Microbial Secondary Metabolites and Their Use in Achieving Sustainable Agriculture: Present Achievements and Future Challenges

Abstract

The agricultural sector faces serious challenges due to climate change, threatening global food security. In addition to economic impacts, decreasing agricultural production jeopardizes nutrition, particularly in vulnerable populations. The implementation of mitigation actions and sustainable alternatives becomes urgent. In this context, microbial secondary metabolites (MSMs) emerge as a promising solution. Some of these molecules have the potential to strengthen soil health, increase plant resistance to pests and adverse weather conditions, and improve nutrient availability, for example, LCOs (lipochitooligosaccharides) to improve legume nodulation and several other physiological changes in the plant, and several pyrazines with biocontrol potential. However, although the potential benefits are clear, the industrial viability of commercially using these compounds has not yet been fully established. In addition, the connection of the academic research on MSMs with their potential role in agriculture as bio-inputs is still limited. This review aims to contribute to filling the gaps by aggregating information on the classification, application, and synthesis of these molecules. Additionally, we discuss strategies and technologies that could enhance the use of MSMs in agriculture.

1. Introduction

Due to its significant vulnerability to parameters such as rainfall and temperature patterns, the agricultural sector is one of the most affected by climate change. Beyond the economic impacts, these events bring decreases in agricultural production and directly threaten the food and nutritional security of the population [1], especially those living in socially vulnerable situations [2,3]. In this context, urgent action is needed to mitigate the effects of climate changes on crops [4]. Additionally, adopting agro-industrial strategies that reduce environmental impacts is essential [5]. Among these alternatives, products containing microorganisms and their metabolites can improve soil health and biodiversity, increase crop resistance to pests and adverse weather conditions, and facilitate nutrient availability to plants [6,7].

Secondary metabolites are adaptive molecules that have appeared during the evolutionary process for purposes beyond primary metabolism [8]. They often present chemical structures distinct from molecules belonging to essential cellular metabolism processes. Among these structures, β-lactam rings, cyclic peptides containing non-protein amino acids, rare sugars and nucleosides, unsaturated bridges of polyacetylenes and polyenes, and large macrolide rings can be highlighted [9]. In microorganisms, unlike primary metabolites, which are essential for survival, secondary metabolites are produced by strains, species, or genera for physiological, social, or predatory reasons, making these molecules closely related to their ecology [8]. Among the described microbial secondary metabolites (MSMs) are antibiotics [10], nematicides [11], fungicides [12], immunosuppressants [13], phytohormones [14], pigments [15], toxins [16], enzyme-degrading toxic waste [17], siderophores [18], among others.

Studies have shown that microorganisms commonly associated with crops produce secondary metabolites that can directly or indirectly benefit the associated crop, increasing its tolerance to abiotic stress and its resistance to pathogens, in addition to greater nutrient assimilation and growth stimulation.

However, the viability of using these compounds on an industrial scale is not yet well established [19]. Additionally, there is an academic production deficit connecting the scientific knowledge generated so far on MSMs with their potential role in agriculture as commercial products. According to the Web of Science (WOS) (https://www.webofscience.com), on the 6 May 2025, there were approximately 6920 scientific articles containing the term “Microbial Secondary Metabolites” published since 1990, of which 3160 were published in the last five years (2021–2025). When associated with the keyword “agriculture”, the number of publications dropped to 2700 since 1990, 1510 of which were published in the last five years.

In this scenario, a deeper scientific understanding of the potential benefits of MSM use in global agriculture is needed. Discussions should also focus on the feasibility of developing large-scale industrial production studies for these molecules.

This review aims to provide information on studies on MSMs, their classification, and application, and on production, separation, and identification techniques. It will also address the potential use of these compounds as agro-industrial or bio-input products.

2. Microbial Secondary Metabolites (MSMs)

Metabolism can be defined as the total set of biochemical reactions carried out by a particular organism. Both the end products of these reactions and the intermediate molecules synthesized in these processes are called metabolites [20]. Some metabolites are essential for the survival, growth, and reproduction of the organisms, being present in all cells and actively participating in cell division processes [21], such as amino acids, lipids, and carbohydrates. However, there is a second group of metabolites that, although may contribute to a higher survival rate, do not play a crucial role in the maintenance and functioning of cellular processes. Based on this difference, the term secondary metabolite was coined by Albrecht Kossel in 1891 [20]. These molecules were defined as non-essential for survival and separated from the primary metabolites.

Although secondary metabolites are derived from the primary metabolism processes, their molecular skeletons are quite distinct and unusual. They include β-lactam rings, cyclic peptides containing non-protein amino acids, rare sugars and nucleosides, unsaturated bridges of polyacetylenes and polyenes, and large macrolide rings [9]. Nevertheless, the distinction between primary and secondary metabolites is ambiguous, since many intermediates of the primary metabolism overlap with intermediates of the secondary metabolism [22], indicating that some metabolic pathways can be shared by both [23].

Secondary metabolites, in general, seem to be formed when large quantities of primary metabolism precursors, such as amino acids, acetate, and pyruvate, accumulate in the cells [24]. For this reason, secondary metabolites can be considered as a “buffer zone”, where excess carbon and nitrogen can be diverted into inactive forms of primary metabolism and then reconverted to active forms according to the organism’s needs [20]. This delicate dynamism and balance between primary and secondary metabolism are highly influenced by cell growth and development and environmental conditions [25].

Regarding microorganisms, microbial secondary metabolites are produced by strains, species, or genera for physiological, social, or predatory reasons, making these molecules closely related to their ecology [8]. There is no consensus in academia regarding the reasons why secondary metabolisms emerged and perpetuated throughout evolution. The most accepted hypothesis is that these molecules increase the survival rate of the producer in hostile environments [8]. This occurs mainly in two ways: by increasing the capacity for growth, dispersion, or reproduction, or by synthesizing molecules for protection, competition, or predation [26]. The fact that secondary metabolites vary in quality and quantity according to environmental conditions, biodiversity, and nutritional availability, especially in stressful conditions [27], is one of the arguments supporting this hypothesis [28].

MSMs can be classified according to their biological role into four main categories [29] as follows:

• Promoting defense against competitors or pathogens;
• Increasing adaptability to adverse environmental conditions;
• Modulating the interaction and symbiosis with other organisms;
• Inducing signaling and communication among other participants in the ecosystem where the microorganism is inserted.

From the perspective of MSM applications in human, animal, and plant nutrition and health, they can be classified according to their biological function. They can be grouped into antibiotics, antivirals, antifungals, antitumoral, pigments, chelating compounds, growth hormones, toxins, enzyme inhibitors, among others. Some examples of these compounds can be seen in Figure 1.

It is worth mentioning that many biosurfactants—surface-active molecules used in agro-industrial processes, bioremediation, and biofilm removal—come from the secondary metabolism of bacterial species [30]. Similarly, several biopolymers synthesized by microorganisms—such as cellulose, alginate, and xanthan gum—have applications in the fuel, food, medicine, and cosmetics industries [31].

2.1. Classification of MSMs

Recently, Reddy and collaborators [21] estimated the number of identified bioactive MSMs at 200,000. They are commonly classified according to their structure, function, and biosynthesis into five main groups: peptides, polyketides, volatile compounds, terpenes/steroids, and growth regulators [32] (Figure 2).

2.1.1. Peptides

Peptides derived from MSMs are soluble molecules and often present as cyclic compounds or are associated with diketopiperazines [21]. They are widely involved in antimicrobial activities, commonly as bacteriocins [33].

Bacteriocins are a heterogeneous group of antimicrobial peptides produced by ribosomal synthesis associated with several microbial genera, such as Bacillus sp. [34]. Subtilisin (Figure 3), produced by Bacillus subtilis, is a well-studied bacteriocin of Subclass I (lantibiotics), which acts in interbacterial communication and antibiotic resistance processes.

Generally, peptides are synthesized through enzymatic condensation reactions or as extensions of diketopiperazine rings [21]. In addition to antimicrobial activity, peptides can act on the degradation of bacterial toxins, such as anionic surfactants [35].

Figure 3. Subtilisin (peptide) structure [36].

Figure 3. Subtilisin (peptide) structure [36].

2.1.2. Polyketides

Polyketides are a structurally heterogeneous group of MSMs, synthesized through processes similar to fatty acid biosynthesis (decarboxylative condensation of malonyl CoA), guided by multifunctional proteins known as polyketide synthases [21].

Commonly, polyketides are classified into the following three types:

Type I: Macrolides synthesized by multimodular megasynthases. Macrolides have a wide range of biological activities, such as bactericidal, antifungal, and immunosuppressive [37]. Rapamycin (Figure 4A), synthesized by the actinomycete Streptomyces hygroscopicus, is a macrolide with antifungal action against phytopathogenic fungi such as Aspergillus fumigatus and Fusarium oxysporum [37].

Type II: Aromatic compounds produced by dissociative enzyme action. In general, they are molecules with known antibiotic action, such as anthracyclines, angucyclines, and tetracyclines [37]. Oxytetracycline (Figure 4B), a widely used broad-spectrum antibiotic, was first isolated from Streptomyces rimosus [38].

Type III: Small aromatic compounds, mostly synthesized by fungal species with especially antimicrobial action [39]. Spirolaxin (Figure 4C) is a Type III polyketide produced by the fungus Sporotrichum laxum with action against Helicobacter pylori, a bacterium that causes gastrointestinal diseases in humans [40].

Figure 4. Microbial polyketides structures. (A) Rapamycin (Type I) [41]; (B) oxytetracycline (Type II) [42]; (C) spirolaxin (Type III) [43].

Figure 4. Microbial polyketides structures. (A) Rapamycin (Type I) [41]; (B) oxytetracycline (Type II) [42]; (C) spirolaxin (Type III) [43].

Polyketides are the most abundant MSM in nature, produced by a wide range of microorganisms and predominantly showing antibiotic action [29].

2.1.3. Volatile Compounds

Volatile compounds, commonly synthesized by soil microorganisms, are a diverse group of low molecular weight molecules with high vapor pressure and low boiling points [44]. These physicochemical characteristics allow these metabolites to evaporate easily and diffuse among the pores present in the soil and rhizosphere. For this reason, volatile compounds from secondary metabolism are the main characters in antagonist and competitive interactions among microorganisms [29]. The most found and characterized volatile compounds are as follows:

• Pyrazines: Well-studied secondary metabolites known for their antimicrobial properties. Typically produced by Bacillus spp. and Pseudomonas spp. [45], such as 2-methylpyrazine (Figure 5A), produced by Pseudomonas putida, a molecule with antifungal action against Magnaporthe oryzae, a pathogen of rice (Oriza sativa) [46].
• Indole: A signaling molecule synthesized by both Gram-positive and Gram-negative bacteria [47]. Volatile indoles can also stimulate plant growth, such as some produced by Proteus vulgaris [48].
• Sulfur-containing volatile compounds: Dimethyl sulfite (Figure 5B), dimethyl bisulfite, and dimethyl trisulfide metabolites that contain sulfur in their composition. They are known for their action as fungal growth inhibitors and as key components in microorganism–plant interactions [49]. Furthermore, dimethyl sulfite produced by Serratia odorifera was described to cause negative effects on the growth of Arabidopsis thaliana [50].

Figure 5. Microbial volatile compound structures. (A) 2-methylpyrazine (pyrazine) [51]; (B) dimethyl sulfite (sulfur-containing volatile compound) [52].

Figure 5. Microbial volatile compound structures. (A) 2-methylpyrazine (pyrazine) [51]; (B) dimethyl sulfite (sulfur-containing volatile compound) [52].

2.1.4. Terpenoids/Steroids

Terpenoids are large structurally bioactive molecules derived from isoprene polymers, while steroids have a tetracyclic carbon skeleton [53]. Many of the terpenes and steroids from secondary metabolism possess antimicrobial activity and are produced by endophytic microorganisms [54]. Albaflavenone (Figure 6) is a well-known example of a terpenoid produced by several Streptomyces species, such as Streptomyces coelicolor and Streptomyces spectabilis, and is biologically active against bacteria such as B. subtilis [55]. Geosmin and y-terpinene are other MSMs in this category with proven antibiotic action [56,57].

2.1.5. Growth Regulators

Produced at very low concentrations, growth regulators belong to a different category of MSMs. These molecules are mostly synthesized by a specific set of plant-associated microorganisms, with an emphasis on bacteria, also known as plant growth-promoting bacteria (PGPB). PGPB comprise a heterogeneous group of soil bacteria that, through their metabolic and physiological activities, directly or indirectly affect plant growth and development. Through their association (symbiotic or not) with plants of different families and genera, PGPB play a central role in balancing forest and agricultural ecosystems [59]. Growth regulators produced by PGPB can affect plant growth and development in several ways and can also increase plant resistance to biotic and abiotic stresses [60,61].

Among the growth-regulating MSMs are phytohormones, such as auxins (glucosinolate), cytokinins, jasmonic acid (alkaloids), gibberellins, abscisic acid (terpenes), salicylic acid (phenol), and brassinosteroid (polyhydroxy steroid) [29]. Phytohormones, in general, play important roles in all stages of plant growth and development [14].

Auxins are directly related to cell expansion and apical dominance processes, while cytokinins stimulate cell division, and protein and RNA synthesis, and act in processes of plant senescence [62,63]. Gibberellins play an important role in breaking seed dormancy and inducing flowering processes. Salicylic acid increases plant resistance to pathogens and is involved in transpiration, photosynthesis, and ion transport [29]. Brassinosteroids assist in consolidating plant immunity and reproductive processes. Abscisic acid stimulates molecular signals of leaf abscission and is often associated with the development of strategies for plant responses to abiotic stresses, in continuous antagonism with cytokinins [64]. Finally, jasmonic acid acts in plant defense against phytopathogen attacks [29].

• LCOs (lipochitooligosaccharides)

An intriguing example of growth regulators derived from secondary metabolism with proven beneficial effects on plants are LCOs (lipochitooligosaccharides). Also known as Nod factors, they are synthesized by the bacterial group collectively called rhizobia.

Rhizobia, especially alpha-rhizobia, a class encompassing several genera, such as Bradyrhizobium, Rhizobium, Sinorhizobium, Mesorhizobium, and Azorhizobium, are composed of hundreds of species [65]. These bacteria share a very peculiar characteristic: they symbiotically associate with the roots of several leguminous plants. The result of the symbiosis is the formation of highly specialized structures called nodules, usually on roots, within which the atmospheric nitrogen fixation process takes place [66,67].

The process of nodule formation is highly complex and specialized, depending on a delicate molecular “dialogue” between the bacteria and the roots of the host plant. Generally, this dialogue occurs in two steps: First, the host legume releases molecules, mainly flavonoids, into the rhizosphere. These molecules are then perceived by the specific rhizobia and activate the second step of the interaction, the synthesis and release of LCOs [67,68].

The structure of LCOs is quite diverse, with dozens of molecular arrangements [69,70,71]. As a general characteristic, they are structures formed by three to five 1–4 β-linked acetylglucosamine residues with the N-acetyl group of the terminal non-reducing sugar replaced by an acyl chain [66,68]. Their molecular skeletons depend on the producing species and environmental conditions at the time of synthesis and are essential for the host specificity of rhizobia [70,71,72].

LCOs promote significant physiological changes in the host plant [67,68], including the deformation of root hairs [73], induction of genes essential for infection thread formation [74], and division of cortical cells [75]. Changes in the balance of plant hormones in the rhizosphere have also been reported to be orchestrated by LCOs. Martinez-Abarca and collaborators [76] described that LCOs can reduce the level of salicylic acid in the roots, which would aid in suppressing the defense responses of the host plant, facilitating infection by rhizobia.

Surprisingly, studies have shown that the effects of LCOs go beyond the molecular signaling necessary for the establishment of symbiosis between rhizobia and legumes. Even at very low concentrations—in the nanomolar range—the presence of LCOs induces physiological changes and promotes growth in both host and non-host plants [70,71,77].

In addition to the widely studied LCOs produced by rhizobial bacteria, in the last two decades, similar molecules have been described in various fungal species, particularly mycorrhizal fungi. These fungal LCOs are referred to as Myc-LCOs and appear to play an important role in the establishment of symbiosis between plants and arbuscular mycorrhizal fungi [78]. Previous studies suggest that bacterial and mycorrhizal symbioses share a highly conserved metabolic pathway, known as the common symbiosis signaling pathway (CSSP) [79,80]. The CSSP is activated in plants in the presence of both bacterial LCOs and Myc-LCOs. However, these symbioses employ distinct molecular mechanisms after root colonization [81]. The activation of CSSP allows root colonization by microorganisms, which results in the formation of nodules and mycorrhization, respectively [80]. Due to its high conservation in both leguminous and non-leguminous plants, the CSSP may be responsible for enabling the beneficial effects of bacterial LCOs observed in non-host plants.

Girardin and colleagues described that genes encoding Myc-LCO receptors involved in mycorrhization in tomato (Solanum lycopersicum L.) and petunia (Petunia hybrida L.) are active when inserted into the genome of Medicago truncatula and Lotus japonicus—both legumes—promoting functional nodulation. These data suggest that the system for detecting LCOs in plants is ancestral. It was initially involved in mycorrhization and later recruited during the evolution of rhizobial symbiosis in legumes [82].

The effects of LCOs on plants may be, at least in part, related to the fact that these molecules indirectly affect photosynthesis and stimulate meristematic mitotic activity in leaf tissues [83]. Moreover, Liang and collaborators [84] inferred that LCOs can suppress innate immune responses, facilitating microbial interactions.

2.2. Extraction, Purification, Quantification, and Characterization of MSMs

Due to their wide diversity and structural characteristics, the production, extraction, and characterization of MSMs can require complex processes.

External factors such as temperature, pH, aeration, and nutrient availability greatly influence the diversity and quantity of MSMs produced [85]. As the first step in the extraction and characterization processes, each of these parameters needs to be optimized to achieve the highest possible productivity [86]. In many cases, mimicking a stress condition, such as increased salinity [87] or decreased nutrient availability [20], in the culture medium can enhance metabolite production.

2.2.1. Extraction

Generally, the extraction is performed by adding solvents, either individually or in combination. The choice of solvent for the extraction is determined by the solubility of the target metabolite.

Polar solvents such as ethanol, acetone, methanol, and acetonitrile are used for the extraction of polar MSMs, such as flavonoids and auxins, and most polyketides, terpenes, and steroids. On the other hand, non-polar solvents such as hexane, dichloromethane, and chloroform are used for the extraction of non-polar molecules, such as lipids and some flavonoids and steroids [88]. Furthermore, the concentration of polar solvents used in the extraction processes of some secondary metabolites appears to be relevant, with concentrations of 1:1 (solvent/water) being more effective than extractions using pure water or solvent separately [89]. Once the extraction process is complete, the extracts are usually separated from the solvent by filtration and then undergo a drying process using a rotary evaporator, resulting in crude metabolite extracts [90].

Recently, new MSM extraction techniques have been developed and improved, such as the use of enzyme-, microwave-, and ultrasound-assisted extraction. In the enzyme-assisted extraction of microorganisms, various enzymes are used to break down the cell wall and release the compounds of interest. Some of the most common enzymes are carbohydrases (such as cellulase and xylanase), proteases, and pectinases, which are selected based on the composition of the microorganism’s cell wall [91].

Microwave-assisted extraction is a technique that uses microwave energy to heat solvents and extract compounds from plants or microorganisms. It is considered a very efficient and economic method, as the microwave energy increases analyte solubility and solvent diffusion into the matrix, increasing the metabolite yield [92].

Finally, ultrasound-assisted extraction uses ultrasonic waves to enhance the extraction of compounds from microorganisms such as bacteria, fungi, or yeast. It is based on the principle of cavitation, where ultrasonic waves create bubbles that expand and collapse, generating high local energy that disrupts cell walls and facilitates the release of intracellular compounds [93]. Smirnou and colleagues achieved a high purity level of schizophyllan, a well-known anti-inflammatory produced by the fungus Schizophyllum commune, using ultrasound in a culture broth. The use of the technique reduced the viscosity of the broth, which facilitated the filtration processes and reduced losses [94].

In many cases, one or more extraction techniques can be combined, increasing productivity and lowering the production costs of a given metabolite. Esquivel-Hernandéz and colleagues used ecological solvents associated with microwaves to extract bioactive compounds produced by the cyanobacteria Arthrospira platensis, achieving high yield levels [92].

2.2.2. Thin-Layer Chromatography

TLC is generally used as the first step for isolating MSMs from a crude extract. Alkhulaifi and collaborators [95] employed TLC to determine the similarity of intracellular and extracellular MSMs among different fungal species found in soils.

Using TLC, the compounds present in the extract can be separated according to their molecular weight, polarity, and each molecule’s affinity for the mobile/stationary phase. Silica gel is commonly used as the stationary phase of TLC for isolating MSMs, especially those produced by endophytic fungi [96].

TLC is also widely employed for the extraction of compounds with antimicrobial potential. Liu and colleagues [97] searched for metabolites with antibiotic activity from the crude extract of Bacillus amyloliquefaciens strain LWYZ003. Similarly, Ramadhana and collaborators [98] extracted metabolites produced by endophytic fungi to evaluate their effect against bacteria related to gastrointestinal diseases in humans. Moreover, Parera-Valadez et al. [99] combined two chromatographic techniques—TLC and ultra performance liquid chromatography (UPLC)—in bioassays testing the antimicrobial activity of crude extracts of MSMs from marine bacterial strains against Staphylococcus aureus and Pseudomonas aeruginosa.

A more advanced and precise derivation of TLC is called high-performance thin-layer chromatography (HPTLC). It is a chromatographic technique with enhanced versatility and higher accuracy, especially when associated with other analytical tools such as biochemical and biological assays [100]. Jamshidi-Aidji and collaborators [101] employed HPTLC coupled with different bioassays (HPTLC-EDA) for the analysis of extracts of lipopeptides produced by Bacillus spp. strains with potential antimicrobial activity. HPTLC is used for the control of biotechnological processes involved in the production of bioactive metabolites, which are often sensitive to external influences [101].

2.2.3. High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is widely used for MSM purification. Its main advantage is gradient elution, which allows changes in the mobile phase composition during analysis. This enhances the separation of compounds based on their affinity for mobile and stationary phases [96].

In addition to purifying molecules, HPLC can also be used to quantify MSMs. For this, a standard solution of the purified molecule at a known concentration is required [96]. The results of the HPLC analysis of the standard solution can be used to estimate the concentration of the target molecule present in the purified extract [102].

Enyi and Ekpunobi [103] employed HPLC for the extraction and identification of MSMs with demonstrated antimicrobial and antioxidant activities—benzylpyridine B and aurantiamine—from Trichoderma spp. Wang and colleagues [104] determined, using HPLC, the amounts of asarinin and indole-3-acetic-acid (IAA) present in the rhizosphere of Asarum heterotropoides plants, utilizing standard calibration curves for these molecules. Similarly, El-Sayed and collaborators [105] inferred the purity and concentration of an extract previously separated by TLC containing epothilone B—a potent anticancer drug—produced by Aspergillus fumigatus using HPLC and a standard epothilone solution.

HPLC is a powerful tool for analyzing and separating MSMs in complex matrices, offering relatively simple sample preparations. However, it cannot detect all MSMs in a single run [106], often requiring the use of complementary analytical methods.

2.2.4. Gas Chromatography–Mass Spectrometry (GC-MS)

GC-MS is an analytical method that combines two technologies to identify different substances contained in a sample [107]. By comparing the results obtained with standard samples, it is possible to determine which MSMs are present in the analyzed extract. The GC-MS methodology is particularly suitable for identifying volatile organic compounds [85].

It is highly reliable technology in terms of reproducibility, with robust reference databases, allowing the identification of thousands of metabolites [108]. On the other hand, non-volatile compounds, large-sized molecules, or compounds sensitive to high temperatures cannot be detected through this analytical tool [106].

In many cases, GC-MS is employed in samples previously extracted and separated through chromatographic techniques. Parera-Valadez et al. [99] used GC-MS in the identification of metabolites from marine actinomycete strains first separated by HPLC and TLC.

GC-MS can also be used in bioprospecting studies of bioactive compounds of interest. Nas et al. [109], using GC-MS, detected 56 different metabolites in crude extracts obtained from the cultivation of halophytic Bacillus spp. strains. Similarly, Chakraborty et al. [110] used GC-MS combined with other analytical methods for the bioprospecting and profiling of metabolites from the marine bacterial species Streptomyces levis strain KS46. Maulidia et al. [111] analyzed, using the same technique, metabolites extracted from cultures of the endophytic bacteria B. thuringiensis with the aim of identifying compounds with potential nematicidal activity against the nematode Meloidogyne sp.

2.2.5. Other Techniques for the Identification and Characterization of MSMs

Beyond those previously cited, numerous other analytical methodologies exist for the purification, identification, and characterization of industrially relevant MSMs.

NMR spectroscopy is a non-destructive analytical technique that provides detailed information about the chemical structure of unknown compounds and the molecular dynamics of metabolites. It is particularly useful for the identification of small molecules and complex mixtures [112]. However, due to its limited sensitivity, NMR is predominantly employed for the identification and characterization of metabolites present in abundance within the analyzed samples [106]. Guo and Zou [113] isolated four compounds from secondary metabolisms, two of which are yet to be documented, from the endophytic fungus Monosporascus eutypoides, utilizing NMR in conjunction with other analytical techniques. Similarly, Orfali and collaborators [114] elucidated the chemical structure of five molecules, one of which is unknown, synthesized by the fungus Aspergillus sp. isolated from the rhizosphere of Phoenix dactylifera.

In molecules that crystallize, it is also possible to use X-ray diffraction techniques to determine their three-dimensional molecular structures [115]. Chen et al. [116] and Song et al. [117] employed X-ray diffraction, along with other analytical tools, for the identification and characterization of MSMs produced by endophytic fungi.

FTIR spectroscopy provides information about the functional groups present in metabolites based on their absorption of infrared radiation. Sivarajan and collaborators [118] investigated the bio-efficacy of culture extracts from a marine species of Streptomyces sp. as larvicide and antibiotic activity, utilizing FTIR for the characterization of the MSMs present in the extract.

There are not many studies comparing analyses of MSMs using FTIR and XRD. In one study of crystallinity of three different types of cellulose, from banana rachis and commercial or bacterial cellulose, both methods were effective [119], although some information was clearer in each of the methods. Therefore, as the complexity of the molecules and matrices increases, there is no doubt that good results will be achieved only by integrating techniques.

2.2.6. Metabolomics

Several microorganisms, including archaea, bacteria, and fungi, have been recently identified as producers of MSMs, with potential applications in the fields of agriculture, medicine, and industry [107]. However, genetic sequencing of both cultivable microorganisms and non-cultivable microbiomes indicates that much of their potential remains inaccessible for identification and study [120]. It has been inferred that over 90% of the products from MSM gene clusters are not expressed under laboratory conditions [121,122]. There are others that are very difficult to extract and identify with conventional techniques [120].

In this context, metabolomics techniques emerge as highly effective alternatives in exploring and discovering new MSMs. Metabolomics is generally defined as the study of small molecules synthesized within a specific biological system. Its results provide direct quantification and/or qualification of MSMs produced by a microorganism of interest [120].

Metabolomics uses the already described techniques for the extraction and isolation of MSMs associated with bioinformatic and algorithmic tools, resulting in high-resolution and sensitivity analyses of complex samples [123].

Metabolomic analysis of MSMs typically takes place through two main approaches: targeted and untargeted metabolite identification [124]. Targeted identification is employed when the aim is to identify a specific group of metabolites (about 20) that are previously known. On the other hand, untargeted identification aims to identify a broad range of microbial metabolites, both known and unknown [125], often used in bioprospecting studies.

Sun and collaborators [126] induced the production of MSMs by Aspergillys sydowii through co-cultivation with B. subtilis. The metabolic profiles generated during co-cultivation were subsequently analyzed using metabolomic techniques that enabled the identification of 25 compounds from the secondary metabolism, 7 of which were previously undescribed.

Due to the enormous amount of data generated by metabolomic analysis, it is necessary to use powerful analysis tools capable of deciphering the results generated and providing satisfactory answers. In this context, chemoinformatic is widely used, as it is a computational data analysis tool designed to handle gigantic amounts of data, generating chemometric models capable of predicting in silico the biological behavior of known or unknown compounds [127].

However, being a relatively recent analytical tool, it still has a higher economic cost than conventional techniques. Through the integration of various methodologies, metabolomic analyses frequently demand a diverse range of instruments and equipment [125], also making it more expensive [128]. Additionally, metabolomic analyses often generate a large amount of data, making it difficult to sort and analyze them without powerful bioinformatic tools and updated databases that may not be available or affordable [123].

3. Actual and Potential Applications of MSMs in Agriculture

Climate change increasingly impacts agriculture, while population growth drives the demand for food. These factors pressure the agricultural science sector to develop new technologies that enhance crop productivity. More importantly, they must ensure the sustainability of agriculture under adverse environmental conditions. This is essential for ensuring food security for the population in the medium and long term [63].

Nowadays, the indiscriminate use of chemical fertilizers and pesticides is a major concern. Furthermore, the ineffective use of water resources and of unsustainable soil and crop management practices need to be addressed. These agricultural practices must be reconsidered and reformed towards new regenerative agriculture. The goal is to reverse the productive and economic losses that are beginning to be observed globally and to decrease environmental impacts.

Faced with this great challenge, the use of PGPB and their metabolites emerges as an important strategy for creating sustainable and economically viable agricultural practices [129]. The various secondary metabolites synthesized by PGPB and released into the rhizosphere may not only contribute to increased plant growth and production but also to increased plant resistance to pathogens and biotic and abiotic stresses [130]. Some examples of MSMs produced by PGPB can be seen in Figure 7.

In addition to the aforementioned phytohormones, molecules acting in the nodulation process of legumes, iron chelation (siderophores) [18], phosphate solubilization, and induction of systemic resistance in plants [131,132], along with compounds acting as antibiotics [10], nematicides [11], and fungicides [12], are also products of PGPB secondary metabolism.

One of the most remarkable characteristics of some PGPB or even specific groups of secondary metabolites is their broad-spectrum mechanisms of action, which can be employed for both pest control and plant growth promotion.

Bacterial species of the genus Bacillus are major examples of microorganisms that can be utilized as agricultural bio-inputs with diverse objectives [133,134]. As biopesticides, the presence of certain Bacillus species can suppress the growth of phytopathogens through antagonism and/or competition [135]. Apart from competing for space and nutrients, the synthesis of secondary metabolites from various other classes—such as hydrolytic enzymes, siderophores, and bacteriocins—has been described [136,137].

The genus is also reported as a significant producer of surfactin, iturin, and fengycin, all antibiotics belonging to the lipopeptide class [138]. Ongena and colleagues evaluated the effect of surfactin produced by a strain of B. subtilis on common bean (Phaseolus vulgaris L.) and tomato plants infected with the pathogen Botrytis cinerea. The study results showed a 33% disease suppression rate compared with non-inoculated plants [139].

Additionally, Bacillus species exhibit plant growth-promoting action. There are reports of the production of secondary metabolites that facilitate nitrogen fixation [140] and phosphate solubilization [141], along with plant hormone molecules such as auxins, cytokinins, gibberellins, ethylene, and abscisic acid [16]. B. amyloliquefaciens, for instance, is known as a producer of IAA, a phytohormone associated with plant growth promotion [142].

The genus Pseudomonas spp. also include species of PGPB. This group has been described as a producer of non-ribosomal cyclopeptides (CDPs), which promote lateral root growth in A. thaliana [143]. It also produces N-acyl homoserine lactone (AHL), a molecule primarily involved in quorum sensing. However, AHL also has a root growth-promoting effect [144,145]. Pseudomonas fluorescens, one of the most well-known PGPB, is a significant producer of the antibiotic 2,4-diacetylphloroglucinol (DAPG), which has broad-spectrum antibiotic activity, being effective against various pathogenic fungi and bacteria [146]. This compound is abundant in soils associated with wheat roots [147]. In addition to its antibiotic action, DAPG also promotes plant growth by increasing auxin production and altering root architecture [148]. P. fluorescens can also produce siderophores, which enhance iron absorption by plants [149] and induce their systemic resistance system (ISR) [150].

Azospirillum spp. are another bacterial genus known to promote plant growth in various ways [14,151]. The benefits associated with inoculating crops with Azospirillum spp. include, among others, the production and secretion of phytohormones such as auxins [152], known as growth promoters, and salicylic acid [153], which are involved in plant defense responses, by activating systemic acquired resistance (SAR) and induced systemic resistance (ISR) [151,154]. Furthermore, Azospirillum spp. synthetize metabolites that can enhance biological nitrogen fixation [155] and phosphate solubilization [156].

When discussing the broad-spectrum activity of secondary metabolite groups as plant growth promoters, LCOs have great potential. The presence of these molecules in the rhizosphere confers benefits to plants in various ways. In soybean (Glycine max L.) crops, it has been described that the application of LCOs led to increased grain productivity [87] and seed germination rates [66], along with reduced oxidative stress and increased resistance to abiotic stresses [157]. Interestingly, by supplying specific flavonoid inducers of nodulation genes that will synthesize the LCOs, benefits on nodulation and plant growth are also observed [68,158].

Furthermore, despite playing a fundamental role in the specificity of communication between the bacteria and their host plant, and in the formation of viable nodules [159], LCOs also appear to exert observable beneficial effects on non-host plants. For example, Bomfm and collaborators [160] described positive effects on the growth and productivity of soybean inoculated with commercial inoculants containing Bradyrhizobium spp. supplemented with extracts containing LCOs from Rhizobium tropici CIAT 899, a symbiont of common bean. Prithiviraj et al. [66] reported that LCOs increased seed germination rates and stimulated early seedling growth in various non-host crops such as maize (Zea mays L.), cotton (Gossypium hirsutum L.), and cucumber (Cucumis sativus L.). Similarly, Marks et al. [161] reported increased foliar N content and grain productivity in maize inoculated with PGPB and supplied with LCOs.

LCOs are also known to induce cell division and stimulate genes responsible for the cell cycle, accelerating embryonic processes and seed germination. De Jong and colleagues [162] and Egertsdotter and von Arnold [163] described this effect in carrot (Daucus carota L.) and Norway spruce (Picia abies L. Karst.), respectively. Khan and colleagues reported increases of 33% in length and 76% in the root surface area of Arabidopsis spp. grown in a culture medium enriched with LCOs of Bradyrhizobium japonicum [164]. Similar results were reported by Tanaka and colleagues in maize, reinforcing the theory that LCOs are growth regulators in non-leguminous plants [165]. Increases in root size and surface area caused by the presence of LCOs enhanced the plant’s capacity to absorb nutrients and water from the soil [166].

Changes in CO₂ exchange processes in plants also appear to result from the presence of LCOs in non-host plants. Prithiviraj and colleagues observed a significant increase in dry mass in Arabidopsis sp. treated with LCOs, along with an eightfold increase in CO₂ fixation rates and lower CO₂ loss through respiration [66].

However, one of the most notable characteristics of LCOs seems to be their ability to ‘mimic’ plant hormones, such as cytokinins, stimulating germination and promoting biomass accumulation [166]. Chen and colleagues reported increased flowering and fruit production in tomato plants treated with foliar applications of LCOs in both greenhouse and field trials [167]. The authors infer that the observed benefits—increased nutrient translocation, stimulation of pollination, and higher productivity—are due to LCO activity, similar to phytohormones such as naphthaleneacetic acid, auxin, and gibberellin [167]. The alteration of phytohormone balance caused by LCOs is also linked to improved photosynthetic rates and increased resistance to biotic and abiotic stresses [83,168].

LCOs also appear to induce the ‘priming effect’ in some plant species. Plants in a ‘primed state’ can respond more quickly and effectively to stressful environments. Lucas and colleagues observed the activating of the priming effect in tomato plants inoculated with LCOs and exposed to high levels of UV radiation [169]. The authors linked the positive results to the activation of non-enzymatic antioxidant systems. This includes the production of tocopherols, ascorbic acid, and glutathione promoted by LCOs. Inoculated plants showed lower levels of oxidative stress and, consequently, greater resistance to UV radiation [169].

The priming effect induced by LCOs also seems to enhance plant resistance to salinity. Nandhini and colleagues found that black gram (Vigna mungo L.) seeds inoculated with LCOs had higher germination rates in saline environments compared with non-inoculated seeds [170]. The authors attribute these benefits to increased cell division rates, improved oxygen uptake, enhanced alpha-amylase activity, and greater nutrient mobilization from the cotyledons to the embryonic axis due to LCO presence [170].

In soybean, the foliar application of LCOs has been reported as beneficial under water deficit conditions, significantly reducing production losses [171]. When applied to roots, LCOs appear to increase soybean resistance to the pathogen Microsphaera difusa [172] and improve root development in an acidic pH (below 4) [173].

From the opposite side of the LCO–plant biochemical interaction, some studies have shown that root exudates from non-leguminous plants can activate LCO synthesis genes in rhizobia present in the rhizosphere [174]. Although the reasons for this induction are not yet fully understood, the increase in LCOs in the rhizosphere can promote seed germination and plant growth in non-host species.

To date, many studies have described the benefits to crops of many other classes of MSMs produced by PGPB. Increases in seed germination rate [66], plant development [175], and grain yield [87,161,176]; increased resistance to pests [177,178], drought [179,180,181], and salinity [35,182]; reduced oxidative damage [157]; and production of phytohormones [182,183] are usually the most well-observed and documented effects. A list of some of these studies is shown in

Table 1. List of information on MSMs produced by PGPB and their benefits to crops of agricultural interest.

PGPB	MSM	Benefit	Culture	Reference	Readiness for Commercialization
Curtobacterium albidum	ACC-deaminase/Indole-3-acetic-acid	Salinity resistance	Rice (Oriza sativa)	[47]	No
Rhizobium tropici	LCOs	Increased productivity	Maize (Zea mays), Soybean (Glycine max)	[87,161]	Yes [184,185]
Azospirillum brasilense	Salicylic acid Abscisic acid	Salinity resistance	Maize	[14,182]	No
Rhizobium tropici; Bradyrhizobium diazoefficiens	LCOs	Increased grain quality and yield Increased root activity	Soybean	[157,176]	Yes [186,187]
Bacillus amyloliquefaciens	Auxins Abscisic acid Gibberellins	Salinity resistance	Rice	[188]	No
Serratia nematodiphila	Gibberellins	Cold resistance	Pepper (Capsicum annuum)	[183]	No
Azospirillum brasilense	Salicylic acid	Drought resistance	Arabidopsis thaliana	[189]	No
Bacillus pumillus; Bacillus subtillis	Indole-3-acetic-acid	Salinity resistance Drought resistance	Tomato (Solanum lycopersicum)	[190]	No
Bradyrhizobium japonicum	LCOs	Increased seed germination rate	Maize Rice Soybean Common bean (Phaseolus vulgaris)	[66]	Yes [186,187]
Streptomyces sp.	Fungal cell wall-degrading enzymes	Increased resistance to phytopathogenic fungi	Peanut (Arachis hypogaea)	[177]	Yes [191]
Arbuscular mycorrhizae	Strigolactones	Drought resistance	Tomato, Lettuce (Lactuca sativa)	[181]	No
Rhizobium tropici CIAT 899	LCOs	Increased productivity Plant growth promotion	Soybean	[160]	Yes [186,187]

.

The evidence presented in these studies demonstrates the significant potential of using MSMs, particularly LCOs, as powerful bio-inputs. The use of these molecules to prevent damage caused by abiotic stresses, which are often uncontrollable and unpredictable, offers a sustainable alternative. This approach promotes environmentally friendly agriculture practices.

Field Applications of MSMs

Although the beneficial effects of MSMs, especially those produced by PGPB, on agricultural plants have been reported for decades, most studies have focused on in vitro or greenhouse experiments. However, field studies have been conducted, particularly with LCOs. For example, Chen and colleagues observed that the foliar application of LCOs from B. japonicum significantly increased flowering and fruit production in field-grown tomato plants [167].

It is worth mentioning that the number of reports of successful applications of MSMs, combined or not with other molecules and microorganisms under field conditions, is increasing.

Marks and colleagues reported increased dry weight, total N concentration, and maize productivity in field experiments inoculated with A. brasilense and LCOs from R. tropici [161]. Similarly, Moretti and colleagues observed a significant reduction in oxidative damage in soybean plants inoculated with a consortium containing Bradyrhizobium spp., A. brasilense, and LCOs of R. tropici and B. diazoefficiens compared with plants inoculated only with Bradyrhizobium spp. over two cropping seasons [157]. In a related study with the same consortium, soybean plants showed increased yield and grain quality across three cropping seasons [176].

Under field conditions, the effectiveness of biological molecules depends not only on the MSMs, but also on various factors, such as timing and mode of application, climate, soil conditions, and plant status [192]. Therefore, while these studies suggest the potential benefits of MSMs in crops, more research focusing on the application under field conditions and with different crops is necessary. This will help to better define the role of MSMs in promoting plant growth, increasing the interest in their industrial-scale production.

4. Industrial Production of MSMs for Agriculture Use

4.1. First Steps

It is important to note that the industrial production of MSMs for use in agriculture is preceded by several laboratory and field validation stages (Figure 8). The duration of each of these steps varies enormously and the entire process can take years, which makes it the first bottleneck in the industrial production of MSMs [193].

The industrial production of MSMs requires a fermentation process, followed by the preparation of the crude extract containing the molecule(s) of interest, and, finally, their isolation and identification. The synthesis of MSMs begins during the idiophase (stationary phase), the fermentation period immediately following the trophophase (exponential phase). During the idiophase, there is a progressive accumulation of MSMs [27]. This accumulation is inversely proportional to the availability of essential growth nutrients (Figure 9), emphasizing that MSMs are produced under stress conditions [194].

Liquid (or submerged) fermentation and solid-state fermentation have been the most used bioprocesses for MSM production. The main difference between the techniques is the type of substrate used [112].

4.2. Liquid (Submerged) Fermentation

Submerged fermentation for MSM production—both batch and fed-batch—uses culture broths as substrates for microbial fermentation. Generally, this type of fermentation is employed when the metabolite(s) of interest are secreted into the medium, facilitating their subsequent extraction and recovery [112].

In general, the inoculum containing the microbial culture is cultivated in flasks in the laboratory and then scaled up to larger bioreactors in a controlled condition [20]. Several parameters are controlled during submerged fermentation: culture medium composition, pH, aeration, agitation, and temperature. Additionally, in this type of fermentation, it is possible to add compounds to the medium that are known to induce the synthesis of the metabolite of interest, and to restrict compounds that inhibit molecule production. Certain flavonoids, for example, are described as inducers of LCO synthesis in nitrogen-fixing bacteria [195,196].

One of the major advantages of liquid fermentation is the possibility of remote automation and control of all parameters, greatly facilitating MSM production on an industrial scale [27]. However, although many microorganisms have expressive growth rates in liquid fermentation, they are not capable of synthesizing significant amounts of MSMs during the process [27]. It is inferred that this occurs because the production of secondary metabolites is highly dependent on external signals present in their habitat. During submerged fermentation, these signals may disappear, greatly hindering the production of MSMs [197].

4.3. Solid-State Fermentation (SSF)

Solid-state fermentation (SSF) can be defined as a microbial culture that develops on the surface and inside a solid matrix in the absence of free water [20]. Depending on the goal to be achieved during the process, SSF can have one or two types of substrates [198].

The production of specialized biomass-degrading enzymes is broadly carried out by SSF [199,200]. Similarly, this type of fermentation is preferably used for the synthesis of MSMs by filamentous fungi [27,112]. In general, bacterial cultivation using this production process is not as productive as fungal and yeast cultivation [27], due to the greater demand for free water by most bacteria.

Despite this apparent disadvantage, bacterial fermentation through SSF has been reported in several studies. Shengping and colleagues optimized the production parameters of a biofertilizer from Bacillus circulans via SSF using food waste as the substrate [171]. Additional studies report similar success in SSF for other Bacillus species: B. amyloliquefaciens [201] and Bacilus sphaericus [202]. Similarly, Zhao and colleagues reported the production of Rhizobium leguminosarum via SSF using wheat (Triticum aestivum L.) straw and charcoal as substrates [203].

Solid-state fermentation offers advantages over submerged fermentation for MSM production, like lower energy consumption and higher production levels in a shorter time [126,203,204]. Additionally, sterile fermentation conditions and the use of sophisticated equipment are often unnecessary, purification procedures are relatively simple, and there is less waste and effluent generation [112,205]. Finally, one of the greatest advantages of SSF is the possibility to utilize industrial and agricultural residues as substrates [27], creating an efficient circular economy system.

Regardless of all the advantages described, the industrial production of MSMs by SSF also faces some important challenges. Significant changes in substrate composition, including chemical composition, particle size, water retention capacity, and surface area, directly affect the fermentation parameters [206]. Downstream processes are considered the most significant bottleneck of SSF. Separating the product of interest from the solid matrix is often technically and economically unfeasible. Typically, this type of industrial fermentation is used to produce secondary metabolites that do not require a high level of purity [203,204].

4.4. Challenges in the Industrial Production of MSMs

4.4.1. Upstream Processes

• Scalability

The scaling stage is often the first bottleneck encountered by industries. To achieve the rapid production of the target molecule, the inoculum must have a high density of microbial cells, which is not always feasible [27]. In some cases, this may require high-tech bench-scale bioreactors [207], which tend to be expensive.

For fungal MSM production in SSF systems, it is highly recommended that the inoculum consists of spores. Spores mix more easily with the substrate and have a higher viability over longer periods. However, producing spores takes longer and has a higher incidence of unwanted mutations [208].

Scaling processes are highly specific and vary depending on the microorganism and the MSM of interest. Currently, information on biomass generation parameters and optimization of industrial production scaling processes for each microorganism is still scarce [27].

2.

Production Levels

In natural environments, MSMs are often produced at very low concentrations [77]. This pattern tends to replicate and, in some cases, worsen under artificial cultivation conditions [85].

For industrial production to be viable, it is essential to optimize parameters such as temperature, pH, and nutrient availability, and to use inducers and/or repressors. Attempting to mimic natural environments is also crucial [86]. However, optimizing MSM production can be time-consuming, especially if the metabolite of interest and/or the producing microorganism are not well-known or studied.

It is crucial for the matrix used in SSF processes to be homogeneous. Material heterogeneity results in gradients of temperature and oxygen, leading to lower production levels [209]. Moreover, the design of bioreactors used in MSM production appears to play a critical role in achieving high production levels [210,211]. Despite this, little information currently links bioreactor design to high biomass and MSM production.

4.4.2. Downstream Processes

• Extraction and isolation

Choosing the ideal solvent for MSM extraction is a critical step in the production process.

In fermentations using solid matrices, the extraction stage is often quite rudimentary and tedious since the product of interest is distributed throughout the substrate and is difficult to recover [27].

In isolation processes, chromatographic techniques are commonly used in industry when a high degree of purification is required. However, this stage is a significant bottleneck in the industrial process, consisting of several steps that increase the product’s cost. For example, Kaaniche and colleagues isolated seven compounds synthesized by Streptomyces cavourensis through SSF using a complex chromatographic purification system. The process included silica gel column steps, followed by medium-pressure liquid chromatography using different elution gradients, resulting in four compounds, and a final chromatographic step using Sephadex LH20 to obtain the remaining three compounds [212].

The costs of extraction and isolation processes vary depending on the type of fermentation employed and the level of purity required. In liquid fermentations, purity levels are typically higher, as the separation of the target molecule is easier and faster. For this reason, this type of fermentation is mostly used for producing compounds in the food and pharmaceutical industries, which are regulated by stricter biosafety laws [213].

2.

Residue generation

Especially in industrial processes for MSM production, a significant bottleneck that arises at the end of the process is the generation of residues. If the final product is not the fermented substrate itself, the treatment costs for the generated waste can be high, significantly increasing the final product cost [214]. Depending on the regulations governing industrial activity and the type of waste generated, it may require disinfection and/or decontamination processes and proper disposal. These activities are typically handled by specialized industrial waste management companies. Generally, waste management costs are included in the final product price, making it less accessible and competitive [27].

4.5. Solutions and Alternatives

Considering the production bottlenecks, various solutions and alternatives have been discussed. Different stakeholders have proposed them for the evolution and development of industrial MSM production for agricultural use. Following, we highlight some important points.

4.5.1. Basic Research

To achieve satisfactory production levels, it is crucial to fill the knowledge gap regarding microorganisms and their interactions with the environment and other microorganisms. This includes characterizing molecular structures, clarifying metabolic pathways involved in the synthesis of the MSM of interest, and updating databases.

Elucidating the microbial processes for synthesizing compounds, identifying inducing and/or repressing molecules, and understanding the role of the microbiome in MSM synthesis are key issues for optimizing production and reducing costs [215]. In this context, collaboration between academia and industries is essential

4.5.2. Optimization of Parameters

High production levels of MSMs can be reached through the optimization of parameters [27].

In many cases, additional techniques are necessary. Notable among these are the co-cultivation of microorganisms and the OSMAC (one strain many compounds) approach.

Microbial co-cultivation (or mixed fermentation) involves cultivating two or more microorganisms together under controlled conditions. This technique can induce the expression of genes related to MSM synthesis, increasing production and allowing for the discovery of new molecules [216].

The coexistence of microbial species in the fermentative environment mimics a more competitive natural growth environment. This competitive pressure stimulates the emission of signaling molecules related to quorum sensing and auto-regulation, resulting in increased production of secondary metabolites [217].

Wakefield and collaborators applied the microbial co-cultivation technique in the fermentation of the fungus Aspergillus fumigatus with two strains of the actinobacterium Streptonyces leeuwenhoekii, resulting in significant increases in the production of the metabolite chaxapeptin and the discovery of secondary metabolites never before described for this fungal species [216].

The OSMAC approach involves modifying relatively simple cultivation parameters for a particular microorganism. Similar to co-cultivation, the OSMAC approach aims to mimic natural growth conditions of the microorganism, activating metabolic pathways for the synthesis of MSMs [218].

Paranagama modified the fermentation conditions of Paraphaeosphaeria quadriseptata, using tap water instead of distilled water to prepare the growth medium, leading to the discovery of six previously undescribed MSMs [219].

Moreover, the OSMAC approach can be used to induce stress in cultivation conditions, resulting in the increased production of MSMs. Thermal shock has been identified as an inducer of the production of the antibiotic validamycin [220]. In the same way, the limitation of alanine in the growth medium induced the production of methylenomycin by Streptomyces coelicolor [221].

4.5.3. Strain Improvement and the Use of Biofactories

Numerous studies have demonstrated that the use of genetically improved strains can yield MSM production levels up to ten times higher compared with wild-type strains [222,223].

Currently, classical techniques of non-directed mutation are still employed for strain improvement using physical and chemical methods. El Sayed and collaborators observed an increase in the production of cobalt ferric (an antimicrobial and antitumoral agent) nanoparticles by the gamma radiation-mutated fungus Monascus purpureus [223].

In actinobacteria, methods such as chemical mutagenesis using N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), or nitrous acid (NA) and physical mutagenesis (UV radiation or X-rays) are widely used to obtain highly efficient Streptomyces spp. strains for secondary metabolite production [224].

The main advantage of using physico-chemical methods for strain improvement is that they do not require an in-depth knowledge of all factors involved in the process. This lack of requirement accelerates the development of simple reproducible protocols [224].

However, in some cases, neither parameter optimization nor traditional mutagenesis strategies are sufficient to enhance MSM production [225]. In these situations, advanced molecular biology techniques prove to be powerful tools.

Currently, there are various ways to increase the production levels of a molecule of interest through the manipulation of its genetic material. It is possible to replace promoters or groups of promoters involved in the synthesis of the molecule [226], to overexpress genes that code for transport proteins to the extracellular environment [226], and to modify transcription regulatory genes [227].

Other techniques used in strain improvement include optimizing the synthesis of enzymes involved in the synthesis of the molecule of interest, manipulating regulatory genes (both positive and negative), modifying transcriptional and translational machinery, and deleting competing genes [145].

Depending on the goal to be achieved, structural modifications in the molecule of interest may also be performed [225], like the combinatorial biosynthesis technique. It alters the biosynthetic pathway of a specific molecule to obtain new products, and it may also accelerate the evolution of metabolic pathways for synthesizing target compounds [228].

In addition, the use of biofactories for producing molecules of interest is becoming increasingly popular. The biofactory concept involves introducing genes that code for biosynthetic pathways of a particular metabolite into ‘host microorganisms’ to increase the levels of production of the molecule [145].

One major advantage of this technique is the ability to produce molecules that are normally synthesized in low quantities or in non-cultivable microorganisms [229]. The host microorganisms are generally easier to manipulate, grow quickly under laboratory conditions, allow for efficient production optimization, and are genetically more stable than the producing microorganisms. Commonly used host organisms as biofactories include Escherichia coli, Pseudomonas putida, B. subtilis, and Streptomyces spp. [230].

4.5.4. Design of Efficient Bioreactors for Solid-State Fermentation

The design and technology of the bioreactors used to produce MSMs can significantly influence the industrial and commercial viability of the target molecule.

Solid-state fermentations present characteristics that are challenging to control and adjust, which can affect MSM production. Heat dissipation, moisture and oxygen distribution in the substrate, and biomass transfer are some examples [27].

However, considerable effort has been dedicated to designing efficient bioreactors for this type of fermentation [209]. Examples of more advanced bioreactors successfully used in SSF include rolling bed fermenters [210], continuous stirred tank reactors [231], and plug flow reactors [232].

4.6. Circular Economy Applied to Industrial MSM Production

According to Intergovernmental Panel on Climate Change (IPCC) data, the agricultural sector is responsible for 24% of global greenhouse gas (GHG) emissions [233]. Paradoxically, the same agricultural sector is one of the most affected by the direct consequences of climate change.

Periods of extreme drought, floods, increasing average annual temperatures, desertification processes, increased soil salinity in coastal areas, population explosions of crop pests, and massive losses of soil biodiversity are among the most significant (but not the only) consequences of climate change that directly affect agricultural production [234].

According to data published in 2016 by the Food and Agriculture Organization (FAO), if current GHG emission rates do not decrease, a significant decline in the production of major cereal crops will be observed by 2100, estimated at 20–45% in maize (Zea mays L.), 5–50% in wheat, and 20–30% in rice (Oryza sativa L.) crops [2].

Taking this reality into account, the concept of a circular economy applied to industrial systems is a promising alternative. It aims to mitigate and optimize processes and production. This approach has gained prominence in recent years, particularly in China and European countries [235].

The goal of the circular economy is to minimize or eliminate the use of non-renewable production sources and maximize the reuse of raw materials, products, and by-products within the same system [236].

In industries and factories, the concepts of circular economy are being established through regulations and legislation, such as UNE-EM ISO 14006:2011. This international standard provides guidelines for incorporating ecodesign into environmental management systems. In practice, the focus is on the product life cycle, waste management, and calculating the environmental impact generated by its use and/or application [184].

The application of circular economy principles in agriculture and crop management is already a reality in many areas. The use of bio-inputs containing bacteria and their metabolites represents an economically viable and, primarily, environmentally attractive option [185]. Their production can greatly benefit from these principles.

In the production of MSMs, the circular economy can be highly beneficial and sustainable. The use of bagasse and crop residues as solid fermentation substrates is an example of process circularization. In this case, the waste from one process or production chain becomes the raw material for the development of others. Pinheiro and colleagues utilized waste from rice processing and beer fabrication for the production of gibberellins by Gibberella fujikuroi through SSF [236]. Similarly, Slivinski and colleagues achieved high levels of surfactin production, a biosurfactant, by using sugarcane bagasse and SSF with Bacillus pumilus [237]. The production of surfactin through SSF using waste and bagasse has also been reported in two other Bacillus species: B. amyloliquefaciens, fermented on rice straw [238], and B. subtilis, fermented on agricultural and food waste [239].

Also, the circular economy can be applied more broadly than just using waste as fermentation substrates. The residue generated at the end of the process can be transformed into compost and used as a biofertilizer, closing the cycle (Figure 10).

Composting can convert waste into a resource. Its impact is so significant that it is considered one of the strategic trends in combating climate change. This is due to its ability to reduce greenhouse gas emissions and sequester CO₂ [185].

Using compost as a biofertilizer adds organic matter and humic and fulvic acids to the soil. This enhances soil fertility and diversity. Furthermore, its application decreases the need for chemical inputs, promotes plant growth, and increases plant resistance to biotic and abiotic stresses [240].

These initiatives promote better resource utilization, reduced waste generation, increased energy efficiency, and savings in natural and financial resources [5], along with assisting in soil fertility preservation and biodiversity enhancement [241]. Additionally, from a commercial and industrial perspective, applying circular economy concepts to bio-input production leads to energy and financial savings. This increases the feasibility of manufacturing and commercialization.

4.7. Industrial Production of Agricultural Interest MSMs: Challenges and Future Perspectives

To date, thousands of MSMs with potential benefits for human, animal, and plant health and nutrition have been identified. For example, the genus Streptomyces spp., commonly found in soils, had 7600 secondary metabolite molecules identified by 2005 [242]. Additionally, computationally predicted estimates suggest that the genus could synthesize up to 150,000 distinct antimicrobial agents [243].

Due to their immense variety and biological activity, secondary metabolites emerge as a possible tool for mitigating the impacts of climate change on agriculture by increasing plant resistance to abiotic and biotic stress, and by improving agriculture productivity with higher sustainability. Although the total viability of using these compounds on an industrial scale is not yet clear [19], there are already registered commercial products. For example, Signum [186] and NodPro [187], both contain LCOs, and other fermentation products of agrochemical importance, such as abamectin, milbemectin, and spinosad [244].

The large-scale manufacture of MSMs still faces some production challenges. Issues such as low production [8,218], lack of definition of suitable and financially viable production parameters [19,205], and difficulties in isolating and purifying these molecules [19,203,245] are among the main bottlenecks in the production of these compounds.

For these reasons, MSMs are still less commercially explored by industries than molecules synthesized by plants, which already have better known and more structured production and identification technologies [246]. However, it is worth mentioning that some authors argue that industrial-level MSM production has advantages compared with plant metabolite production. Among the advantages are the faster and relatively simple growth and downstream processes, which would make the manufacturing of these compounds more economically advantageous [19,27].

Within this context, the partnership between academia and industries is mandatory. Developing microbial strains more adapted to the industrial environment and optimizing production and isolation processes to each species are priority areas of focus. Advanced techniques, such as genetic editing of strains, cloning and expression of metagenomic DNA from soil and rhizosphere, and genomic mining are gaining ground and becoming possibilities for increasing production. The commercial product RETURN^®, from the company Pivot Bio, is based on a genetically modified strain of Klebsiella variicola. This mutant strain has its biological nitrogen fixation capacity constantly activated, increasing the availability of this nutrient to the associated crops [247]. However, we must consider that biological safety is a major concern when defining strains, as horizontal gene transfer is frequently reported in soils, majorly in the tropics. Additionally, further research in technology and engineering and the application of circular economy concepts are essential for increasing the viability of the industrial-scale production of agricultural interest MSMs.

5. Concluding Remarks About the Greatest Challenge in Agriculture in the Next Decade

The adverse effects of climate change on crops are becoming increasingly evident. and the following consequences are clear: progressive decreases in cultivable areas and an increase in food insecurity on all continents. This will probably be the major factor limiting agriculture in the next years, but there are certainly other limitations.

Faced with this worrying scenario, it is more than necessary to seek alternatives that mitigate or, at least, slow down the impacts of abiotic and biotic stresses on agriculture. According to the information presented in this review, microbial secondary metabolites are emerging as a potential solution. They offer alternatives for developing more sustainable agricultural techniques and may help to mitigate the effects of climatic changes.

However, despite the scientifically well-supported results of benefits of applying MSMs in agriculture, there is still a long road ahead. Production challenges and bottlenecks remain evident, requiring attention and technological development.

Bio-inputs containing MSMs are thus fundamental for implementing more sustainable and economically efficient agricultural practices. The use of these products transcends geopolitical boundaries and is key in achieving and maintaining food security for both current and future generations. In addition to promoting increases in production and reducing dependence on agrochemicals, their role in advancing circular economy cycles solidifies them as essential components in the agricultural landscape.

For all these reasons, despite the challenges, it is expected that, with increased research, government support, and financial incentives, the number of products containing agriculturally relevant MSMs will grow significantly in the coming years, contributing to the establishment of more sustainable agricultural systems.