of granules, powder, or liquid. The bioinoculant is composed of a consortium of bacteria (Bacillus spp. and Pseudomonas spp.) and fungi (Aspergillus spp. and Trichoderma spp.). The biofertilizer is said to be applicable to plant seeds in general [13].

In 2022, the Indian Motherhood University filed patent IN202211019835-A, which presents another major trend in the bioinoculant product sector. This patent has both the function of being a biofertilizer and a bioinsecticide, more specifically a biofungicide. The bioproduct consists of five strains of the bacterium Pseudomonas sp., which can benefit plant growth and protect against fungal pathogens present in the soil. The formulation can help control the fungal pathogen Fusarium sp., which causes root diseases in cultivated plants, through the production of peptides and metabolites that antagonize fungal growth, as well as stimulating plant growth through improved nitrogen fixation, phosphate solubilization activity, and zinc solubilization activity [153].

Another example of a patent, IN202241027681-A, also filed in 2022, by the company FIB-SOL Life Technologies Pvt. Ltd., is a biogel with biofertilizing properties. It comprises inorganic ions, salts and/or proteins, a water-soluble and/or biodegradable polymer such as polyvinyl alcohol (PVA), xanthan gum and/or starch, and at least one bacterial cell (Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azotobacter vinelandii, and Rhizobium leguminosarum) or fungal cell (Aspergillus, Fusarium, Penicillium, Piriformospora, and Phoma). Combined microorganisms can provide means of improving nitrogen fixation, phosphorus, iron, zinc, and sulfur solubilization, and potassium mobilization. The gel can coat seeds, be applied to soil, used hydroponically, or for foliar spraying, either individually or in combination [154].

In the second year after the pandemic, in 2023, the Basf Company filed patent WO2023088791-A1, which continued the trend of combining bioinsecticides with fertilizers in a single product. The patent concerns the use of strains of the bacterial genus Paenibacillus for use in production agriculture. Applications include using it as a biological pesticide to protect plants from fungi and harmful bacteria, and to enhance plant growth by producing phytohormones and mobilizing nutrients, including biological nitrogen fixation and inducing systemic resistance. Among the substances with a biopesticidal effect against plant growth pathogens, the patent mentions polymyxins, tridecaptins, polypeptins, paenylipoheptins, octapeptins, olipeptin, gavaserin, and saltavalin [14].

That same year, in 2023, patent WO20232462-A1, filed by the company CO₂ Revolution, once again applied the trend of uniting a consortium of microorganisms to improve agricultural crops. The invention is a composition for coating seeds, in which there will be a mycorrhizal fungus, a bacterium of the genus Pseudomonas, and a fungus of the genus Trichoderma. The application improves seed germination and survival, leading to enhanced plant growth, productivity, and resilience against environmental stresses. The invention asserts that these impressive results stem from the microbial consortium’s ability to enhance the plant’s capacity to assimilate essential nutrients, including nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, zinc, copper, manganese, molybdenum, boron, and chlorine. Furthermore, it promotes more efficient utilization of phytohormones, such as auxin, gibberellins, and cytokinins [15].

In 2024, the search for efficient bioinoculants continues, but with a strong tendency to help mitigate environmental problems. Newleaf Symbiotics Inc. has filed patent WO2024015850-A2 to launch a bioinoculant that improves agricultural production and captures methane gas from the environment. The invention uses a strain of methanotrophic bacteria, of the Methylocystis hirsuta species, which is applied to a plant, part of a plant, or seed. The document mentions that it can be applied to rice, wheat, corn, soybeans, peanuts, barley, alfalfa, millet, sorghum, oats, and rye plants. In this way, the bioinoculant allows plants to use the methane present in the soil, atmosphere, or groundwater as a source of carbon, oxidizing it and allowing for better efficiency in the use of nitrogen. This is due to the presence and expression of central niF genes in the genome of methanotrophic bacteria [155].

Patent IN202321064354-A, filed by Saurashtra University in 2024, describes a liquid solution that produces indole-acetic acid (IAA) using a plant growth-promoting rhizobacterial strain. The bacterium in the invention is of the Priestia filamentosa species, which helps with nitrogen fixation, phosphorus solubilization, and the synthesis of phytohormones. IAA is a phytohormone that acts on cell growth, division, differentiation, and genetic regulation. Bacteria that produce IAA can help the host plant absorb nutrients to the maximum. The document mentions that a good plant for such an application would be wheat. In this way, agricultural crops can be created in places with abiotic stresses or infertile soils [156].

5.3. Current and Future Market Possibilities

Market reports from 2022 have indicated that the size of the global biofertilizer market is valued at around USD 1.5–2.8 billion and that its compound annual growth rate (CAGR) will be 10.9–12.8% between 2023 and 2032. In contrast, the synthetic fertilizer market had a higher global market value, reaching USD 202 billion, but with a lower CAGR of 2.7–3.3% for the next decade [43].

This economic outlook suggests that demand for organic farming inputs, like biofertilizers, will grow each year as consumers become more environmentally conscious and as environmental issues, such as groundwater contamination and soil fertility loss, increase. An important factor to note is the rising prices of food produced with synthetic fertilizers, which is driving increased consumption of biofertilizers in the coming years [157].

It is important to highlight that, beyond promoting sustainable production, biofertilizers significantly lower agricultural input costs through enhanced production efficiency. Microorganisms offer the same benefits as synthetic fertilizers while requiring fewer substrates, which are often readily available in the crop’s environment. This not only boosts productivity but also fosters a more sustainable approach to farming. This application is carried out using precision agriculture technologies, where geographic information system software coupled with GPS-guided equipment and sensors optimizes the efficiency of the amount of biofertilizer to be used, avoiding waste and inappropriate use [5].

All of this results in more efficient production, lowering production costs and avoiding environmental contamination. Biofertilizers can also function as bioinsecticides, helping crops resist pathogens and cope with dry weather, poor soil, and water shortages without needing additional products [158].

This can be seen from the patents mentioned above. Many inventions, in addition to acting as biofertilizers, also have the active ingredient of being biopesticides, protecting the plant against phytopathogens or weeds. Of the 114 patents registered from 2020 to 2024, 94 have nitrogen-fixing microorganisms as their property, 53 phosphate solubilizers, 37 phosphorus solubilizers, 29 potassium mobilizers, and 14 zinc solubilizers. It is also worth noting that 54 patents featured both the application of biofertilizer and bioinsecticide, mainly regarding the control of bacterial or fungal phytopathogens. These research results can be seen more clearly in Figure 2.

6. Regulation and Quality Control of Inoculants in the World

The growing population’s demand for food and the need for sustainable production have heightened the agro-industrial sector’s interest in producing inoculating microorganisms, such as biofertilizers. Several other factors have also contributed to the increased demand for biofertilizers in the market. The COVID-19 health crisis exacerbated the supply issues of agricultural inputs, and territorial conflicts between Ukraine and Russia have disrupted the global supply of fertilizers for agriculture [160].

Therefore, national regulations play a crucial role in controlling the quality of these products. However, existing legislation still has gaps, and there is a need for international agreements, particularly regarding the standardization of product labeling [161]. Some countries, such as India, have strict and comprehensive quality criteria, including the concentration and identification of microorganisms, purity, and effective application conditions. These conditions consider the type of carrier material, pH, humidity, and particle size. Additionally, India includes quality requirements for the use of microbial consortia in its list of biofertilizers, which is not common in the regulations of many countries that focus on using only one type of microorganism. Canada’s standards incorporate detailed criteria regarding the type of microorganisms, their concentration, and specific tests that statistically demonstrate their effectiveness over a period that accounts for seasonal and climatic variations. Uruguay’s regulations include unique temperature criteria for storing the product, distinguishing them from other countries (

Table 4. Quality requirements established by regulatory standards in different countries.

Country	Biofertilizer	Carrier Material	Storage Condition	Efficacy Time (Minimum)	Seed Size versus Concentration MOs	Viable Cell (Minimum)	pH	Moisture	Shelf Life	Contamination (Dilution Factor)	Reference
India	Rhizobium, Azotobacter, Azospirillum Phosphate-solubilizing bacteria, Mycorrhizal, potassium mobilizing, zinc solubilizing, Acetobacter and consortium	Peat; lignite; peat soil; humus; wood charcoal	No	No	No	5 × 107 to 5 × 108	5.0–7.5	8–40%	n.s.	10−4 a 10−5	[162]
Canada	Only Rhizobium species	n.s.	No	1 to 2 years	Yes	103 to 105	No	n.s.	6 months	10−6	[163]
Australia	Only Rhizobium species	Peat; bagasse; manures, sugarcane filter mud; coconut coir dust; perlite; clays	No	No	Yes	105	No	n.s.	n.s.	10−6	[161]
Uruguay	Only Rhizobium species	Peat; others (n.s.)	4 °C	No	No	1 × 109	No	n.s.	n.s.	n.s.	[164]
Brazil	Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium, Azorhizobium Bacillus subtilis, and Frauteria aurantia	Peat; others (n.s.)	No	No	No	1 × 109	No	n.s.	6 months	1 × 10−5	[165]

n.s., not specified.

).

India has one of the most comprehensive legislations regarding biofertilizers [166]. In 2006, a section on the registration and quality requirements of biofertilizers was included in the Essential Commodities Act, 1955 (10 of 1955), under the Fertilizer (Control) Order, 1985. This regulation broadly defines biofertilizers as products based on live microorganisms (solid or liquid) containing a carrier that contributes to increasing soil and/or crop productivity through nitrogen fixation, phosphorus solubilization, or nutrient mobilization. For the commercialization of biofertilizers in the country, these products must be registered and authorized by the competent authorities and meet specified quality standards [162].

The quality parameters for biofertilizers in India include several requirements: (i) form (liquid, tablets, or root), (ii) minimum concentration of viable cells (ranging from 5 × 10⁷ to 5 × 10⁸ cell g⁻¹ or mL⁻¹), (iii) absence of contamination at a dilution factor ranging from 1 × 10⁻⁴ to 1 × 10⁻⁵, (iv) pH levels according to the type of microorganism, (v) particle size, (vi) percentage of moisture by weight, which varies from 8% to 40%, and (vii) efficiency of each type of biofertilizer [162]. The quality parameters are shown in

Table 5. Quality standards for biofertilizers in India based on microorganism type and carrier materials.

Biofertilizer	Carrier Base	Viable Cell (Minimum)	pH	Particles Size	Moisture Percent by Weight
Rhizobium Azotobacter Azospirillum	Peat, lignite, peat soil, humus, and wood charcoal	5 × 107 cell/g	6.5–7.5	0.15–0.212 mm IS sieve	30–40
		1 × 108 cell/mL
Phosphate-solubilizing bacteria	Peat, lignite, peat soil, humus, and wood charcoal	5 × 107 cell/g	6.5–7.5
		1 × 108 cell/mL	5.0–7.5
Mycorrhizal biofertilizers	n.s.	100 propagules/g of finished product	6.0–7.5	90% should pass through 250 micron IS sieve	8–12
Potassium-mobilizing biofertilizers	Peat, lignite, peat soil, humus, and talc	5 × 107 cell/g	6.5–7.5	0.15 to 0.212 mm IS sieve	30–40
		1 × 108 cell/mL	5.0–7.5
Zinc-solubilizing biofertilizers	n.s.	5 × 107 cell/g	6.5–7.5	0.15 to 0.212 mm IS sieve	30–40
		1 × 108 cell/mL	5.0–7.5
Acetobacter	Peat, lignite, peat soil, humus, and wood charcoal	5 × 107 cell/g	5.5–6.0	0.15 to 0.212 mm IS sieve	30–40
		1 × 108 cell/mL	5.5–6.0
Carrier-based consortia	n.s.	5 × 107 cells/g All microorganisms	n.s.	0.15 to 0.212 mm IS sieve	30–40
Liquid consortia	n.s.	5 × 108 cells/ mL All microorganisms	5.0–7.0	n.s.	n.s.

n.s., not specified; IS, International Standard. Source: [162].

.

An aspect of this regulation is the inclusion of microbial consortia, both carrier-based and liquid, which must contain two or more microorganisms, such as Rhizobium, Azotobacter, or Azospirillum. These consortia must have a minimum concentration of 1 × 10⁷ CFU/g to 1 × 10⁸ CFU/mL, respectively [162].

Most soil microbial inoculant products available on the market are based on unique species and strains of microorganisms. Despite being little explored in the market, the use of microbial consortia, where inoculant products contain two or more genera or species, shows promise and offers multiple functionalities, allowing them to be applied to various crops and different geographic regions. However, there is no general agreement on the use of a mixture of microorganisms, and evidence of their effectiveness is scarce [62].

Canada prioritizes the quality of inoculating microorganism supplements containing rhizobia and highlights the long-term benefits of these supplements. The requirements outlined in the Canadian Food Inspection Agency (CFIA) regulations include the following: (i) microorganism species Rhizobia (Rhizobium sp., Bradyrhizobium sp., Mesorhizobium sp., and Sinorhizobium sp.) should be the only active microorganism in the product; (ii) the characteristics of the crop, including the type of legume (e.g., alfalfa, clover, trefoil, sainfoin, beans, peas, and soybeans), seed size, and the relationship between product weight and the number of viable cells of the nodule-inducing species; (iii) production methods; (iv) quality control procedures to ensure consistency in production, product purity, and absence of contaminants; (v) strict biosecurity requirements for genetically modified microorganisms is not permitted; (vi) minimum time and number of tests for national registration, requiring at least six efficacy trials for a rhizobia inoculant over a minimum period of two years to account for seasonality and environmental conditions such as soil moisture, precipitation, and soil and air temperature to demonstrate seed survival during the proposed period; furthermore, products must show statistical efficacy in at least 60% of trials; and (vii) compatibility and viability of inoculants with additional seed treatments, such as pesticides [163]. The requirements are shown in

Table 6. Quality standards for inoculant microorganisms containing rhizobia in Canada for legumes of different seed sizes.

Crop	Seed Size	N° of Seeds per kg	Number of Viable Cells per Seed
Alfalfa, clover, Birdsfoot Trefoil	Small	>200,000	103
Sainfoin	Medium	200,000–30,000	104
Beans, peas, soybeans	Large	<30,000	105

Source: [163].

.

In Australia, quality standards for high-quality inoculant microorganisms were implemented in 2010 and are outlined in the Australian National Code of Practice “Quality Trademark for Microbial Inoculant Products.” The standards for inoculating microorganisms in Australia consider the type of inoculant product (peat, liquid, granules, or freeze-dried), fresh count, expiration count, and expiration time (

Table 7. Quality standards for inoculant microorganisms in Australia based on the type of inoculant product.

Product	Fresh Count	Expiry Count	Expiry Time (Months)
Peat (CFU/g)	≥1 × 109	≥1 × 108	12–18
Liquid (CFU/mL)	≥5 × 109	≥1 × 109	6
Granules (MPN/g)	≥1 × 107	≥1 × 106	6
Freeze-dried (CFU/vial)	≥1 × 1012	≥5 × 1011	6

Source: [161].

). Contaminants in these products must be absent at dilution factors of 10⁻⁶ [161].

The evolution of standards regulating the quality standards of inoculant microorganisms in Australia is the result of an analysis of problems associated with the use of peat inoculant over 50 years of industrial production of these microorganisms for vegetable crops. These problems were related to the low number of viable cells and survival rate of microorganisms due to climate change, water availability, soil salinity, temperature, and sterilization treatments applied due to high levels of contamination [161,167].

Australian standards also use the relationship between the minimum concentration of microbial cells and seed size as a quality criterion. For small seeds (<2 mg), such as Biserrula and white clover, a concentration of 500 cells per seed is recommended, while for large seeds (>10 mg), such as lupine, pea, and soybean, a concentration of 10⁵ cells per seed is recommended. For the application of live cells per area, inoculants must provide 1 × 10¹⁰ live cells per hectare within the shelf life of the inoculant [161].

The Regulation (EU) 2019/1009 establishes a regulatory framework for the use of microorganisms as inoculants in fertilizers, ensuring safety, efficiency, and transparency in the European fertilizer market. The regulation includes these microorganisms within the definition of “fertilizer product” and sets specific standards for microbial biostimulants, which consist of one or more microorganisms aimed at optimizing plant physiological processes, such as nutrient uptake and abiotic stress tolerance. Among the authorized microorganisms are Azotobacter spp., mycorrhizal fungi, Rhizobium spp., and Azospirillum spp. These biostimulants must ensure the absence of pathogens such as Salmonella spp., Escherichia coli, Listeria monocytogenes, Vibrio spp., Shigella spp., and Staphylococcus aureus. For other microorganisms, such as Enterococcaceae, anaerobic microorganisms, molds, and yeasts, maximum allowable counts are set at 10, 10⁵, and 10³ CFU/g or mL, respectively. Additionally, the regulation mandates that the labeling of these products must list all microorganisms present, with their concentrations expressed in CFU/g, allowing for a 15% variation in the declared microbial concentration [168,169].

Although there is no global standard for quality criteria for inoculating microorganisms, a minimum number of rhizobia per seed is recommended, depending on the seed size [170]. India and Mercosur countries do not establish parameters for this relationship between concentration and seed size.

In 1998, Mercosur countries, Argentina, Brazil, Uruguay, and Paraguay, recognized the need to regulate standards to facilitate the production and commercialization of inoculants among member countries through Resolución Mercosur/GMC/RES Nº 28/98. The commercialization of inoculants is only permitted for products registered in one of the countries defined in this agreement. Regulatory bodies are responsible for registering and inspecting these products. They also recommend the type of strain to be used, which must be formulated on a sterile support, free from contaminants, and must meet the minimum microorganism concentrations compatible with the specific needs of each state throughout the product’s shelf life. Quality criteria for inoculant products include microorganism concentration, purity, and identity. Additionally, products should not be sold in bulk [171].

Uruguay has had regulatory standards for inoculants since 1981, established by Decree No. 546 of October 28, 1981. This decree mandates that all inoculants be registered and inspected by the Ministry of Agriculture and Fisheries [164]. Registration is granted for a period of three years, provided the manufacturer complies with the specifications, including using Rhizobium strains recommended and supplied by the Soil and Inoculants Control Microbiology Laboratory.

The quality requirements for inoculants used in Uruguay are as follows: (i) type of inoculant: peat-based and can be applied in dry, liquid, or granular form; (ii) for liquid inoculants, the concentration of microbial cells must be at least 1 × 10⁹ CFU/mL and free from contaminants; and (iii) inoculants may contain more than one species of Rhizobium, but each species must be prepared separately and mixed identically at the time of packaging. The use of polyvalent inoculants containing Rhizobium strains for different legumes is not permitted; (iv) the minimum concentration of bacteria per peat impregnated in the soil must be 5 × 10⁹ CFU/g of soil; and (v) inoculants must be stored at 4 °C.

In Brazil, regulation for the use of inoculating microorganisms in agriculture has been in place since 1980, based on Law No. 6894 of 16 December 1980. This law established guidelines for the inspection and supervision of the production and trade of fertilizers, correctives, inoculants, biofertilizers, remineralizers, and plant substrates intended for agriculture. It was updated in 2014 by Decree No. 8384 of 29 December 2014 [172,173].

Brazilian legislation provides legal definitions for both inoculants and biofertilizers. Inoculants are products containing microorganisms designed to promote plant growth and can be applied in pure form or on a carrier. Biofertilizers are active products that can directly or indirectly enhance all or part of cultivated plants, thereby increasing their productivity [172].

Inoculants must be registered with the relevant regulatory authority before being sold. The manufacturer is required to submit all necessary documentation, including details on the microorganisms present in the product, an efficacy study under recommended use conditions, toxicological data, and appropriate labeling (BRASIL, 2014).

For registration purposes, Brazilian standards for the production, import, and commercialization of inoculants require the following: (i) Type of inoculant: it is recommended to use inoculant products containing nitrogen-fixing bacteria for symbiosis with legumes, with a minimum concentration of 1 × 10⁹ CFU per gram or milliliter of the product, ensuring product efficacy until its expiration date. Other inoculants are permitted if formulated with associative bacteria and plant growth-promoting microorganisms. The microorganism concentrations must be specified in the product registration. (ii) Type of carrier: inoculants must be produced on a sterile support that maintains the necessary conditions for microorganism survival, with solid products requiring a dilution factor of 1 × 10⁻². (iii) Freedom from contaminants: inoculants must be free from non-specified microorganisms at a dilution factor of 1 × 10⁻⁵. (iv) Product shelf life: the shelf life of the product must be at least 6 months from the date of manufacture [165].

Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium, and Azorhizobium are among the genera recommended by Brazilian legislation to produce inoculants used in cultivating over 45 species of legumes, including peanuts, soybeans, alfalfa, and chickpeas. For eucalyptus crops, Bacillus subtilis and Frauteria aurantia are approved as inoculants, while Azospirillum brasilense is permitted for use in rice, corn, and wheat crops. The use of other inoculating microorganisms requires approval from the relevant regulatory authority [165].

7. Gaps for Applications and Performance in the Field

The use of agricultural inoculants in field applications, including both bacterial and fungal types, has been growing worldwide, driven by the increasing demand for sustainable and green agricultural practices. Government support for bio-based agricultural inputs and advances in biotechnology have been promoting the substitution of chemical-based fertilizers for biological solutions. In Brazil, the use of inoculants to replace chemical fertilizers in the field has gained widespread applications in soybean cultivation to promote biological nitrogen fixation. According to the National Association of Inoculant Producers and Importers (ANPII), 85% of the 2022/2023 soybean harvest was based on the use of inoculants, particularly Bradyrhizobium and Azospirillum. Usually, less than 10% of soybean plantations where inoculants were applied require additional nitrogen supply in the form of urea. The cost of application in soybean seeds ranges from BRL 60 to 70 (around USD 10–12) per hectare, and this biological solution for nitrogen supply has proven to be the most cost-effective to the producer. Annually, Brazil saves around BRL 25 billion (or USD 4.5 billion) by avoiding the use of chemical nitrogen fertilizers [174,175].

The use of biological inoculants, however, presents some challenges and gaps that can affect their effectiveness and widespread field application. Even successfully registered biological agents have faced difficulties in this transition from the lab to the field, due to the lack of standardized scale-up protocols [176]. The first obstacle is related to their biological nature, which requires special handling to preserve viability and shelf life. Bacterial inoculants are living microorganisms, and their viability can decrease over time, especially if not stored under adequate conditions. Maintaining their viability during storage and transportation is crucial, and sometimes, producers are not familiar with these special requirements.

There are also challenges related to the application and post-application procedures. The method of application (e.g., seed coating, soil application, or foliar spray) can directly influence the effectiveness of bacterial inoculants. These biological agents are sensitive to environmental conditions such as temperature, pH, and moisture levels. Inadequate conditions can reduce their survival and ability to colonize plant roots effectively. For example, in the case of soybean, the inoculated seed should be handled with care, especially in dry and very hot weather. Even though cellular protectors are employed in product formulations to reduce cell mortality, the exposed bacteria can dehydrate. Therefore, sowing should be conducted within a maximum of 12 h. Another concern is that some agricultural practices involve the use of chemical fertilizers, pesticides, or herbicides, which can affect the viability or effectiveness of the bacterial inoculants [175,177,178].

Finally, there are gaps related to knowledge dissemination and public awareness about the cost-effectiveness, safety, and benefits of biological inoculants. Because of the specificities mentioned above, sometimes, the inadequate handling of inoculants in the field can lead to inefficient results, which in turn leads to the false perception that biological products are not effective enough. Another public concern is about the specificity and safety of biological agents in agriculture, which primarily involve potential risks to human health, non-target organisms, and the environment. Could the introduction of exogenous microorganisms disrupt the ecological balance and potentially affect beneficial organisms such as soil microorganisms, natural pest control agents, and pollinators? To address these issues, ecological studies should be conducted and disseminated, promoting transparency and encouraging the development of technologies that are both efficient and safe for the environment. By ensuring safety assessments and implementing appropriate risk management strategies, bacterial inoculants can be used responsibly, in adherence to regulatory guidelines, to promote sustainable agricultural practices.

8. Conclusions

This review comprehensively covered the significant advancements and various applications of bioinoculants in the field of agriculture. It highlighted the benefits of biological agents in improving the sustainability of agriculture. Bioinoculants enhance soil health, boost crop yields, and decrease the need for chemical fertilizers, making them a promising option for sustainable farming. This review emphasizes the need to integrate biological solutions into modern agriculture for better environmental care and sustainable productivity. While bioinoculants offer promising solutions to reduce chemical fertilizer dependency and improve crop productivity, current research still faces challenges. These include scaling up production, optimizing field application efficiencies, and ensuring environmental interactions are well understood. Regulatory frameworks in various countries need better alignment to support global trade and the use of bioinoculants, despite being strong in some areas. Future research should focus on improving the long-term stability of bioinoculants and their integration into different agricultural systems. Researchers should also focus on developing more cost-effective production methods and expanding the range of microorganisms that can be used. Investigating consortium-based strategies and the synergistic interactions between bioinoculants and crops will be essential for future advancements in this area, offering a deeper understanding of sustainable agricultural biotechnology.