Applied Microbiology for Sustainable Agricultural Development

Abstract

Background: Developments in biology, genetics, soil science, plant breeding, engineering, and agricultural microbiology are driving advances in soil microbiology and microbial biotechnology. Material and methods: The literature for this review was collected by searching leading scientific databases such as Embase, Medline/PubMed, Scopus, and Web of Science. Results: Recent advances in soil microbiology and biotechnology are discussed, emphasizing the role of microorganisms in sustainable agriculture. It has been shown that soil and plant microbiomes significantly contribute to improving soil fertility and plant and soil health. Microbes promote plant growth through various mechanisms, including potassium, phosphorus, and zinc solubilization, biological nitrogen fixation, production of ammonia, HCN, siderophores, and other secondary metabolites with antagonistic effects. The diversity of microbiomes related to crops, plant protection, and the environment is analyzed, as well as their role in improving food quality, especially under stress conditions. Particular attention was paid to the diversity of microbiomes and their mechanisms supporting plant growth and soil fertility. Conclusions: The key role of soil microorganisms in sustainable agriculture was highlighted. They can support the production of natural substances used as plant protection products, as well as biopesticides, bioregulators, or biofertilizers. Microbial biotechnology also offers potential in the production of sustainable chemicals, such as biofuels or biodegradable plastics (PHA) from plant sugars, and in the production of pharmaceuticals, including antibiotics, hormones, or enzymes.

1. Introduction

Microbial biotechnology, focusing on microorganisms, plant extracts, and minerals, is crucial for sustainable development by reducing reliance on harmful chemicals and improving environmental health. It offers solutions like green chemistry, biopesticides, bioherbicides, bioinsecticides, and effective microorganisms (EMs) for sustainable agriculture. However, ethical concerns exist, including the release of GMOs, and increased resistance in weeds, diseases, and pests, highlighting the need for further research into their potential and limitations [1,2].

Sustainable agriculture employs various farming systems to minimize environmental impact, adhering to economic, social, and environmental pillars. Common systems include:

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Perennial crops: Reduce energy, water, and fertilizer use, lowering greenhouse gas emissions [1,2].

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Mixed cultivation: Increases biodiversity and reduces crop loss risk [1,2].

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No-tillage: Improves water retention, reduces soil erosion, and increases soil organic matter.

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Agroforestry: Boosts biodiversity and positively impacts water, climate, and ecological balance [3].

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Precision farming: Optimizes resource use (water, energy, fertilizers, plant protection products) through accurate monitoring [4].

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Organic farming: Minimizes environmental impact using natural methods like composting, green manures, and biological pest control [1,2,3,4].

Alongside these, technologies like farming automation and robotics enhance productivity and reduce costs [5,6], and algae to enhance plant productivity and health while protecting the environment. Key applications include:

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Biostimulants: Microorganism-based substances that stimulate plant growth and resistance by affecting metabolism [6,7,8].

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Biofertilizers: Live microorganisms that improve nutrient uptake, soil quality, and crop yield [3,6,7,8].

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Biopesticides: Microorganisms (bacteria, viruses, fungi) that control pests and diseases, reducing the need for chemical pesticides [7,8,9].

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Mycorrhiza: A symbiotic relationship where fungi provide plants with nutrients and plants supply carbohydrates to the fungi [5,6,7,8,9].

These systems offer benefits like increased yields, enhanced plant resistance, reduced environmental impact, and lower production costs. However, risks such as potential health impacts on humans and animals, environmental pollution, food contamination, lack of effectiveness, undesirable interactions, and the development of microbial resistance necessitate careful application according to best practices and recommendations [3,6,7,8,9,10,11,12,13,14,15,16,17].

Soil microorganisms are vital for sustainable agriculture, participating in pathogen reduction and nutrient transformation [1,4,7]. They constitute a significant portion of environmental biomass and the carbon cycle [1,6]. The soil is a rich reservoir of diverse microorganisms, leading to efforts in developing microbiological preparations for soil fertility [1,5]. These biopreparations, often containing symbiotic microorganisms, improve plant growth and yield [1,6].

Microorganisms play a pivotal role in agriculture, horticulture, and animal production such as in biofertilizers, bioherbicides, bioinsecticides, and growth bioregulators [7,8,9,10,11,12,13,14]. Recent research also explores combining EM applications with herbal extracts [6,7,8,9,10,11,12,13,14]. Continued research in agricultural microbiology is crucial to understanding microbial interactions with soil, plants, and the environment, unlocking their full potential for increased yields, enhanced plant resistance, improved nutritional value, and, ultimately, food security and sustainable development [2,3,5,6].

Hence, the aim of this study is to highlight the key role of microorganisms in the natural environment, with particular emphasis on their use in agriculture, horticulture, and animal production in the form of biofertilizers, bioherbicides, bioinsecticides, and growth bioregulators and in denitrification processes [4,7,8,9,10,11,12,13,14]. This review aims to synthetically present the current achievements of microbiology in the development and use of biopreparations in sustainable agriculture, such as biostimulants, biopesticides, and biofungicides, and effective microorganisms and other microbiological organisms [1,2,3,4,5,6]. Additionally, the manuscript indicates new directions of research in agricultural microbiology, focusing on increasing plant resistance to diseases, improving the biodiversity of microbial species contributing to crop growth, as well as the impact on plant health and their nutritional and energy value, which is crucial for food security and sustainable development [2,4,5,6].

This paper puts forward an alternative hypothesis to the null hypothesis:

H0. 

Assumes no significant effect of microorganisms on the efficiency and sustainability of agricultural production.

H1. 

Alternative hypothesis, according to which the targeted use of specific groups of microorganisms (e.g., bacteria, fungi) in biopreparations (biofertilizers, biopesticides, biostimulants) significantly improves plant health, increases yields, reduces dependence on chemical plant protection products and artificial fertilizers, and thus contributes to the sustainable development of agriculture, minimizing the negative impact on the environment and human health [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16].

2. Materials and Methods

Articles used in this literature review were retrieved from PubMed/Medline, Scopus, Embase, Web of Science, Google Scholar, and ScienceDirect. No restrictions were applied to the year of publication or language in order to cover the widest possible range of literature. A total of 165 publications were included. The search was conducted using relevant keywords such as “fermentation,” “bioactive compounds,” “health benefits,” and the names of specific plants and microorganisms. Articles were selected based on the criteria of (1) relevance to the review topic, (2) publication in peer-reviewed journals, and (3) methodological soundness and substantive contribution. Preference was given to newer studies, while older key papers were also included. Titles and abstracts were reviewed independently by the authors; full texts were assessed in detail and any discrepancies were resolved through discussion. The review provides a comprehensive summary of current knowledge on the use of microorganisms and biotechnology in agricultural science and practice.

3. Review

3.1. Agricultural Microbiome

The soil microbiome is a complex community of microorganisms, including bacteria, fungi, viruses, protozoa, and other single-celled organisms that inhabit the soil layers. The soil microbiome plays a key role in soil processes such as mineralization, degradation of organic matter, nitration, phosphorylation, and others. The microbes in the soil are also important for maintaining plant health as they aid in the process of nitrogen transformation and nutrient absorption. The soil microbiome is very diverse and can vary greatly depending on soil type, climate, geographic location, and other abiotic and biotic factors. The different microbial species in the soil are also linked to different soil processes, meaning that the soil microbiome has a significant impact on the functioning of ecosystems and on agricultural productivity [1].

Changes in soil cultivation methods, related to the introduction of modern agriculture, chemicalization, and intensive fertilization, have contributed to significant changes in the soil microbiome. The introduction of modern agricultural technologies, modern, high-performance machines, which, however, destroy the soil structure, mineral fertilizers, pesticides, and growth regulators caused a change in the composition of soil microorganisms. In particular, the use of mineral fertilizers and chemical pesticides can lead to a reduction in the number of soil microorganisms, especially saprotrophic bacteria and fungi, which are responsible for the decomposition processes and processing of organic matter in the soil [2].

Compared to intensively fertilized and chemically treated soils, poorly fertilized or organic soils are usually more diverse in terms of microorganisms. Microorganisms such as nitrogen bacteria and organic-matter-processing microorganisms predominate in such soils and contribute to soil fertility by converting organic matter into plant nutrients. The introduction of modern agricultural technologies may lead to some negative effects on the soil microbiome; at the same time, they may contribute to increasing the efficiency of agricultural production. It is important to find a balance between increasing the efficiency of agricultural production and protecting the soil microbiome and ensuring sustainable agricultural production in the long term. The introduction of modern, intensive agriculture, chemicalization, and intensive fertilization can significantly change the soil microbiome. Although these activities are aimed at increasing plant production, they can lead to disturbances in the microbial balance in the soil [1,2].

The use of chemicals in agriculture, especially the use of pesticides, plant growth regulators, and artificial mineral fertilizers, may lead to a decrease in the diversity and number of microorganisms in the soil. For example, pesticides can kill beneficial microorganisms, such as nitrogen bacteria, which take nitrogen from the air and convert it into nutrients. Intensive cultivation can lead to changes in the soil microbiome. Reducing plant diversity in farmland can also lead to bacterial poverty, as different plant species differ in ways that are beneficial to different microorganisms. In addition, changing the cultivation method can also affect the composition of the microorganisms, as different plant species support different populations of microorganisms. Climate change also affects the interactions between microorganisms and between microorganisms and plants in direct and indirect ways. Consequently, they have an impact on soil ecosystem functions such as carbon storage, productivity, and fertility. Climate change may also affect the species composition of the microbiome inhabiting plant roots, as well as the activity of mycorrhizal fungi and rhizosphere bacteria (plant-growth-promoting rhizobacteria (PGPR)). It causes numerical and qualitative changes in microbial populations. The processes affecting the biological activity and fertility of soils may have affected intensification or inhibition [2,15,16].

Compared to intensively fertilized and chemical-treated soils, weakly fertilized and organic soils usually have a higher number and greater variety of microorganisms. These soils contain more organic matter, which promotes the development of soil microorganisms. As the content of organic matter in the soil increases, the variety of microorganisms increases, which affects plant efficiency and general soil quality. The introduction of modern agriculture, chemigations, and intensive fertilization leads to changes in the soil microbiome, which are unfavorable for plant performance and general soil health. Compared to poorly fertilized and organic soils, intensively fertilized and chemical-treated soils usually have a smaller number and variety of microorganisms, which can lead to a reduction in quality and efficiency of crops [3,4].

The variety of soil microbiomes in arable soils around the world is very diverse and depends on many factors, such as climate, soil type, crop plants, and cultivation practices, for example: in Europe, soil microbiomes are strongly associated with the history of crops and types of crops. In regions with a long cultural history, soils usually have a lower variety of microorganisms. For example, in the regions of cereal cultivation in Central and Eastern Europe, soils usually have a lower number and variety of microorganisms compared to soils in vegetable cultivation regions. Asia has various soil environments, which leads to the differentiation of soil microbiomes in different regions. For example, in Southeast Asia, where rice is grown, soil usually has a high number and variety of nitrification bacteria, which are crucial for the nitrogen cycle in soil. In China, soil in tea-growing areas usually has a higher variety of fungi than soil in other regions. Africa usually has low organic matter and pH soils, which affects the composition of the soil microbiome. In the regions of wheat and corn cultivation, soil usually has a lower variety of nitrification bacteria compared to soils in vegetable cultivation regions. In North and South America, soils in vegetable cultivation regions usually have a higher number and variety of nitrification bacteria and other microorganisms compared to soils in cereal cultivation regions [3,4,5,6,7,12,13,14,15,16]. To sum up, in brief, the variety of soil microbiomes in arable soils is very diverse around the world and depends on many factors. Looking at the diversity of microorganisms in arable soils in different continents, countries, and regions allows for a better understanding of the complexity of interaction between soil, plants, and microorganisms, which is crucial to increasing the efficiency of crops and soil protection.

3.2. Sustainable Green Chemistry

The term “green chemistry” was used for the first time first used by P.T. Anastas in 1991 in the US Environmental Protection Agency “green chemistry program.” It is a discipline that aims to design and develop chemical products and processes that reduce or completely eliminate the use of hazardous substances and the generation of waste. The approach to “green chemistry” is also known in the literature under other terms: “environmentally benign chemistry”—chemistry that is gentle to the environment, “clean chemistry,” “atom economy”—economy of atoms, “benign by design chemistry”—gentle chemistry by design [6,7]. Green chemistry is defined as environmentally friendly chemical synthesis or as alternative methods of chemical synthesis, preventing environmental contamination, and its methodological distinctiveness, unlike traditional modern chemistry, in the search (design), development, and implementation of new technologies and chemical materials, considering the consequences related to the use of materials [7,15,16,17,18,19,20,21,22,23,24,25,26].

Microbial biotechnology can be used to produce sustainable chemicals for a variety of purposes, such as pest control and fertilization, by utilizing microorganisms that naturally produce these chemicals or by genetically modifying microorganisms to produce specific chemicals [6].

One example of the use of microbial biotechnology in sustainable green chemistry is the production of biopesticides, which are pest control products made from natural materials such as microorganisms, plant extracts, and minerals. They are generally considered safer and more environmentally friendly than synthetic pesticides because they are often biodegradable and have less toxicity to non-target organisms. Microbial biotechnology can be used to produce fertilizers and biopesticides by using microorganisms that produce toxins that are harmful to pests, such as Bacillus thuringiensis (Bt), or by genetically modifying the microorganisms to produce specific toxins [12,16]. Bacillus sp., especially B. thuringiensis, are widely used on the biopesticide market around the world due to their ability to effectively combat pests in economically important crops [16]. Traditional fertilizers are often derived from non-renewable resources such as fossil fuels. Both their production and use may have a negative impact on the natural environment. Microbial biotechnology can be used to produce fertilizers from renewable resources such as plant residues and animal manure by using microorganisms that convert these materials into nutrients that can be taken up by plants. As an example, nitrogen-fixing bacteria, such as rhizobia, can be mentioned, which can change atmospheric nitrogen into a form that is easily absorbed by crop plants, or phosphorus-dissolving bacteria, which increase the availability of insoluble forms of phosphorus for plants [6,7].

Green technology has a significant, beneficial effect on increasing the efficiency of high-end equipment manufacturing (HEM) in medical use. Microbial biotechnology can also be used to produce other sustainable chemicals such as biofuels, biodegradable plastics, and polyhydroxy alkenoates (PHAs) by fermenting plant sugars or other organic materials. And finally, microorganisms can be used to produce various pharmaceuticals such as antibiotics, hormones, and enzymes through fermentation processes [12,13,14,15].

Microbial biotechnology in sustainable green chemistry can bring numerous benefits, such as reducing our dependence on non-renewable resources, reducing greenhouse gas emissions, and improving the sustainability of the production and use of chemicals. However, there are also various challenges and ethical issues to consider, such as the potential for GMOs to be released into the environment. Therefore, further research is needed to better explain the strengths and weaknesses of this technology and to develop safer ways to use it [6,7].

3.3. Biofertilizers

The problems of hidden hunger in the world can be solved with the help of new farming systems and methods, and one of them is biofortification and the use of biofertilizers in food crops [27,28,29,30,31,32]. The classification of biofertilizers based on their functions and nature is shown in Figure 1.

Column 1 shows microorganisms capable of converting atmospheric nitrogen (N2) into forms available to plants (e.g., ammonia, ammonium salts) via a reduction process. For example: free-living Azotobacter spp. (e.g., A. chroococcum), Clostridium spp.; symbiotic: Rhizobium spp. (for legumes), Bradyrhizobium spp. (for soybeans), Frankia spp. (for actinorhizal plants, e.g., alder). Associative symbiotic microorganisms include Azospirillum spp. (for grasses and cereals, inhabit the rhizosphere), Gluconacetobacter diazotrophicus (endophytic for sugar cane). Key mechanisms include activity of nitrogenase enzyme, formation of nitrogen receptors (e.g., root nodules), and production of phytohormones promoting root growth and colonization (Figure 1).

Column 2 shows phosphorus-solubilizing microorganisms capable of transforming hard-to-reach, bound forms of phosphorus (e.g., calcium, iron, aluminum phosphates) into soluble and easily assimilable forms for plants. For example, bacteria: Bacillus spp. (e.g., B. subtilis, B. megaterium, B. circulans), Pseudomonas spp. (e.g., P. putida), Enterobacter spp., Burkholderia spp.; fungi: Aspergillus spp. (e.g., A. awamori, A. niger), Penicillium spp. (e.g., P. bilaii). Key mechanisms include the production of organic acids (e.g., citric, oxalic, gluconic) that lower pH in the rhizosphere and secretion of phosphatase enzymes (e.g., phytase, acid/base phosphatase) that hydrolyze organic phosphorus compounds (Figure 1).

Column 3 shows phosphorus-mobilizing (symbiotic) microorganisms that form symbiotic relationships with plant roots to increase the range of soil exploration and the efficiency of the uptake of phosphorus (and other nutrients) from a larger volume of soil. Examples: arbuscular mycorrhizal fungi (AMF): Glomus spp. (e.g., G. intraradices), Rhizophagus spp. (formerly Glomus), Gigaspora spp., Acaulospora spp. (occupying 80% of crop plants); ectomycorrhiza: Laccaria spp., Paxillus spp., Amanita spp., Boletus spp. (typical for forest trees) (Figure 1); ericoid mycorrhiza: Pezizella spp., Hyaloscypha spp. (for ericaceous plants, e.g., blueberries, rhododendrons); orchid mycorrhiza: Rhizoctonia solani, Tulasnella spp. (essential for orchid germination and growth). Key mechanisms include formation of an extensive network of mycelial hyphae outside the root depletion zone, increased surface area for water and nutrient absorption, production of enzymes that degrade organic sources of phosphorus, and modulation of plant physiology to increase stress tolerance (Figure 1).

Column 4 shows micronutrients and other functions, including microorganisms that improve the availability of micronutrients (Fe, Zn, Cu, Mn) and perform other beneficial functions for plants and soil. Examples: silicate dissolvers: bacteria (e.g., Bacillus spp., Pseudomonas spp.) and fungi (e.g., Aspergillus spp.) that break down silicate minerals, releasing silicon, potassium, and other elements; siderophore-producing microorganisms: many bacteria (e.g., Pseudomonas spp., Bacillus spp., Azotobacter spp.) that produce iron-chelating compounds, facilitating its uptake by plants under conditions of deficiency; oxidizing/reducing microorganisms that change the oxidation state of elements (e.g., Mn, Fe), affecting their solubility. Key mechanisms involve the secretion of organic acids (similar to phosphorus), production of siderophores (highly specific iron chelates), increasing cation exchange in the rhizosphere, and improving soil structure, which affects the availability of microelements (Figure 1).

Column 5 illustrates plant-growth-promoting microorganisms that stimulate plant growth and development through direct or indirect mechanisms, independent of primary N fixation or P mobilization. Examples are plant-growth-promoting rhizobacteria (PGPR): Pseudomonas spp. (e.g., P. fluorescens, P. putida), Bacillus spp. (e.g., B. amyloliquefaciens), Azotobacter spp., Azospirillum spp., Burkholderia spp., as well as endophytic fungi, including many types of non-symbiotic fungi that live inside plants.

Their key mechanisms are production of phytohormones: indole-3-acetic acid (IAA—auxins), gibberellins, cytokinins—stimulating cell division and root and shoot growth, as well as production of ACC deaminase enzyme, which reduces ethylene levels in the plant, relieving stress.

Pathogen biocontrol involves antagonism towards pathogens through the production of antibiotics, siderophores, lytic enzymes, or competition for space/nutrients (induction of systemic resistance—ISR) with improved resistance to abiotic stress (drought, salinity, heavy metals) through various mechanisms (e.g., osmolytes, antioxidants) (Figure 1).

Preferred mechanisms for improving plant growth include increased nutrient availability (i.e., N, P, K, Mg, Zn, and S), phytohormonal modulation, biological management of phytopathogens, and stress mitigation (e.g., drought stress, salt stress) [33,34].

Biofertilizers are substances containing a variety of microorganisms that have a high ability to absorb nutrients in association with plants due to the colonization of the rhizosphere, and thanks to this, they are easily available to plant root hairs. They are a good alternative to synthetic, chemical fertilizers. There are different types of microbial biofertilizers, including symbiotic and free-living nitrogen fixers, solubilizers, and phosphorus mobilizers. Their formulas and applications and the latest approaches to the development of next-generation biofertilizers are extremely important for sustainable agriculture [32,33,34]. In the future, it is expected that biofertilizers will contribute to a significant reduction in the application of both chemical fertilizers and pesticides. According to Mathur et al. [34,35] inoculation of crop plant seeds with an effective plant–arbuscular mycorrhizal fungi (AMF) vaccine or spores of obscure mycorrhizal fungi is an effective way to increase the symbiosis of plants with AMF in the soil [35,36]. The AMF symbiosis enhances the transport of water and nutrients from the soil to the host plants, which then allows the transit of carbohydrates from the photosynthesis pathway to an arbuscular mycorrhizal fungus [36,37].

Phosphate biofertilizers are one of the most important groups of beneficial microorganisms, which play an important role in the preparation of nutrients for crops. They are recognized as phosphate suppliers for various cropping systems. They can also provide other cropping systems with macro- and micronutrients. Both fungi and bacteria form the main groups of phosphate biofertilizers. They can live freely and independently or as symbiotic organisms in cultivated soils. Mycorrhiza is the so-called symbiont fungus that increases the uptake of phosphates, nitrogen, and micronutrients by plants. In addition, it improves the soil structure by creating a very extensive, dense network of mycelium connected to the roots of plants. Phosphate-dissolving microorganisms, on the other hand, are usually free-living, capable of dissolving insoluble phosphate compounds in the soil by releasing organic acids as well as chelating metabolites. The effectiveness of these microorganisms is significantly influenced by abiotic factors and farmland management practices. Tillage has a negative impact on the activity of mycorrhizal fungi. Also, the application of synthetic fertilizers reduces the survival and effectiveness of phosphate biofertilizers [31,37,38].

Plant extracts or metabolites are absorbed by weed seeds, which first initiates cell membrane damage, DNA damage, and mitosis, increases amylase activity and other biochemical processes, and delays or inhibits seed germination. The production of reactive oxygen species (ROS) and stress-related hormones increases, including the effects of antioxidants. Enzymes and toxic substances secreted by microbes degrade the seed coat of weeds and they use the endosperm for survival, which inhibits seed germination. Microbes grow through the intercellular spaces, reaching the root core, and the toxins deposited in the cells affect cell division and their functions. Metabolites of harmful microorganisms cause disease, necrosis, and chlorosis, which in turn contribute to inhibition of germination and growth of weed seeds by inhibiting photosynthesis and gibberellin activity and enhancing ROS, abscisic acid, and ethylene (Figure 2).

Figure 2 shows a comprehensive approach to weed control that combines the use of natural chemicals, microbial bioherbicides, and potentially modified chemical derivatives (such as organophosphates). The key mechanism of microbial bioherbicides is the use of microorganisms (or their metabolic products) to directly control weeds, potentially through the production of herbicides such as organophosphates. This points to future strategies that integrate biological control methods (microorganisms) with natural chemicals, offering an alternative to synthetic herbicides and supporting the concept of sustainable agriculture.

3.4. Development of EM and Biofertilizer Formulation

The development of an effective microorganism (EM) formulation that ensures good survival for all strains in a consortium is a challenge due to the different optimal conditions for individual species [31,39,40,41,42]. Recent research focuses on microencapsulation and prebiotics to improve the stability and synergism of strains in complex EM formulations, enabling their better survival under variable environmental conditions and ensuring long-term efficacy in the field [43].

Rhizobium, a bacterium widely used in the production of biofertilizers, is an example of the most environmentally friendly options [29]. Biofertilizers, including those based on fungi, contribute to the reduction of plant diseases by inhibiting the development of infections and limiting the biological functions of pathogens [31,39,40,41,42]. These fungi are seen as key contributors to biological life in the soil. Nitrogen fixation strategies, using both bacteria and fungi, focus on increasing plant nutrient utilization and reducing the dependence on synthetic inorganic fertilizers [31,39,40,41,42]. Particular attention has been paid to endophytic bacteria and fungi that fix nitrogen within plant tissues, which may open new possibilities for non-legumes [44].

Biofertilizers increase soil and plant nutrient content through a number of processes, including nitrogen fixation, phosphorus solubilization, and plant growth biostimulation [28,29,30,31]. Recent studies have shown that arbuscular mycorrhizal fungi (AMF) can reduce the structural and functional damage in the PSII and PSI photosystems induced by drought stress. For example, the colonization of D. moldavica roots by AMF promotes the growth of plants of this species by activating the antioxidant system and reducing the production of reactive oxygen species (ROS) [42,45,46,47,48]. Another novelty is the use of mycorrhizal fungi in combination with other biopreparations, which enhances the synergy and complex resistance of plants to many abiotic and biotic stresses [49]. Microbes associated especially with plants from the Fabaceae family (legumes) have great potential in solving environmental problems. Rhizobium, a bacterium used to produce biofertilizers, lives in symbiosis with the roots of legumes in structures called root nodules. These nodules enable the assimilation of atmospheric nitrogen in the form of ammonium, available to plants [30]. Rhizobia are common in soils and “attracted” by lectins secreted by the roots of legumes and penetrate the hair cells via an infection thread. Infection of the adjacent epidermal cells of the root occurs, growth hormone is secreted by the plant, and finally a root nodule is formed. During the period of intensive atmospheric nitrogen fixation (the flowering phase of the plant), the nodule takes colors from light pink to red, while white or brown colors indicate the cessation of this process. A protein called leghemoglobin, biochemically similar to animal hemoglobin, is responsible for the pink or red color of the nodules, as well as for maintaining appropriate oxygen conditions inside the nodule. Legume nodules differ anatomically and physiologically, and each plant species is usually capable of symbiosis only with the appropriate strain of bacterium. For example, beans form symbiosis with the bacterium Rhizobium leguminosarum by phaseoli, clover with Rhizobium leguminosarum by trifolin, etc. Some wild Rhizobium strains can coexist with many legume species and then have the ability to cross-infect plants. Soybean, on the other hand, has the ability to coexist only with the bacterium Bradrhizobium japonicum, which does not naturally occur in Europe. Therefore, it is necessary to inoculate it with a vaccine with this bacterium [31,39,40,41,42].

The source of phosphorus in biofertilizers is often ash and bones, and microorganisms, e.g., Bacillus megaterium [3,40], are used for its solubilization. For example, the only high-quality bacterial soil preparation on the Polish market, which enriches the soil with easily assimilable phosphorus from the decomposition of complex and insoluble forms, enhancing plant growth, is based on a single, carefully selected strain of Bacillus megaterium spp. phosphatic bacteria. Fungal biopreparations, e.g., Nod, support plant growth and protect them against pathogens. Thanks to this, it is possible to increase the efficiency of legume cultivation, reduce the intensity of synthetic nitrogen fertilization, and protect crops against pathogens in a sustainable way [43]. A novelty in this field is research on the use of endophytic fungi other than mycorrhizal fungi, which can also affect the availability of phosphorus and potassium, as well as plant resistance [44].

Research is being conducted to obtain biopreparations that protect and stimulate plant growth. For example, it has been shown that the active ingredients of biopreparations derived from fungi (i.e., Fusarium spp., Trichoderma spp., Tortorella spp.) and bacteria (e.g., Pseudomonas luteoma, P. fluorescens) stimulate crop growth and the absorption of phosphorus and potassium from soil minerals (apatites, phosphates). Bioprotective biopreparations against phytopathogens have been developed, containing non-pathogenic fungi from the genera Fusarium, Trichoderma, and Tortorella or inducers of plant resistance pathways. It has been shown that plants treated with these biopreparations are resistant to phytopathogens [40,41,42,43]. Many studies in this area are covered by patent law. For example, plants treated with biopreparations developed by the Polish team of Jaroszuk-Ściseł from Maria Curie-Skłodowska University in Lublin are resistant to phytopathogens because biopreparations activate systemic resistance pathways that are quickly activated in the event of contact with pathogens. This innovative invention has been submitted for patent protection (patent application P.428832) and uses natural biological methods of plant protection without the need to resort to chemical fungicides. Copper and its compounds, e.g., copper sulfate, copper oxychloride, are substances with fungicidal (fight fungi) and bactericidal (fight bacteria) effects. They are widely used in agriculture, horticulture, and forestry to combat plant diseases caused by fungi (e.g., apple scab, downy mildew) and bacteria. Currently, there is a growing interest in precise sequencing of microorganism genomes in order to identify and optimize genes responsible for the production of biostimulants and protective compounds, which opens the way to designing “tailor-made” biopreparations [50]. Recently, seaweed extracts (SwEs) have been used for the strengthening and fertilization of plants under drought stress conditions, mainly from brown macroalgae, due to their composition and richness in polysaccharides, betaines, macro- and microelements, and phytohormones, which are considered as beneficial signaling molecules for improving plant response [43]. The application of SwE under favorable or critical conditions, such as drought stress, is associated with physiological and biochemical processes concerning the stimulation of enzymes involved in carbon and nitrogen metabolic pathways, the Krebs cycle and glycolysis, stimulation of phytohormones, and enhancement of mineral absorption and accumulation by biostimulated plants due to modification of root morphological structure [43,44]. The use of SwE can be considered as an appropriate and sustainable approach to overcoming agricultural problems caused by anthropogenic climate change. It is worth adding that the development of extraction and fractionation technologies allows for obtaining more concentrated and specific bioactive compounds from seaweed, which increases their efficiency and precision of action [51].

3.5. Biopesticides

The principles of green chemistry, related to research and the search for safe chemical products, indicate a growing interest in and introduction of new pesticides to agrochemical practice [44,45,46,47]. New chemical products introduced onto the market, including pesticides, should meet the basic requirements of green chemistry, i.e., principal no. 10, according to which after use they should not remain permanently in the environment but degrade into products harmless to ecosystems. Biopesticides are a special group of active substances used in plant protection. They usually occur naturally or are produced synthetically but include natural substances. These compounds usually decompose quite quickly, and semi-chemical pesticides are used in very low doses [44,45,46,47]. Biopesticides or biological pesticides are methods of pest and disease control that use predatory compounds, parasitism, and their chemical action [45,46,47,48].

Historically, the definition of biopesticides has been related to the biological control and manipulation of living organisms. Public opinion can also influence the perception of this issue and its regulatory significance. Thus, in the European Union, biopesticides are defined as “a form of pesticide based on microorganisms or natural products”, while in the USA, the Environmental Protection Agency (EPA) states that biopesticides include “pesticides of natural origin (biochemical pesticides), microorganisms, bacterial pesticides, and pesticides produced by plants with added genetic material (Protectants or PIPs)” [45,46,47,48]. Typically, such products are produced by culturing and concentrating naturally occurring organisms or their metabolites, including bacteria and other microorganisms, as well as proteins, fungi, nematodes, etc., which are recognized as important components of integrated pest management programs and considered as substitutes for chemical plant protection products [45,46,47,48]. The development of new fermentation technologies, including fermentation in bioreactors, allows for scaling up biopesticide production and reducing their costs, which increases their availability on the market [52]. There are three main groups of biopesticides: microbial pesticides, biochemical pesticides, and plant-incorporated protectants (PIPs) [45,51,52]. Microbial pesticides refer to microorganisms such as bacteria, pathogenic fungi, or viruses and sometimes metabolites produced by bacteria or fungi. Entomicidal nematodes are often classified as microbial pesticides, although they are multi-cellular organisms. Biochemical (or herbal) pesticides are naturally occurring substances used to control pests and diseases caused by various microorganisms or for monitoring in the case of pheromones [53]. The novelty in this area is the identification and synthesis of new, highly specific semiochemicals and pheromones that act at very low concentrations, minimizing the impact on the environment [54]. Plant-incorporated protectants (PIPs), which are produced by plants that have incorporated genes from other species into their own genetic material (GMOs), are problematic due to controversy, especially in European countries [45,51,52]. Despite the controversy, research on gene editing (e.g., CRISPR/Cas9) in plants to increase their natural resistance to pests and diseases, without introducing foreign genetic material, is a promising new avenue in the development of PIPs that may be more socially acceptable [55]. Biopesticides, although their biological activities, especially against fungi, nematodes, insect pests, and other organisms, are well documented, have no known function in photosynthesis, growth, or other aspects of plant physiology [45,51,52,55]. All vascular plant species have developed a unique, integrated chemical structure that protects them from pests. These biodegradable and renewable alternatives to conventional pesticides have been successfully used, especially in organic farming systems [20]. In order to explain the mechanisms of pesticide action, genetics and molecular physiology are used as tools to overcome plant resistance to new generations of chemicals and antimicrobials [44,48,49]. Human and animal exposure to pesticide residues involves substances originating from various sources, such as the environment or food. The adverse effects of such combinations can occur unexpectedly and manifest themselves in different ways, leading to a change in the assessment of health risks [3,46,50,51,52]. According to the latest research and international and European regulatory changes in this area, the analysis of pesticide residues used in plant protection products (PPPs) aims to determine the risk and precisely establish maximum pesticide residue limits and plan their control [8,44,50,51,52,53,54]. The novelty here is the development of fast and precise analytical methods (e.g., mass spectrometry techniques), which allow monitoring a wide spectrum of pesticide residues in real time, which is crucial for ensuring food safety [56]. Kowalska et al. [45] proved the high potential of 19 isolated strains of yeast-like microorganisms and their antagonism towards the causative agent S. sclerotiorum at different temperatures. They also found a high protective potential of Saccharomyces cerevisiae and its suitability for protective treatments against gray mold in strawberries and reduction of symptoms of Phytophthora infestans on potato plants, which proved useful for dressing planting material and increasing the shelf life of vegetables. In addition, these authors proved the differential survival of P. oligandrum and B. subtilis in different temperature ranges and their suitability for controlling the main potato pathogens in field conditions in strategies using them as an alternative to copper preparations.

Pathogenic soil microorganisms can cause diseases or damage to crops. These microorganisms can also be used to control diseases, weeds, and pests. Biopesticides are a group of active substances used in plant protection. They occur naturally or are synthetic products identical to natural substances. Biopesticides are a type of pesticide derived from natural materials such as bacteria, plants, animals, and some minerals. They include many living organisms (so-called biocontrol organisms) [45,48,49,50,51,52,53,54,55]. They are usually less toxic than conventional pesticides. Their effects are typically low risk to non-target organisms. They tend to degrade quite quickly, and some semiochemicals are used at very low doses. Their mechanisms of action and effects on human and animal health are divided into several groups or classes: biochemical biopesticides, microbial biopesticides, and plant-incorporated protectants (PIPs), which are pesticidal substances that plants produce from genetic material that has been added to plants [4,5,10,11,12,13,14,15,48,51,55]. Various natural substances, including plant extracts, are classified as biopesticides, including: semiochemicals from insect pheromones; various fermentation products such as spinosad (a macrocyclic lactone); chitosan; natural products of plant origin (alkaloids, natural phenols, terpenoids, and other secondary metabolites); some vegetable oils, e.g., rapeseed oil, as well as products made from plant extracts, such as garlic, onion, and nettle. They have been registered in the EU and elsewhere and can be used in plant protection. Pesticide residue limits in food are regulated by the Regulation (EC) on official controls of pesticide residues in food, both for products manufactured in the EU and imported [46]. Microbial biopesticides consist of a microorganism (e.g., bacterium, fungus, virus, or protozoan) as an active ingredient and can control many different types of pathogens, but each active ingredient is specific to its target pest. For example, there are species of fungi that control certain species of weeds or fungi that kill certain species of insects. This allows for the improvement of physicochemical and biological properties of the soil, increases plant resistance to pathogens, and allows for the proper selection of biologically active compounds [53,54,55,56]. For example, Bacillus thuringiensis subsp. tenebrionis destroys Leptinotarsa decemlineata larvae [44,55,56]. It is worth emphasizing that the new generation of microbiological biopesticides is designed considering the specificity of pathogen–host interactions at the molecular level, which allows for precise pest control with minimal impact on non-target organisms (Figure 3) [16,48].

Figure 3 shows the key role of microorganisms in sustainable agriculture. The main elements are in a healthy plant, roots are surrounded by symbolic beneficial microorganisms, such as bacteria and fungi, indicating their role in plant nutrition and health. In the background, crop fields are outlined with visible features of sustainable practices, such as mixed cropping or agroforestry, symbolizing the broader context of application. In the margin, there is a small, schematic diagram of a biopesticide attacking a pest, visualizing the application of biological control.

Entomopathogenic fungi are micropesticides that attack insects and cause diseases inside their bodies, ultimately leading to death [36,37,38,39]. Two species of fungi are known to be used as micropesticides: Beauveria bassiana, which causes white muscardine, and Metarhizium anisopliae, which causes green muscardine [36,37]. B. bassiana, a filamentous fungus from the class Deuteromycetes (imperfect), is used against Colorado potato beetles, apple fruit moths, and American cuticle moths. It attacks the hemocoel of insects via spores. Once the spores attach to the insect cuticle, the fungus germinates and its hyphae penetrate the insect cuticle, forming an appressorium and a penetration peg. At the same time, they secrete chitinases, lipases, and proteases that can dissolve the cuticle. The fungal hyphae enter the hemolymph, multiply, and colonize the insect, releasing blastospores. Death of insects occurs due to depletion of nutrients in the hemolymph or poisoning due to the secretion of toxic metabolites [36,37,38,39]. New approaches also include the selection and engineering of entomopathogenic fungi strains with increased virulence, UV resistance, and longer survival time in the environment, which is crucial for their commercialization [33,34,35]. The combined use of biologicals in combination with non-chemical weed control methods significantly improves soil structure, increases earthworm numbers, and reduces soil carbon dioxide emissions but still contributes to a decrease in soil enzymatic activity [42]. The implementation of integrated weed, disease, and pest control in commercial agriculture has ultimately led to an increased interest in biopesticides, which in turn will reduce food safety risks [56]. Biochemical biopesticides are naturally occurring substances that control pests through non-toxic mechanisms that include reproductive disruptors such as insect sex pheromones, as well as various fragrant plant extracts that in turn attract insect pests to traps [53].

3.6. Bioherbicides

The priority of sustainable agriculture is food security and environmental security [53,57,58,59]. Already in 2001, Lasiodiplodia theobromae MTCC 3068 was used as a potential bioherbicide in pre- or post-emergence treatments, including Parthenium hysterophorus L. from the Asteraceae family, Datura stramonium from the Solanaceae family, and Sida prickly. The mycoherbicide produced from Lasiodiplodia pseudotheobromae C1136 is suitable for controlling both dicotyledonous and monocotyledonous weeds [23]. Pathogenesis induced by phytotoxins of pathogenic fungi can cause physiological, biochemical, and metabolic changes in the host plant, and this leads to abnormalities in the translocation of storage materials, which in turn ends in chlorosis and aging of the leaves at the sites of infection [20,57,58,59]. This process usually leads to the degradation of genetic material in plant cells and growth inducer [24,25,50].

The recently discovered effect of phytotoxic metabolites produced from bioherbicide isolates may significantly prevent environmental threats in the future [60]. Therefore, further research into bioherbicides and all aspects of the safety of phytotoxic metabolites produced from wild and genetically modified strains of L. pseudotheobromae and their bioherbicide effect on various species of weeds is justified.

Microbial biotechnology can be used to produce bioherbicides through the use of microorganisms that produce toxins that are harmful to weeds or through the genetic modification of microorganisms to produce specific toxins [23]. One example of the use of microbial biotechnology for the production of bioherbicides is the use of herbicide-tolerant microorganisms. Many plants produce compounds that are toxic to weeds, and these compounds can be extracted and used as bioherbicides. Microbial biotechnology can be used to produce plant extracts with herbicidal properties through the fermentation of plant material or through the genetic modification of microorganisms to produce specific compounds [23,24]. In addition to the use of microorganisms and plant extracts, microbial biotechnology can also be used to produce bioherbicides from minerals. For example, sulfur, a naturally occurring mineral, is commonly used as a bioherbicide to control fungi and bacteria in agriculture, forestry, and horticulture. Sulfur compounds (SO₂ and H₂SO₄) are often used as bioherbicides to reduce fungal and bacterial diseases in orchards (e.g., apple scab, black spot, or downy mildew). This is about sulfur compounds as fungicides, not about their use as herbicides in relation to diseases. There is a slightly misleading formulation used, as sulfur is not a typical bioherbicide for weeds but rather a fungicide. Overall, the use of microbial biotechnology for the production of bioherbicides has the potential to provide numerous benefits, such as reducing the reliance on synthetic herbicides, improving the safety of weed control, and reducing the environmental impacts of weed control [23,30,38].

One challenge that needs to be dealt with when applying microbiological biotechnology in the production of bioherbicides is the possibility of developing resistance in various weed populations. Like synthetic herbicides, bioherbicides can select for the survival of weeds that are resistant to the toxins or other compounds used in the bioherbicides. This can lead to the development of weeds that are immune to bioherbicides, which can reduce their effectiveness over time.

In the system of biodynamic and organic agriculture, there are no effective methods of combating plant diseases and pests [55,57,58,59,60,61]. Vaitkeviciene [56], Levickienė [60,61] and Vlahova [62] used the biodynamic preparation BD 500 in organic cultivation. It has been proven that some cultivars have different abilities to reduce weed infestation when grown in varietal mixtures compared to varietal monoculture [63,64]. For example, some tomato varieties are more tolerant than Cuscuta spp. [65,66].

In the case of bioherbicides, in the vast majority of cases the pathogen is a fungus, as preparation of mycelium, conidia, or spores. Only a few live microbial bioherbicides have been approved for commercial use and have been placed on the market, but their use has unfortunately been very limited for a number of reasons, mainly economic. Examples of bioherbicides are listed in

Table 1. Examples of microbial bioherbicides.

Microbe/References	Weed Target(s) and Status	Trade Name	Year of Introduction or Registration/State
Albifimbria verrucaria (formerly Myrothecium verrucaria)	Conyza canadensis (glyphosate-resistant)—Experimental	-	2023
Alternaria cassia Bannon, 1988	Cassia obtusifolia C. coccidentalis Crotalaria spectabilis—Never commercialized	Casst™	Never
Alternaria destruens Bewick et al., 1984	Cusucta spp.—Discontinued	Smolder™	2005
Ascophyllum nodosum (seaweed)	Various weeds—Available	BioWeed USA	USA
Chondrostereum purpureum Hintz 2007	Populus and Alnus spp.—Unknown	Chontrol™	2004
Citronella oil (22.9%)	Jacobaea vulgaris	Barrier H	2015 (EU, Japan, USA)
Colletotrichum acutatum Morris 1989	Hakea sericea—Discontinued	Hakatak	1990
Colletotrichum echinochloae	Echinochloa crus-galli—Experimental	-	2023
Colletotrichum gloeosporioides f. sp. aeschynomene Cartwright et al., 2010	Aeshynomene vigrinica—Available on demand	Collego®	1982
Colletotrichum gloeosporioides f. sp. aeschynomene	Aeschynomene virginica—Available	LockDownTM	2006 (USA)
Colletotrichum gloeosporioides f. sp. Malvae Boyetchko et al. 2007	Acacia mearnsii and A. pycnantha—Discontinued	Stumpout™	1997
Cylindrobasidium laeve. Morris et al., 1999	Acacia mearnsii and A. pycnantha—Discontinued	Stumpout™	1997
Fusarium oxysporum f. sp. strigae	Striga hermonthica—Under development	Kichawi Kill™	2023 (Kenya)
Lasiodiplodia pseudotheobromae, Macrophomina phaseolina, Parkinsonia aculeata—Available	Neoscytalidium novaehollandiae (Di-Bak® Parkinsonia)	Di Bak®	2019 (Australia)
Pelargonic acid (natural fatty acid)	Grassy and broadleaf weeds	Katana®	2016 (USA)
Phoma macrostoma	Broadleaf weeds—Available	Bio-Phoma™	2016 (Canada)
Phoma macrostoma Bailey et al., 2011	Many broadleaves weed species—Available	Bio-Phoma™	2016
Phytophthora palmivora Ridings 1986	Morrenia odorata—Discontinued	DeVine®	1982
Pine oil + sugar formula	Herbaceous and grassy weeds—Available	Bioweed™	Australia/West Asia
Pseudomonas fluorescens Kennedy et al., 2001	Bromus tectorum—Discontinued	D7®	2014
Puccinia canaliculata Phatak et al., 1983	Cyperus esculentus—Discontinued	Dr. Biosedge™	1987
Puccinia thlaspeos Knopp et al., 2002	Isatis tinctorial—Discontinued	Woad Warrior®	2002
Sclerotinia minor Watson 2018	Taraxacum officinale—Discontinued	Sarritor®	2009
Several fungi Gale and Goutler 2013	Parkinsonia aculeate—Available	Di-Bak®	2019
Solanum habrochaites—plant extract	Various weeds	Available WeedLock	2017 (Malasya)
Streptomyces hygroscopicus (Bialaphos)	Broad-spectrum post-emergence herbicide	Bialaphos®	2016 (East Asia)
Streptomyces scabies O’Sullivan et al. 2015	Several grass and broadleaf weeds—Never commercialized	Opportune™	2012
Tobacco mild green mosaic vírus Charudattan and Hiebert 2007	Solanum viarum—Available	SolviNix™	2014
Trichoderma koningiopsis and others	Euphorbia heterophylla, Bidens pilosa, Conyza bonariensis—Experimental	-	2023
Xanthomonas campestris pv. poae Imaizaumi et al., 1999	Poa annua—Discontinued	Camperico™	1997

Source: own.

.

Table 1 shows the growing diversity of bioherbicide sources. These include:

• Traditional microorganisms (e.g., Fusarium, Colletotrichum, Phoma, Streptomyces), soil microorganisms and parasitic fungi, natural plant extracts (Solanum habrochaites, pelargonate, essential oils), and even seaweed products (Ascophyllum nodosum). This reflects the global trend away from synthetic herbicides towards natural, biodegradable, and environmentally safer compounds.
• Commercialization status and regionality: Some bioherbicides are available locally (e.g., Di-Bak^® in Australia, Bio-Phoma™ in Canada, Kichawi Kill™ in Kenya). Many products are “experimental” or “discontinued,” which shows the difficulties with their sustainable commercialization, including problems with mass production, field efficacy, and registration. There is still a lack of international standardization, and registration is often limited to regions where weed is a problem.
• Practical challenges: Bioherbicides are also accompanied by practical challenges, as they are often less fast and selective than synthetic agents; they can act slower and require specific conditions (humidity, temperature). Some of them only act on selected species (e.g., Colletotrichum only on Aeschynomene). Storage problems and shorter shelf life are often mentioned as barriers.
• Ecological and agrotechnical importance: Bioherbicides as a key tool in sustainable agriculture or in integrated plant protection (IPM) and in organic production systems. They can also reduce the problem of herbicide resistance, e.g., in the case of Conyza canadensis (resistant to glyphosate), Albifimbria verrucaria is the answer to this challenge.
• Promising research directions: Microorganisms such as Trichoderma, Phoma, and Fusarium oxysporum are intensively studied as carriers of new bioactive metabolites. There is also growing interest in multi-component products (e.g., Di-Bak^®—a mixture of three fungi) and target application systems (e.g., capsules, implants, nanoformulation).

Table 1 therefore reflects the dynamic development of bioherbicides but also real implementation barriers. This proves that, despite their great potential, most products require further research, optimization, and regulatory support.

The advantages and disadvantages of live bioherbicides are presented in

Table 2. Advantages and disadvantages in technologies with microorganisms.

Advantages	Disadvantages
Increasing food production: Some microorganisms are beneficial to plants and help in their growth and production. They can also help produce food by fermenting and processing food.	Uncontrolled growth of microorganisms: In some cases, microorganisms can grow uncontrollably and become harmful to plants, soil, and human health.
Plant protection: Some microorganisms are able to fight plant diseases and pests, reducing the use of harmful pesticides and other chemicals.	Environmental pollution: Some microorganisms, such as E. coli bacteria, can cause soil and water pollution, which is a public health risk.
Improving soil quality: Microorganisms can help enrich the soil with nutrients and improve its structure, which positively affects the health of plants.	GMO risks: Some microbial technologies, such as genetic engineering, can lead to genetically modified organisms (GMOs), which raise social and ethical concerns.
Sustainable agriculture: The use of microorganisms can aid sustainable agriculture by reducing the use of harmful chemicals and improving soil quality.	Costs: Some systems and technologies using microorganisms can be expensive, which is a barrier to their widespread use.

. If the bioherbicide is not genetically modified or contains any synthetic chemicals, it can be accepted by organic farmers and such a product must not leave any chemical residues in the crop [53,54,55,56,62,66,67,68]. Another issue is the safety of the tested strains of bacteria for humans [60,61,67,68].

Varieties of plants with high tolerance to weeds are important in special situations where chemical control of weed infestation is impossible. Such species or varieties are characterized by strong allelopathic properties [67,68,69,70]. Secondary metabolites involved in weed control include phenols, flavonoids, and alkaloids [63,64,69,71]. Such properties of some species and varieties of crop plants are extremely valuable, especially in integrated farming systems, by inhibiting the development of weeds [70,72]. In conclusion, microbial biotechnology can play a significant role in the production of bioherbicides through the use of microorganisms, plant extracts, and minerals. However, various challenges need to be considered, such as the potential for resistance development in weed populations and the need to better understand the environmental impact of bioherbicides.

3.7. Bioinsecticides

Bioinsecticides are insecticides made from natural materials, such as microorganisms, plant extracts, and minerals. Microbial biotechnology can be used to produce bioinsecticides through the use of microorganisms that produce toxins that are harmful to insects or through the genetic modification of microorganisms to produce specific toxins [68,71,72,73,74,75,76,77].

One example of the use of microbial biotechnology for the production of bioinsecticides is the use of Bacillus thuringiensis (Bt). Bt is a soil bacterium that produces toxins that are harmful to certain insects, such as caterpillars and mosquitoes. These toxins are called delta-endotoxins and are toxic to insects when ingested but are generally considered to be safe for humans and other mammals [77,78,79,80,81].

Biopesticides are one way of biological control. The active ingredients of biopesticides include microorganisms, plant extracts, or naturally occurring chemicals (e.g., potassium bicarbonate). As a result, some pest control methods (modes of action or MOAs) differ from traditional, artificial chemical pesticides. Biopesticides with MOAs can counteract insect pests (e.g., preparations containing Beauveria bassiana) or act against plant diseases (e.g., those containing Paraconionthyrium minitans strain CON/M/91-08). Many biopesticide preparations with MOAs contain fungal spores. These spores germinate only when they are on an insect or on a pathogen that causes disease (Figure 4) [78,79,80,81].

Biopesticide preparations with MOAs contain fungal spores. These spores only germinate when they are on an insect or on a disease-causing pathogen.

A biopesticide containing viable fungal spores (blue) that must be on an insect pest or plant pathogen (yellow rectangle) is shown in Figure 4. They then germinate, attack, and grow, eventually killing the pest (Figure 3). Another example is the use of plant extracts with insecticidal properties as bioinsecticides. Many plants produce compounds that are toxic to insects, and these compounds can be extracted and used as bioinsecticides. For example, pyrethrum, a compound produced by Chrysanthemum sp. flowers, is used as a bioinsecticide to control a variety of insects, including mosquitoes, flies, and beetles. Microbial biotechnology can be used to produce plant extracts with insecticidal properties through the fermentation of plant material or through the genetic modification of microorganisms to produce specific compounds [68,81,82].

However, there are still various challenges and ethical issues to consider, such as the possibility of GMOs being released into the environment. In addition, an important challenge is the possibility of developing resistance in insect populations. Like synthetic insecticides, bioinsecticides can select for the survival of insects that are resistant to the toxins or other compounds used in the bioinsecticides [9,10,11,12,13,14,15]. This fact may lead to faster development of insect populations resistant to bioinsecticides, which in turn may reduce their effectiveness. To mitigate this risk, it is important to use integrated pest management (IPM) strategies that incorporate multiple control measures, such as the use of diverse bioinsecticides, cultural practices, and natural predators, to reduce the selection pressure for resistance. Kowalska et al. [45] demonstrated the huge role and the scope of usefulness of beneficial microorganisms that occur naturally or are deliberately introduced into the environment to ensure the well-being of plants in the ecological farming system. Among others, studies showed a highly competitive potential of the antagonistic fungus T. asperellum used for spraying potatoes, strawberries, rapeseed, and lemon balm and used in storage against Botrytis cinerea and S. sclerotiorum [43,83]. Saccharomyces cerevisiae has high protective potential and suitability for protective treatments against gray mold in strawberries and reducing the symptoms of Phytophthora infestans on potato plants [45].

Viral insecticides (baculoviruses) are pathogens that attack insects and arthropods. Viral pesticides are used to control Lepidoptera larvae such as Helicoverpa and Spodoptera sp. on cotton, maize, sorghum, and tomatoes. Baculoviruses are a commonly used viral biopesticide. They are microscopic in size and consist of double-strand DNA. The genus Baculovirus includes three subgroups: nuclear polyhedrosis viruses (NPVs), granuloviruses (GVs), and non-occluded viruses. NPV enters the insect body through ingestion and infects midgut cells through membrane fusion. NPV develops in the cell nucleus and passes through the intestinal epithelium of the insect, causing a systemic infection of the hemocoel [26,45].

Another challenge is the need for further research to better understand the environmental impacts of bioinsecticides [84,85,86]. Therefore, it is important to carefully assess the environmental impacts of bioinsecticides and to implement appropriate measures to mitigate any potential negative impacts. In conclusion, microbial biotechnology has the potential to play a significant role in the production of bioinsecticides through the use of microorganisms, plant extracts, and minerals. It is important to carefully assess the risks and benefits of using bioinsecticides and to implement appropriate measures to ensure their safe and effective use.

3.8. Effective Microorganisms

In the face of drastic climate changes, the growing world population, and growing interest in safe food, there is a growing demand for substances capable of replacing or eliminating conventional pesticides [56,84,87,88,89,90,91,92,93,94,95,96,97,98,99,100]. This is of crucial importance for both consumers and producers striving to reduce the use of chemicals in agriculture, e.g., by introducing safe preparations that are an alternative to synthetic fertilizers and conventional plant protection products. Such an alternative is effective microorganisms (EMs). EMs are a mixture of many microorganisms, such as bacteria, fungi, actinomycetes, and yeasts, commonly found in the environment (Figure 5) [57,58,59,101,102,103,104,105,106,107,108,109,110,111,112].

Recent studies focus on precise characterization of the composition of EM consortia using metagenomic techniques and identification of key microbiological interactions that are responsible for their synergistic effects and effectiveness in different soil environments [55].

3.8.1. Microorganisms in the Influence of Plant Resistance

The use of microbiological preparations in plant production raises many controversies in the world. There are reports of both benefits from their use [57,58,59] and their ineffectiveness [113,114]. Modern approaches aim to standardize application methods and environmental conditions in which EMs show the highest effectiveness in order to reduce the variability of field results [104].

The main strategy of biological plant protection is induced plant resistance. Induced plant resistance is defined in two ways: (1) as induced systemic resistance (ISR) when induced by rhizobacteria (PGPR) and (2) as systemic acquired resistance (SAR) when induced by other factors (e.g., pathogen infection). SAR is expressed at its highest level when the inducing organism causes necrosis, whereas ISR (induced by PGPR) does not show necrosis symptoms in the host plant. Both ISR and SAR are based on the activation of dormant immune mechanisms [70]. The development of molecular techniques such as transcriptomics and metabolomics allows for a deeper understanding of the specific signaling pathways and molecules involved in ISR and SAR, which enables more precise design of biostimulants [115].

Race-specific avr gene products (elicitors) are active only in varieties with complementary resistance genes (R). They then determine the occurrence of race-specific resistance (full resistance). In the case of acquired systemic resistance, i.e., in order to activate the defense system in uninfected parts of the plant, inhibitors are used., plant defense proteins exhibiting inhibitory activity, e.g., polygalacturonase (PGIP). A total of fourteen plant proteins has been identified [71,103,104], mainly with bactericidal activity and also involved in apoptosis. These are gluconases, chitinases, osmotins, protease inhibitors, proteinases, lysozymes, and peroxidases. Numerous interactions are known between the cell walls of fungi and plants, and the expression of the encoding genes depends on the elicitor, which is captured by specific receptors, signaling their induction via kinases. The typical cell wall of dicotyledons contains cellulose (β-1,3; 1,4-glucans), hemicellulose—30% (xyloglucans), pectins—35%, and proteins—1–5%. The typical cell wall of grasses (monocotyledons) contains cellulose—25% (β-1,3; 1,4-glucans), hemicellulose—55% (arabino-glucuronoxylans and glucomannans), hemicellulose—30% (xyloglucans), pectins—35%, and proteins—1–5%. In turn, the cell wall of fungi contains: β-1,3-glucans—30–65%; β-1,6-glucan—1–5%, chitin—2–40%, CWP proteins—30–50% [102]. Common and race-specific elicitors are distinguished. The former (i.e., β-glucans, chitin, polypeptides, glycolipids) are produced by pathotypes of the pathogen. They are responsible for inducing the so-called race-non-specific immunity (field, horizontal, partial). Race-specific elicitors (products of an avirulent gene (avr)) are active only in varieties with a complementary resistance gene (R). They are responsible for the development of “gene-to-gene” immunity, i.e., race-specific (vertical, complete) [71,103]. Elicitors trigger a cascade of defense reactions. This leads to an increase in the content of phenols, flavonoids, and the formation of secondary metabolites (phytoalexins). They can be toxic to pathogens [103]. The signaling cascades of (1) salicylic acid (SA) and (2) jasmonic acid (JA) are shown in Figure 6.

This schematic diagram shows the complex interactions between two major signaling pathways in plant defense: the salicylic acid (SA) pathway and the jasmonic acid (JA)/ethylene (ET) pathway. On the left side, the SA pathway is activated by biotrophic pathogens or elicitors of acquired systemic resistance (SAR). It leads to the activation of pathogenesis-related genes (PR) and consequently to resistance to biotrophic pathogens. On the right side, the JA/ET pathway is induced by necrotrophic pathogens, herbivorous pests, or elicitors of inducible systemic resistance (ISR). This results in the activation of defense genes and resistance to necrotrophy and pests. Figure 6 also highlights the antagonistic interactions between these two pathways (marked with dashed lines with minus signs), which are crucial for the fine-tuning of plant defense responses. The central element shows that environmental signals and abiotic stresses also affect both pathways, modulating their activity. General components, such as MAPK cascades and transcription factors, involved in these processes are also identified.

The main consequence of pathogen/elicitor recognition by a plant cell is rapid reactions at the cell periphery that do not require gene expression. They include membrane depolarization, ion flow across the membrane (including K⁺ and Cl⁻ efflux and H⁺ and Ca²⁺ influx), and an oxygen burst. The gene-for-gene theory states that pathogen recognition and activation of defense responses occur only when a plant possessing the appropriate resistance gene R is attacked by microorganisms that contribute to the avirulence gene (avr). A pathogen with the avr gene is pathogenic for plants that do not possess the appropriate R gene because then there is no defense reaction. Studies on the influence of microorganisms on plant immunity processes were undertaken due to the rapid increase in active oxygen species after contact of plant cell membranes with the pathogen. They are cytotoxic, especially for cells infected with the pathogen. This results in cytoskeleton reorganization and increased thickness of cell walls. According to Kowalska et al. [45] and Barbaś et al. [103,104], acquired systemic immunity, activating the defense system, is acquired by the plant in its healthy, uninfected tissues. The signal to initiate this response is usually salicylic acid (Figure 6). At least fourteen classes of plant proteins are involved in this process, such as: chitinases, glucanases, protease inhibitors, lysozymes, peroxidases (including β-glucosidase), osmotins, proteinases, antibacterial proteins, and proteins involved in apoptosis [102,103,104]. In the places of action of microbiological preparations, as a result of biochemical changes, targeted resistance of crop plants may occur. Recently, the demand for microbiological preparations in the agricultural sector has been gradually increasing due to the development of ecological and proecological methods of plant cultivation [46,96,109].

The demand for microbiological preparations is growing in agriculture due to the desire to reduce chemicalization [45,47,109,110,111,112,113]. Preparations with beneficial microorganisms, including EMs, are widely used in Europe, supporting the growth and yield of crops, such as bacterial symbioses with legumes or mycorrhizal vaccines in forestry. However, the registration procedure for these products for organic farming is often too liberal, and there is often a lack of studies confirming their effectiveness [45,47,113].

3.8.2. EMs in Plant Cultivation

The use of bacterial bioproducts is wide and includes:

Supporting the microbiological balance of the soil thanks to the optimal composition of strains [46,98,99,100,101,102,103,104].

Accelerating mineralization and increasing the availability of nutrients, which reduces the need for high doses of mineral fertilizers [105,106,107,108,109].

Decomposition of difficult-to-degrade plant parts and activation of the humification process, which improves air–water relations in the soil, increase nutrient absorption, maintain soil moisture, prevent soil compaction, and protect plants from biotic and abiotic stresses [109,110,111,112,113].

Helping maintain the lumpy structure of the soil.

Biopreparations are crucial for soil fertility, and their production in bioreactors ensures high concentrations of bacteria and full control over the process [98,114,115,116,117,118,119].

EMs are tested in many countries, including Japan, China, and Poland, and their aim is to improve the quality of soil, water, and agricultural products [111,116,117,118,119,120,121,122,123,124]. Studies have shown that EMs with manure improve root growth and photosynthesis [82] and increase the yield and health of potatoes [94,95,96] and wheat [74,75]. Other authors also confirm the positive effect of EMs on yield and plant health [77,78,79,80,81,82,84,125]. Some studies, e.g., Van Vliet et al. [110], did not show a significant effect. Martyniuk [111] criticizes the liberal registration procedure, which indicates the need for further research on the mechanisms of EM action.

EMs in vegetable cultivation increased the yield of onion and pea [104], affecting photosynthesis and water use. Marczakiewicz [125], Mathews & Gowrilekshmi [126], Yan et al. [127], Al-Taweil et al. [128], Szewczuk et al. [129], Solarska [130], Henry et al. [131], Ney et al. [132], Faturrahman et al. [133], Gacka & Kolbusz [134], Tsatsakis et al. [135] and Pniewska et al. [136] observed increased density and mass of green beans. EMs improved growth and physiological parameters and also increased the yield of beans [125,136]. Górski and Kleiber [86] showed a positive effect of EMs on ornamental plants (roses, gerberas) and Trawczyński [79] on the content of nutrients in potatoes. EMs strengthen plant immunity and displace pathogens [45], inhibiting the development of cereal diseases [78,79,80,81,82,121,122] and reducing the occurrence of Fusarium fungi [104,105]. The use of EMs together with nettle extract reduced the number of potato beetle larvae [71].

Microbiological preparations fill the gap in biological plant protection and increase soil microbiological activity [71,72,91,92,96,97,110,111,124,125,126,127,128]. They have yield-forming potential and the ability to transform harmful compounds [71,72,91,92,96,97,110,111,124,125,126,127,128]. They can be used for seed dressing, spraying, and soaking seedlings [51,52,103,104,118,119,122,123,128,129].

Some authors [109,110,129,130,131,132] confirm the positive effect of EM preparations on morphological, physiological, and immune features of plants. Knowledge of this subject comes mainly from commercial studies and fragmentary scientific research. In order to support the fertility and quality of crops, close cooperation and exchange of experiences between agricultural practice and the scientific community are necessary [132,133,134].

Systems and technologies using microorganisms have many advantages and disadvantages that should be considered (Table 2).

Powerful scientific techniques caused a dramatic expansion of genetically modified crops, leading to a change in agricultural practices, which has a direct and indirect impact on the environment (Figure 7).

Despite the increased yield potential, the basic problems to be dealt with are threats and concerns about biological security related to such crops in the community. Among scientists and decision-makers, there is growing interest in the study of unintentional effects of transgenes related to gene flow, the flow of naked DNA, weeds, and chemical toxicity. Current knowledge proves that GMO crops exert a harmful impact on the environment, e.g., by changing the invasiveness of crops, the appearance of herbicides and insecticides, tolerance to salt stress or drought stress, disturbing biological diversity, transgenes, etc. This impact requires confirmation and critical research. Currently, there is not enough data on the significant negative impact of GM plants or their nutritional value. Therefore, the consumption of GM products is considered to some extent safe, with a few exceptions. Meanwhile, Tsatsakis et al. [135] states that they change the existing arrangements and GM crops and their products are divided into target and non-target species and shed new light on the challenges and threats associated with this problem. However, basic research also indicates that the influence of GM crops on biological diversity disorders, immunity development, and evolution somewhat resembles the effects of genetically unmodified crops (Figure 7). The future prospects are also important.

3.8.3. Microbiological Preparations in Composting

Due to the growing problems of managing natural fertilizers and bio-based municipal waste, especially sewage sludge, the methods of their disposal are diverse [10]. The EU is observing the development of legislation on biowaste and the implementation of modern technologies for their management, especially recovery. Among the popular technologies for processing organic waste (including natural fertilizers) are energy recovery and aerobic technologies, such as various forms of composting [106,116,126,132,133,134,135].

A potential application of EMs is composting. EM preparations can improve the quality of compost by increasing the activity of microorganisms, improving the nutrient balance, and reducing unpleasant odors and fly populations [2,94,95,96,97,99,107,127]. Studies by Dacha et al. [107] on composting of sewage sludge with EM addition showed that the thermophilic phase was more intensive without EM addition, and microbiological activity (CO2 emission) was also higher in control compost. Nitrogen losses due to ammonia emissions were 0.04% lower in the EM treatments [107]. The lack of an EM effect could be due to competition with native microflora or parasitism/antibiosis phenomena [107]. Composting, especially of sewage sludge with a low C:N ratio (<10) [117], can lead to gas emissions, therefore it is important to optimize the composition of the mixture by adding materials rich in carbon (e.g., straw, sawdust) [107]. Despite the promotion of EMs in agriculture and municipal economies, their role as a panacea for all problems (fertilization, plant protection, crop quality, feed storage, reduction of ammonia and odor emissions, acceleration of composting) should not be overestimated. Further research is needed to better explain the role of EMs [107].

3.8.4. EMs in Food Processing

EM technology is one of the biotechnologies used in the food industry [96,97,98,106,108,118,119,120,136,137]. It uses microbiological materials (e.g., “Baikal EM1”), such as photosynthetic bacteria, lactic acid bacteria, and nitrogen-fixing bacteria, as well as yeasts and molds [119]. Modern food processing is based on enzymatic and microbiological preparations. More than 100 enzymes are used, and their number is growing [9,10,11,12,13,14,15]. Many microbiological preparations, including EMs, are also used [119,136,137].

The benefits of using enzyme and microbiological preparations in the food industry are various, including accelerating technological processes, the possibility of producing new assortments (including functional foods), increasing the attractiveness and durability of products, increasing the efficiency of raw materials, and reducing production costs [102,122,138,139,140]. This allows us to obtain higher-quality products at lower costs. Some microbiological preparations, such as yeast for wine, beer, and bread production [33,40], or lactic acid bacteria for cheese and dairy products [46,47,136,137,138,139,140,141], are used even in households. Acetic acid bacteria are used for vinegar production [90,102]. EM technology, e.g., in potato cultivation, can reduce digestive system ailments in humans by reducing the absorption of fats when frying potato products and obtaining better-quality raw material [99,119,123].

3.8.5. Mycorrhizal Preparations

Mycorrhizal preparations contain mycorrhizal fungi that inoculate plant roots, creating a symbiosis called mycorrhiza. In this interaction, fungi use photosynthetic products, and the plant receives water and minerals (nitrogen, phosphorus, potassium, microelements) [33,34,35,36,37,38,39]. The key functions of mycorrhiza are the protection of plants against fungal pathogens, increased resistance to frost and salinity, more abundant flowering, better rooting, and improved plant growth and appearance [37,38,39]. Mycorrhizal fungi are applied once, and plants can benefit from mycorrhiza throughout their lives. Early application (e.g., in seedling production) improves plant adaptation after transplanting [71,142].

In the context of climate change and long-term droughts, the use of arbuscular mycorrhizal fungi (AMF) is an effective and sustainable strategy [140]. Correira et al. [142] showed that AM fungi affect osmoregulation in H. serratifolius plants under conditions of moderate water deficit via soluble sugars and amino acids and under severe water deficit via proline and reducing sugars [142]. AM fungi promote greater absorption of phosphorus and nitrates only under conditions of good water supply, increasing dry mass in both water deficit and water supply conditions [142]. Mycorrhizal colonization is directly related to water regime, with well-watered plants showing higher colonization rates and plants with severe water deficit showing lower rates. Recent studies have focused on identifying plant and fungal genes responsible for the efficiency of mycorrhizal symbiosis under abiotic stress conditions, which will allow for the selection of more resistant genotypes [143].

3.8.6. Microorganisms in Aerobic Nitrification and Denitrification

Both heterotrophic nitrifying and aerobic denitrifying microorganisms (HNADMs) can oxidize ammonium nitrogen and then denitrify it in the presence of oxygen [142]. HNADMs may offer better utilization options [132,142]. To improve the efficiency of nitrogen cycling in agriculture, EM can be used simultaneously in nitrification and denitrification (SND) and HNADM processes [126,127,143,144].

Climate change and drought stress pose challenges to water management, food security, and water and wastewater treatment technologies. Türkmen et al., [145]. Monitoring nitrogen pollution, mainly from nitrogen fertilizers, remains a challenge in wastewater treatment [146]. Nitrogen compounds still cause many environmental problems, because their excess leads to deterioration of air quality, eutrophication of waters, and changes in forest processes, which in turn negatively affect water quality, human and animal health, and the environment [144]. In practice, biological methods are more often used. In the technology of biological nitrogen removal, two stages are distinguished: aerobic nitrification and anaerobic denitrification [147].

Denitrification is carried out by heterotrophic microorganisms, and nitrates are first converted to nitrites, and then to NO, N₂O, and finally to N₂ [146,147,148]. The discovery of heterotrophic nitrifying bacteria and aerobic denitrifying bacteria disproved this concept [146,147,148,149]. SND bacteria have the ability to both nitrify and denitrify. SND technology has significantly reduced costs and improved the efficiency of denitrification. Simultaneous heterotrophic nitrification and aerobic denitrification (SND) for nitrogen removal have been gaining increasing public interest due to their high efficiency and low cost [150].

Combined research strategies are proposed for future research directions, including the study of the nitrogen removal mechanism of SND using multi-topic strategies. Understanding this mechanism will allow for more efficient and better utilization of SND for nitrogen removal, e.g., from municipal or pharmaceutical wastewater [127]. Simultaneous nitrification and denitrification reactions can occur spontaneously in practice and are widely distributed in the environment [127,150], therefore simultaneous heterotrophic nitrification and aerobic denitrification (SND) play an important role in wastewater treatment plants and nitrogen removal reactors [151]. For example, Acinetobacter sp. is already used in full-scale A2O operation for nitrogen removal from pig farm wastewater (

Table 3. Isolated species.

Origin of Species	Specification of Species	References
Activated sludge	Acinetobacter sp. ND7	[153]
	Ochrobactrum anthropic LJ81	[147]
	Alcaligenes faecalis NR	[152]
	Achromobacter sp. GAD3	[151]
	Agrobacterium sp. LAD9	[151]
	Acinetobacter sp. SZ28	[127]
	Acinetobacter sp. WB-1	[127]
	Ochrobactrum sp. KSS10	[28]
	Pseudomonas stutzeri CFY1	[152]
	Thauera sp. FDN-01	[154]
	Diaphorobacter sp. PD-7	[127]
Artificial lake	Acinetobacter sp. H36	[148]
	Acinetobacter sp. CN86	[148]
Domestic wastewater	Achromobacter xylosoxidans CF-S36	[155]
	Paracoccus denitrificans ISTOD1	[156]
	Klebsiella pneumoniae CF-S9	[155]
Drinking water reservoir	Zoogloea sp. N299	[157]
Flooded paddy soil	Arthrobacter arilaitensis Y-10	[158]
	Pseudomonas tolaasii strain Y-11	[158]
Laboratory-scale MBR	Acinetobacter calcoaceticus HNR	[159]
	Bacillus methylotrophicus L7	[127]
	Serratia sp. LJ-1	[129]
Laboratory-scale SBR	Acinetobacter junii YB	[160]
	Pseudoxanthomonas sp. YP1	[161]
Landfill leachate	Zobellella taiwanensis DN-	[147]
Seabed sludge	Paracoccus versutus LYM	[162]
Songhua River	Microbacterium esteraromaticum SFA13	[163]
Wastewater system	Acinetobacter sp. YS2	[144]
	Cupriavidus sp. S1	[161]
	Pseudomonas sp. yy7	[152]
	Rhodococcus sp. CPZ24	[152]

Source: own.

) [152,153].

This graph visualizes the relative contribution of different strains of bacteria capable of simultaneous nitrification and denitrification (SND) and nitrogen transformation processes. It shows the contribution of individual strains (A, B, C, D, F) and their overall contribution to denitrification and nitrification, emphasizing the role of these microorganisms in the nitrogen cycle.

Recent research in the area of HNADMs/SND focuses on microbial engineering to select and optimize strains capable of more efficient nitrogen removal under different environmental conditions, as well as on integrating these technologies with existing wastewater treatment systems to maximize efficiency and minimize carbon footprint [153].

3.8.7. EM and Germanium (Ge)

Germanium (Ge) is a semi-metal with atomic number 32. Its organic form is banned in Europe for therapeutic purposes. Ge is associated with oxygen transport in the blood, acting as an excellent oxidant and antioxidant, protecting against aging and degenerative diseases [162]. Some attribute to it the ability to stimulate immune function, deliver oxygen to cells, detoxify, protect against radiation, and inhibit tumor growth [162].

Soil contamination with metalloids such as Ge is a global problem affecting human, plant, and animal health. Even low concentrations are dangerous. Metalloids can significantly affect the structure and biodiversity of soil microbial communities, which can lead to changes in ecosystem functioning [162].

EMs can be used with other techniques, such as germanium. Ge has been shown to have beneficial effects on plants, increasing their growth and stress tolerance. Combining EMs with Ge enhances the uptake and utilization of Ge by plants, leading to increased growth and improved yields [163,164].

Overall, the use of EMs has the potential to bring many benefits to sustainable agriculture, such as improving soil health, reducing dependence on synthetic chemicals, and increasing crop yields. However, ethical issues need to be considered, such as the fear of releasing GMOs into the environment, and there is a need for further research to determine the possibilities and limitations of this technology [163,164].

In summary, microbial biotechnology has the potential to play an important role in the use of EMs in various applications in sustainable agriculture, such as biological crop protection, composting, and improving nitrogen cycling [164]. However, there are still important ethical issues, such as the possibility of releasing GMOs into the environment, and the urgent need for continued research to better understand both the potential possibilities and limitations of this technology. In sustainable agriculture, the assessment of both benefits and risks associated with the use of EM preparations is important for food safety, consumer safety, and the human environment.

3.8.8. Inhibition of the Growth of Various Pathogenic Microorganisms Using EMs

EMs are increasingly used in various areas of life, e.g., in environmental protection [136]. Biological wastewater treatment processes depend on mixtures of microbial communities, including bacteria, fungi, rotifers, and algae. Municipal wastewater contains a wide range of pathogenic and non-pathogenic bacteria, including e.g., Salmonella, which can cause diseases in humans and animals [145,160]. Fungi may also occur in wastewater, the presence of which is highly undesirable. These are multi-cellular, non-photosynthetic organisms, mainly aerobic, but anaerobic species are also known. Due to their different cellular composition, they tend to dominate over bacteria in various wastes and wastewaters poor in nitrogen or with low pH. They may have a filamentous form, which makes their removal by sedimentation techniques difficult [145,146]. A promising technology in wastewater treatment for undesirable microorganisms is EM technology [160].

Mathews and Gowrilekshmi [127] proved that the application of EMs to stagnant rice water changed its physical, chemical, and biological properties. Chemical parameters (pH, TDS, and BOD) showed a decreasing trend. On the other hand, salinity, conductivity, and dissolved oxygen increased. Moreover, it was noticed that, under the influence of EM technology, there was almost a two-fold reduction in the population of Lactobacillus, Actinomyces, and yeasts in the first 2 weeks after EM application [127].

EMs used locally in Iraq [115] proved to be one of the environmental solutions for reducing water salinity and thus improving its quality, both in soil and sewage systems. Mouhamad et al. [115] showed that the effectiveness of EM technology can help farmers use drainage and groundwater for irrigation and make better use of water resources [115].

3.9. Microbiological Organisms in Sustainable Agriculture

Microbiological organisms, including bacteria, fungi, viruses, and yeasts, play an improvement role in sustainable agriculture. They are involved in various processes, such as nutrient cycling, nitrogen fixation and infestation and pest control and also in weed management, disease management, and fertilization management. They can provide numerous benefits, such as improving soil health, increasing crop yields, and reducing the reliance on synthetic chemicals. For example, nitrogen-fixing bacteria, such as rhizobia, are applied in sustainable agriculture to improve the nitrogen content in the soil. These macroelements are essential nutrients for plant growth and development, and nitrogen-fixing bacteria can convert atmospheric nitrogen into a form that plants can use [138,139,141].

This can reduce the need for synthetic nitrogen fertilizers, which can be costly and have negative environmental impacts. Another example is the use of mycorrhizae, a group of fungi that form symbiotic relationships with plants, to improve nutrient uptake and water retention. Mycorrhizae can improve the efficiency of nutrient uptake by extending the root system of plants and by increasing the surface area for absorption. They can also improve water retention by increasing the water capacity of the soil and reducing water loss due to evaporation. In addition to the use of nitrogen-fixing bacteria and mycorrhizae, microbiological organisms can also be used for pest control. For example, bacteria such as Bacillus thuringiensis (Bt) can be used as bioinsecticides to control insect pests, and fungi such as Beauveria bassiana can be used as biofungicides to control fungal diseases [2]. Overall, the use of microbiological organisms in sustainable agriculture has the potential to provide numerous benefits, such as improving soil health, increasing crop yields, and reducing the reliance on synthetic chemicals [1,3].

EMs have a comprehensive effect on the environment of crop plants, protecting them against pests and stimulating their resistance to diseases and unfavorable abiotic and biotic conditions. They can be applied before sowing for seed bioconditioning, and foliar and soil applications can be used [44,88,89,90,92,109,110]. In order to better protect the crop environment against weeds, it is beneficial to propose the use of bioherbicides in weed control, knowing their sources, sources, target weeds, and ecosystems (

Table 4. Herbicides and their respective sources, target weeds, ecosystems, and registered names.

Source	Target Weeds	Ecosystem	Registered Name	References
Alternaria cassiae	Cassia obtusifolia L.	Soy	Recipe development— “CASST”	[54]
Alternaria destruens	Cuscuta spp.	Cranberry field	Assessment—Smolder	[11]
C. purpura	P. Serotina	Forest	Commercialized—Biochon TM	[61]
C. purpura	Populus euramericana	Guinier forest	Commercialized—Chontrol®	[57]
Cephalospprium diospyri	Diospyras virginiana L.	Pastures, pastures	Oklahoma	[53]
Chondrostereum purpureum (Fr.)	Pouz Prunus serotina Ehrh.	Forest, mountains	Commercialized—Mycotech™	[54]
Citrus lime (L.) Osbeck	D. Sanguinalis	Cultivated areas	Commercial herbicide—Avenger®	[51]
Citrus sinensis (L.) Osbeck	Solanum nigrum L.	Crop land, roadside	Commercialized—Green Match™	[62]
Colletotrichum gloeosporioides	Hakea sericea Schrad. & J.C. Wendl.	Mountain meadows	Commercialized—Hakak	[55]
Colletotrichum gloeosporioides	Malvae, Malva Pusilla Sm.	Flax, lentils, horticultural crops	Commercialized—BioMal® [56]	[56]
Colletotrichum gloeosporioidesaeschynomene	Aeschynomene virginica L.	Rice, soybeans	Commercialized—Colle™	[54]
Cylindrobasidium	leave Acacia spp.	Forest, pasture	Commercialized—Stump-Out™	[61]
Cymbopogon citratus (DC.)	Stapf. spp.	Agricultural land	Commercialized—Green Match™ EX	[65]
Phoma macrostoma	Reynoutria japonica Houtt.	Golf courses, agriculture, and agroforestry	Commercialized—Phoma	[59]
Phytophthora palmivora	Morrenia odorata (Hook. & Arn.) Lindl.	Citrus groves	Commercialized—Devine™	[54]
Puccinia thlaspeos C. Shub.	Isatis tinctoria L.	Forest, pastures	-Beloukha® [62]	[64]
S. aromaticum	E. crus-galli	Farmland, rice	Commercialized—Weed Slayer®	[59]
Sclerotinia minor Jagger.	Taraxacum sp.	Turf	Commercialized—Sarritor® [60]	[61]
Streptomyces acidi scabies	Taraxacum officinale L.	Turf	Commercialized—Opportune®	[59]
Syzygium aromaticum (L.) Merr. & LM Perry & Presl. Cinnamomum verum J.	E. crus-galli	Rice, farmland	Commercialized—WeedZap®	[53]
Xanthomonas campestris	Poa annua L.	Turf, athletic fields	Commercialized—Camperico	[55]

).

Soil is a natural habitat for numerous organisms coexisting and protecting each other. Richness in organic and nutritional substances, appropriate air–water ratios, and appropriate reactions create an optimal environment for the life and development of microorganisms, which, with the participation of enzymes, stimulate the transformation of organic and mineral components in the soil. Thus, they participate in the creation and shaping of its fertility and enrich it with biogenic elements, growth substances, antibiotics, and other biologically active substances. This is how they influence the conditions for plant growth and development [1,2,41,54,99]. Ill-considered management of organic substances with microorganisms present leads to destruction, erosion, and decreases in soil fertility [99,111,116]. In order to eliminate unfavorable changes in the soil environment, attention is drawn to the need to increase the number of beneficial microorganisms by inoculating the soil with various microbiological preparations, such as UG-Max [109,110] or EM preparations [105,108,126]. Their development leads to the enrichment of microflora characterizing a given soil [54,60,61].

3.10. Soil and Microorganisms: Basics and Impact of EMs

Soil is a natural environment for countless organisms, coexisting and protecting each other. Rich in organic and nutritional substances, with appropriate air–water relations and proper pH, it creates optimal conditions for the life and development of microorganisms. These, with the participation of enzymes, stimulate the transformation of organic and mineral components in the soil, participating in the formation of its fertility, enriching it with biogenic elements, growth substances, antibiotics, and other biologically active compounds. In this way, they affect the conditions for plant growth and development [1,2,41,54,99]. Improper management of organic substances with microorganisms leads to degradation, erosion, and a decrease in soil fertility [99,111,116]. To counteract this, the need to increase the number of beneficial microorganisms by inoculating the soil with various microbiological preparations, such as UG-Max [109,110] or EM preparations [105,108,126], is emphasized, which leads to the enrichment of soil microflora [54,60,61].

Kaczmarek et al. [89] showed that microorganisms in microbiological preparations increased the number of bacteria, fungi, actinomycetes, and copiotrophic microorganisms in light soil, while inhibiting oligotrophs. Gałązka et al. [54] showed that EM1 and EM2 preparations only affected the growth of cellulolytic bacteria. Kaczmarek et al. [88] proved that EM preparations stimulated the activity of soil dehydrogenases and supported its respiration. Gałązka et al. [54] found that EMs affected the physical and water properties of soil material from arable-humus horizons of mineral soils, causing a decrease in density while increasing porosity [90]. Furthermore, the use of EMs on soils with a heavier mechanical composition improved their natural drainage, allowing for faster drainage of excess gravitational water while maintaining high field water capacity [88]. Kaczmarek et al. [88] observed an increase in the maximum and minimum capillary capacity. The addition of EMs also increases the rate of water filtration in soil with a heavier granulometric composition and reduces it in light soil. These changes have a positive effect on field conditions. A positive effect of EMs on the compressive strength of soil aggregates, an increase in their dynamic and static water resistance, and an increase in the quantity and quality of secondary aggregates were also found, which limit soil surface crusting and erosion [54]. EMs consist of two-layer gel capsules with an anaerobic interior and an aerobic external environment, limited by a zone of facultative microorganisms [1,54]. EMs are absorbed on the surface of mineral particles, and the attached capsules repel soil particles, affecting the loosening of its structure [1,54]. Shah et al. [2] showed that the use of meat and bone meal with EM addition reduces the caking and spraying index and increases the soil structure index. Quiroz et al. [125] noted a beneficial effect of EMs on the chemical properties of the tested soil. Ji et al. [74] claim that EMs, by increasing the number of beneficial microorganisms, contribute to faster mineralization of organic carbon.

Kaczmarek et al. [89,90] report that EMs applied to lessive soil contribute to the increase in the amount of available potassium, magnesium, and mineral nitrogen but cause a decrease in its buffering capacity, which was confirmed by the studies of Pszczółkowski et al. [95,96]. In chernozem, a decrease in the content of macroelements, mineral nitrogen, and hydrolytic acidity was observed, as well as an increase in buffering capacity with the increase in the dose of microbiological preparation. However, Sawicka et al. [98] did not find a significant effect of EMs on the increase in the amount of organic carbon, humus, and pH.

Effective microorganisms fulfill their role especially in heavily degraded, damaged, and poor soils, where there is a lack of microorganisms necessary for the proper development and even survival of plants in unfavorable conditions [103,117,141,142,143,144,145]. According to Sawicka et al. [6], in soils degraded by the mining industry, the use of EMs in perennial legume crops brings good results.

EMs are easy to produce and use, safe for humans, animals, and the environment, are low-cost, are effective, and can contribute to the sustainable improvement of agriculture and the environment [1,6,115]. Cultivation technology using EM preparations is currently used where it has been found that EMs are the best way to replace traditional cultivation and management methods, in agriculture, forestry, horticulture, and in water management, using natural methods [1,2,27,100,115,127,145,164].

Soil Microorganisms Enhance Plant Hea

Many researchers have taken a multi-faceted approach to the role of soil microorganisms, including diatoms [42,43,44]. However, a few scientists have tried to determine the potential and mechanism of action of diatom organisms. Stanek-Tarkowska et al. [63] studied the effect of short-term fertilization with ashes from biomass combustion on microbial communities, mainly diatoms, occurring on the surface of lessive soil. As a result, they identified 23 diatom species, including mainly: Hantzschia amphioxis, Manam atomus, Mayamaea allowis, Nitzschia pusilla, Pinnularia obscura, and Pinnularia schoenfelderi, but the most numerous populations were definitely Stauroneis thermicola. They showed that the main factor determining the biodiversity of diatoms was soil moisture.

The role of beneficial microorganisms, occurring naturally or intentionally introduced into the environment to ensure plant well-being, is indisputable. The mechanism of their action is based on increasing plant tolerance to stress conditions by supporting root growth and plant development and inducing their resistance. Microorganisms occurring in the soil or introduced into it in the form of microbiological preparations are an excellent alternative to conventional, chemical methods of protection in plant cultivation. The results of these authors confirm that plants, under the influence of contact with beneficial diatoms, bacteria, or fungi, produce secondary metabolites. In this respect, this is very important, because diatoms react relatively quickly to chemical changes in the environment [44,106].

Studies on the diversity of diatom communities have shown that these organisms are sensitive to anthropogenic factors, such as soil pollution, plant protection products, fertilization, and plant growth regulators, which disrupt their development. Terrestrial diatoms appear to be sensitive to many environmental factors, such as anthropogenic disturbances, soil pH and moisture, or nitrogen content in the soil. Meanwhile, most aquatic diatom species are well known, and their autecology is well known, qualifying them as biological indicators of water purity. This significant potential has not yet been exploited for terrestrial diatoms [127].

Pioneering discoveries were made on autecological values for soil pH and moisture for common terrestrial diatoms and compared with previous indicator values and tolerances. Data from ecological studies conducted under different climatic conditions were compared and it was found that validation indices improved significantly after removing samples taken from anthropogenic, disturbed habitats. This suggests that anthropogenic disturbance is a major factor determining the occurrence of taxa. International work by scientists in this field has been successful and has now allowed extending the list of terrestrial diatoms with taxa that can be treated as environmental markers in various soil studies. Stanek-Tarkowska et al. [63] discovered a new, undescribed species of the genus Microcostatus in Poland and named it Microcostatus dexteri sp. nov., describing it in the paper “Description of a new diatom species—Microcostatus dexteri sp. nov. from terrestrial habitats in southern Poland.” This species is small (3–9 μm) and rarely encountered. It is the fourth species described by this author recently. Discovering and describing new diatom species is very important for science. Studies on soil diatom communities developing in the surface layer of the soil are niche, extremely rare studies and therefore very valuable for the development of soil science, enriching new knowledge that allows discovering new relationships between abiotic and biotic components of the environment and discovering new diatom species. This research can contribute to the development of environmental sciences and at the same time be a source of knowledge for agricultural practice. In summary, systems and technologies using microorganisms have many advantages, such as increasing food production, protecting plants, and improving soil quality. At the same time, there are also disadvantages, such as uncontrolled growth of microorganisms and the risk associated with GMOs, which should be considered when assessing their use.

4. Discussion

4.1. Criteria for Assessing the Effectiveness of Microbial Technologies

When discussing the effectiveness of microbial technologies in agriculture, it is crucial to consider the following criteria:

-

Targeted Effectiveness: The ability to precisely control weeds, pests, or pathogens while minimizing the impact on beneficial organisms. The measure is related to population/disease reduction and selectivity [75,79,81].

-

Agricultural Efficiency: Measurable impact on crop yield and nutrient efficiency (reduced demand for chemical fertilizers) and increased plant resistance to abiotic and biotic stresses [54,79,80].

-

Soil and Environmental Health: Contribution to improving soil quality (e.g., organic matter content, microbiological activity) and reducing the ecological footprint of agriculture (lower chemical use, lower emissions) [39,45,79].

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Economic and Practical Aspects: Cost-effectiveness for the farmer (cost–benefit ratio), ease of use, and stability of preparations and their compliance with existing agricultural practices and organic certification requirements [123].

The criteria for assessing the effectiveness of microbiological technologies in agriculture are raised and cited by a number of entities depending on the context (science, regulations, agricultural practice).

The main entities raising and citing these criteria are:

-

Research institutions and universities, such as the Institute of Soil Science and Plant Cultivation—National Research Institute (IUNG-PIB) in Puławy, Poland, are key centers that conduct research on microbiological preparations, evaluate their effectiveness, and create guidelines. Their scientific publications and guides for farmers often include detailed criteria [54,104,112].

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Universities of life sciences (e.g., Poznań University of Life Sciences, Maria Curie-Skłodowska University in Lublin, which is mentioned in the text in the context of research on biopreparations) and agricultural and microbiological faculties around the world. Researchers from these institutions publish articles in peer-reviewed scientific journals that present research results in accordance with these criteria.

-

International research centers, including organizations such as the International Center for Tropical Agriculture (CIAT), the International Maize and Wheat Improvement Center (CIMMYT), or the World Agroforestry Center (ICRAF), also conduct extensive research in this area.

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Agricultural and development organizations, such as the Food and Agriculture Organization of the United Nations (FAO), which promotes sustainable agricultural practices and often publishes reports and guidelines on the use of innovative technologies, including microbiological ones, to improve food and environmental security [47].

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As an organization promoting organic agriculture, the International Federation of Organic Agriculture Movements (IFOAM Organics International) sets standards and criteria for products permitted for use in this method of cultivation, which often include detailed guidelines for organic preparations [47].

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In Poland, national ministries of agriculture and regulatory agencies, such as the Ministry of Agriculture and Rural Development, together with subordinate institutions (such as IUNG-PIB), develop and implement regulations on the registration and marketing of microbiological fertilizer products, which require an assessment of their efficacy and safety [50,51,52].

-

In order to obtain registration and introduce products to the market, biotechnology companies and producers of biopreparations must comply with and present data confirming the efficacy of their products in accordance with the guidelines of regulatory bodies. They often cite their own studies and studies of independent institutes [51,52].

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World Organization for Animal Health (WOAH) and World Health Organization (WHO) mainly focus on animal and human health, but their interest in antimicrobial resistance (AMR) makes them indirectly raise the effectiveness criteria for alternatives to antibiotics, including microbiological solutions in agriculture, in order to reduce the use of chemical plant protection and veterinary products that may contribute to AMR [47].

In summary, these criteria are shaped and cited by a full spectrum of scientists, research institutions, regulators, and industry organizations that strive to develop and implement sustainable solutions in agriculture

4.2. Numerical Estimates of the Possible Impact of Using Technology in Various Technological Processes

Here are some estimated impacts and areas where numerical data would be crucial for a comprehensive assessment:

Yield Increase: While not explicitly stated with a universal percentage, studies often report 5–20% or even higher yield increases with effective biofertilizer and biostimulant application, especially under stress conditions or in nutrient-deficient soils. For instance, specific biofertilizers could lead to a 10–15% increase in legume yields due to enhanced nitrogen fixation [80,84,85].

Reduction in Synthetic Fertilizer Use: Microbial technologies, particularly nitrogen-fixing bacteria and phosphorus-solubilizing microorganisms, could lead to a reduction of 20–50% in synthetic N and P fertilizer application without compromising yields, thereby lowering production costs and environmental pollution [29,31,109,110,121].

Reduction in Chemical Pesticide Use: The adoption of biopesticides (bioherbicides, bioinsecticides, biofungicides) could result in a decrease of 15–40% or more in chemical pesticide application, significantly reducing chemical residues in food and the environment [11,12,13,14,15,16,17,18,19,20,21,22,23]. For example, widespread use of Bacillus thuringiensis formulations could dramatically reduce the need for synthetic insecticides in specific crops [11,12,13,48].

Improved Nutrient Uptake Efficiency: Microbial inoculants can enhance the uptake of essential nutrients like potassium, phosphorus, and zinc by up to 25–50%, leading to more efficient resource use by plants [87,117,138].

Soil Organic Matter Increase: While slower to quantify, continuous use of microbial preparations and sustainable farming practices can contribute to an annual increase of 0.1–0.5% in soil organic carbon over several years, crucial for long-term soil health [13].

Water Use Efficiency: Mycorrhizal fungi, for example, can improve a plant’s drought resistance, potentially leading to a 10–20% improvement in water use [41,51,88,89,144].

4.3. Comparative Assessment of the Impact of Existing Technologies on Product Safety and Production Efficiency

Based on the texts provided, we can conduct a comparative assessment of the impact of existing microbial technologies on product safety and production efficiency. The texts clearly contrast the conventional (chemical-based) approach with the microbial approach, highlighting the advantages and challenges of each.

a.

Bioherbicides

• Production Efficiency:

• Potential: The texts indicate that bioherbicides, such as those produced from Lasiodiplodia theobromae or L. pseudotheobromae, can be effective in controlling monocotyledonous and dicotyledonous weeds. This offers a promise for effective control [68].
• Challenges:

○

Resistance: Similar to synthetic herbicides, there is a risk of resistance development in weed populations, which could decrease their effectiveness over time [26,103,115].

○

Limited Commercial Application: Despite promising research, “only a few live microbial bioherbicides have been approved for commercial use and have been placed on the market, but their use has unfortunately been very limited for a number of reasons, mainly economic.” This indicates a barrier to widespread adoption [69,70,77,83].

○

Specificity: In many cases, the pathogen is a fungus, which might imply specificity of action against particular weed species, requiring precise selection [67,103].

• Product Safety:

• Advantages:

○

Natural Origin: Bioherbicides are produced from microorganisms, plant extracts, and minerals (e.g., sulfur, although the text notes sulfur is more of a fungicide than herbicide). This makes them generally “safer and more environmentally friendly” than synthetic pesticides because they are often biodegradable and have less toxicity to non-target organisms [68].

○

No Chemical Residues: Ideally, bioherbicides “must not leave any chemical residues in the crop,” which is crucial for organic and biodynamic farming [24,80,103,130].

○

Human Safety: While emphasizing that “the safety of the tested strains of bacteria for humans” is an important issue, their natural origin generally suggests lower risk [10,11,12,13,14].

b.

Bioinsecticides

• Production Efficiency:

• Potential: High effectiveness is demonstrated. For instance, Bacillus thuringiensis (Bt) produces toxins harmful to caterpillars and mosquitoes, and fungal biopesticides (e.g., Beauveria bassiana) attack insects. Viral insecticides (baculoviruses) effectively control Lepidoptera larvae [12,16].
• Challenges:

○

Resistance: Similar to synthetic insecticides, there is a risk of resistance development in insect populations. “This fact may lead to faster development of insect populations resistant to bioinsecticides” [54,75,164].

○

Environmental Conditions: The effectiveness of preparations containing live fungal spores (e.g., Beauveria bassiana) depends on environmental conditions (humidity, temperature) conducive to germination and infection [68].

• Product Safety:

• Advantages:

○

Natural and Safe: Bioinsecticides “are made from natural materials, such as microorganisms, plant extracts, and minerals” [91,103,120].

○

Low Toxicity to Non-Target Organisms: Bt toxins, for example, are “toxic to insects when ingested but are generally considered to be safe for humans and other mammals” [19,22,25,119].

○

Reduced Chemical Residues: They reduce the need for chemical pesticides, leading to lower or no residues in food [59,97,98,99].

○

Biodiversity Promotion: They can support plant well-being in organic farming systems, e.g., through the use of T. asperellum against Botrytis cinerea and S. sclerotiorum [31,33,124,142].

c.

Effective Microorganisms (EMs)

• Production Efficiency:

• Potential:

○

Improved Soil Microbiological Balance: Thanks to an optimal strain composition [33,92].

○

Mineralization and Nutrient Availability: They accelerate processes and reduce the need for mineral fertilizers [29,33,92,130].

○

Soil Structure: Improved air–water relations, increased nutrient absorption, maintained soil moisture, and prevention of compaction [8,109].

○

Plant Resistance: Strengthened plant immunity and displaced pathogens, e.g., reducing the occurrence of Fusarium fungi [26,55,115].

○

Yield Increase: Studies in Japan, China, and Poland showed “a positive effect of EM on the yield and health of potatoes and wheat,” increased yields of onions, peas, beans, and a positive effect on ornamental plants [28,72,73,104,119,120,121,126].

• Challenges:

○

Controversy and Inconsistent Results: The text frankly admits there are “reports of both benefits from their use and their ineffectiveness.” It notes that “some studies, e.g., Van Vliet et al. [111], did not show a significant effect.”

○

Liberal Registration Procedure: Criticisms of the “liberal registration procedure,” which “indicates the need for further research on the mechanisms of EM action.”

○

Competition with Native Microflora: In the context of composting, the lack of an EM effect could be due to “competition with native microflora or parasitism/antibiosis phenomena.”

○

Not a Panacea: It is emphasized that “their role as a panacea for all problems should not be overestimated.”

• Product Safety:

• Advantages:

○

Natural and Eco-friendly: They are a “safe alternative to synthetic fertilizers and conventional plant protection products” [59,103].

○

Reduced Chemicalization: The main benefit is a decrease in chemical use in agriculture [59,80,130].

○

Potential Human Health Benefits: In the context of potato cultivation, EMs “can reduce digestive system ailments in humans by reducing the absorption of fats during frying potato products” [27,56,57].

d.

Mycorrhizal Preparations

• Production Efficiency:

• Potential:

○

Increased Nutrient Uptake: Mycorrhizal fungi enhance the uptake of water and minerals (nitrogen, phosphorus, potassium, micronutrients) by plants [40].

○

Stress Resistance: Increased resistance to frost and salinity.

○

Improved Plant Growth and Appearance: More abundant flowering, better rooting.

○

Reduced Drought Damage: Arbuscular mycorrhizal (AM) fungi affect osmoregulation in plants under moderate water deficit and promote greater absorption of phosphorus and nitrates under good water supply, increasing plant dry mass [33,34,35,36,40].

○

Long-Term Effect: Applied once, they can benefit the plant throughout its life [36,40,142,143].

• Product Safety:

• Advantages:

○

Natural Symbiosis: Mycorrhiza is a natural symbiosis between fungi and plant roots, making it inherently safe [34,35,36,143].

○

No Chemical Residues: They do not introduce artificial substances into the environment or products.

○

Ecosystem Support: Strengthened natural soil processes and plant resistance, which is beneficial for the entire ecosystem [34,142].

e.

Microorganisms in Aerobic Nitrification and Denitrification (Wastewater Treatment Perspective)

• Process Efficiency (not necessarily agricultural production, but technological processes):

• Potential:

○

High Nitrogen Removal Efficiency: Heterotrophic nitrifying and aerobic denitrifying microorganisms (HNADMs) and simultaneous nitrification and denitrification (SND) processes “have the ability to both nitrify and denitrify,” allowing for effective nitrogen removal from wastewater [85,146].

○

Cost Reduction: SND technology “significantly reduced costs and improved the efficiency of denitrification” [92,146].

○

Practical Application: Examples like Acinetobacter sp. used in pig farm wastewater treatment plants demonstrate their real-world application [146,147].

• Product Safety (more “Environmental Safety” and “Water Quality”):

• Advantages:

○

Reduced Nitrogen Pollution: Effectively remove nitrogen compounds that cause water eutrophication and air quality deterioration and negatively affect human and animal health. This is crucial for aquatic environmental safety [59].

○

Minimized Emissions: These processes convert harmful forms of nitrogen (e.g., ammonia) into harmless nitrogen gas (N₂), reducing pollution [85].

A comparative summary is shown in

Table 5. Impact of Microbial Technologies on Product Safety and Production Efficiency.

Microbial Technology	Impact on Product Safety (Measurable Criteria)	Impact on Production/Process Efficiency (Measurable Criteria)
Bioherbicides	Increases: -Reduction in chemical residues in product: Target: ≥90% reduction compared to synthetic herbicides. -Toxicity to non-target organisms: Target: ≤5% mortality/damage compared to control.	Potentially increases: -Weed control efficacy: Target: ≥80% reduction in weed biomass. -Crop yield: Target: ≥5% yield increase. -Challenges: Resistance development (monitoring), commercialization metrics (market share).
Bioinsecticides	Increases: -Absence/reduction of chemical residues in product: Target: Complete absence or >95% reduction. -Pest specificity: Target: ≤5% impact on beneficial insects. -Safety for humans/animals: Target: WHO Toxicity Class IV (lowest hazard).	Increases: -Pest control efficacy: Target: ≥70% reduction in pest population. -Crop loss reduction: Target: Reduction of losses by ≥10%. -Challenges: Resistance development (monitoring), reliance on environmental conditions (e.g., optimal temp.).
Effective Microorganisms (EMs)	Increases: -Reduction in chemical fertilizer use: Target: ≥15% reduction. -Food quality indicators: Target: ≥5% increase in vitamin/nutrient content (e.g., Vit. C, protein).	Increases (with variable results): -Increase in soil organic matter content: Target: Increase by ≥0.1% annually. -Nutrient availability (P, K, N): Target: ≥10% increase in soil analyses. -Yield increase: Target: Variable (ranging from −5% to +20% depending on crop and conditions). -Controversial issues: Repeatability of results under different conditions.
Mycorrhizal Preparations	Increases: -Absence of chemical residues in product: Target: Complete absence. -Natural symbiosis: Target: Confirmed root colonization (e.g., ≥50% of roots colonized).	Increases: -Nutrient uptake (P, N): Target: ≥20% increase. -Water uptake: Target: Increased drought tolerance (e.g., ≤10% yield reduction under water deficit). -Stress resistance: Target: ≥15% reduction in stress symptoms (e.g., salinity). -Long-lasting effects: Effect maintained for ≥2 seasons.
Microorganisms in Nitrification/Denitrification (Wastewater)	Enhances environmental safety: -Reduction in total nitrogen (TN) concentration in wastewater: Target: ≥90% TN removal efficiency. -Compliance with environmental standards: Target: TN levels fall below permissible limits.	Increases process efficiency: -Nitrogen (N) removal efficacy: Target: ≥95% N removal efficiency. -Treatment cost: Target: Reduction in energy/chemical costs by ≥10%. -Hydraulic retention time (HRT): Target: Shortening of HRT by ≥15%.

Source: own.

.

4.4. Microbiome in the Fight Against Abiotic Stresses: Recent Achievements and Perspectives in Sustainable Agriculture

In the face of increasing climate change, such as droughts and soil salinization, agricultural microbiology is becoming a key tool in ensuring global food security. Modern research is increasingly exploring the interactions between plants and their microbiome complex community of microorganisms inhabiting the soil (soil microbiome) and internal and external plant tissues (plant microbiome, including endophytes and rhizosphere). Recent advances focus on using these microbes to increase plant resistance to abiotic stresses, which is fundamental for sustainable agriculture [60,103].

Rhizosphere and endophytes as a plant fortress: Traditionally, attention has focused on macrosymbioses, such as mycorrhiza or nitrogen fixation by Rhizobium bacteria. Today, with advanced techniques such as metagenomics, metatranscriptomics, and metaphelonomics, we understand that the entire microbial community, from bacteria to fungi and archaea, plays a synergistic role in plant adaptation to harsh conditions [42,49].

4.4.1. Role of the Rhizosphere in Drought Tolerance

The rhizosphere, the zone of soil immediately surrounding plant roots, is a real hotspot of microbial activity. Plant-growth-promoting rhizobacteria (PGPR) and mycorrhizal fungi are key players. Recent studies [32,86,155] show that PGPR in drought conditions can:

-

Increase water availability: By producing exopolysaccharides (EPSs), which improve soil aggregation and water retention. Some strains can also affect root architecture, increasing their ability to explore soil volume.

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Modulate plant hormones: By producing auxins, gibberellins, or cytokinins, which help plants maintain turgor, delay wilting, and stimulate root growth in search of water [76].

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Support water management: The activity of the ACC deaminase enzyme in some PGPR reduces ethylene levels in stressed plants, which alleviates the negative effects of drought [32,155].

4.4.2. Arbuscular Mycorrhizal Fungi (AMF)-Advanced Mechanisms

The great role of AMF is emphasized, but recent studies [41,142,143] deepen this understanding:

-

Change osmoregulation: Many studies have confirmed that AMF affects the accumulation of osmolytes, such as proline, soluble sugars, and amino acids, which protect plant cells from damage by dehydration.

-

Antioxidation: They activate the antioxidant system of plants (increased activity of enzymes such as superoxide dismutase, catalase, peroxidase), reducing the production of reactive oxygen species (ROS) under stress.

-

Increased nutrient uptake under stress: Even with limited water resources, the network of mycelium hyphae is expanded, which can more effectively supply phosphorus, nitrates and microelements, which is crucial for maintaining photosynthesis [34,49].

4.4.3. Endophytes—Hidden Heroes of Immunity

Endophytes are microorganisms living inside plant tissues, without causing disease symptoms. Currently, more attention is paid to fungal and bacterial endophytes [28,49,52,141], which:

-

Improve salinity tolerance: They can reduce sodium absorption by plants, increase tolerance to osmotic stress, and also modify the expression of genes related to ion transport and defense responses.

-

Produce secondary metabolites such as alkaloids, terpenoids, or polyphenols, which not only protect the plant from pathogens but also help it survive drought conditions, e.g., by reducing transpiration [59,71].

-

Induce systemic immunity: By activating signaling pathways such as salicylic acid (SA) and jasmonic acid (JA), similar to PGPR, but acting systemically from within the plant [12].

4.4.4. Latest Achievements and Research Directions

Precise “tailored” biopreparations: Thanks to the development of microbial genome sectioning and gene-editing techniques (CRISPR), scientists are able to identify and optimize genes responsible for the production of biostimulants and protective compounds. This allows the design of biopreparations with much greater specificity and effectiveness, adapted to specific soil conditions, plant species, and types of stress [53,55].

Multi-component microbial consortia: Instead of single strains, consortia of several microorganisms (e.g., combinations of PGPR, AMF, and endophytes) are increasingly being tested, which act synergistically, providing a wider range of benefits and greater resistance to changing environmental conditions [34,49].

Intelligent application systems: The development of microencapsulation technology and the use of prebiotics in biopreparation formulations significantly improve the stability and survival of strains in EM consortia, ensuring their long-term effectiveness in the field. In addition, drones and precision agriculture enable precise application of biopreparations at optimal rates and locations [3,95,113].

Plant priming research: New research focuses on the mechanisms by which microorganism’s “prime” plants for future stress. Plants previously exposed to these microbes respond faster and much more strongly to subsequent drought or salinity [114].

Interactions with other biostimulants: There is currently growing interest in the synergistic effects of microorganisms with other biostimulants, such as seaweed extracts (SwEs). They are rich in polysaccharides and phytohormones and can additionally enhance the response of plants to stress, affect their carbon and nitrogen metabolism, and improve mineral absorption, which is additionally intensified by the modification of root structure by microbes [30,92].

4.4.5. Summary and Outlook

The deepened understanding of the plant and soil microbiome is revolutionizing the approach to sustainable agriculture. Recent advances allow not only the identification of key microorganisms and their mechanisms of action but also the engineering of “smart” bio-based products. Using these innovations to increase plant resistance to drought and salinity is not only promising but is becoming an imperative for ensuring food security in a changing climate. Future research will focus on further integrating these technologies into precision agriculture practices to maximize their efficiency and scalability while minimizing environmental impact.

4.5. Challenges, Knowledge Gaps, and Future Research Directions

In the search for sustainable agricultural solutions, the soil microbiome offers enormous potential, but its full exploitation faces significant challenges. Although we have increasingly better tools such as metagenomics and metatranscriptomics, we still lack a full understanding of “who does what” and “why” in this incredibly complex ecosystem. Integrating the vast amount of data from different sources—from omics (genetic, transcriptional, metabolic) data to soil environmental parameters and plant performance—remains a critical but difficult task.

Predictability and scalability: One of the most pressing challenges is to transform promising laboratory discoveries into reliable and scalable solutions for large-scale agriculture. The success of microbial inoculants or microbiome management strategies is often variable under field conditions. Key factors influencing this variability include:

▪

Soil type: Different soils have different physicochemical properties and native microbial communities, which affect the adaptation and functioning of introduced microorganisms.

▪

Climate: Temperature, humidity, and rainfall dramatically affect microbial activity and survival.

▪

Plant species and variety: Each plant interacts uniquely with the soil microbiome, meaning that solutions often need to be tailored to specific crops.

▪

Type of native microbiome: The existing soil microbiome may resist or interact with new microorganisms in unforeseen ways [92,96,104].

Understanding these interactions and their impact on predictability is essential to developing universal or easily adaptable solutions.

4.6. Regulatory Issues and Farmer Acceptance

The introduction of innovative microbial products also faces numerous regulatory barriers. Regulatory authorities need to develop clear guidelines on safety, efficacy, and environmental impact, which is often a slow and complicated process. Farmer acceptance is equally important. New practices are often more complex than traditional methods and require knowledge of microbiology, which highlights the need for intensive education and support for agricultural producers in adopting these solutions. Without understanding the benefits and how they work, even the most innovative technologies will remain untapped.

The Potential of New Technologies: The Role of Artificial Intelligence and Modeling: In the context of the enormous complexity of the microbiome and the need to integrate multi-dimensional data, the potential of new technologies becomes crucial.

Microbial Gene Editing: If ethically, legally, and safely permissible, microbial gene editing could play a huge role in the future. This would allow for precise modification of metabolic pathways of bacteria or fungi, increasing their ability to fix nitrogen, solubilize nutrients, produce biostimulants, or even increase their resistance to harsh environmental conditions. This opens the door to creating “super-microbes” tailored to specific agricultural needs.

Artificial Intelligence (AI) and Modeling: These tools are absolutely essential to dealing with the “big data” generated by microbiome research.

Interaction Prediction: AI algorithms can analyze vast genomic, metagenomic, and environmental data sets to predict complex interactions between microbes, as well as between microbes and plants and soils. This would allow for the identification of key microbes and metabolic pathways responsible for efficient nitrogen use.

Strategy Optimization: AI can be used to model scenarios and optimize microbiome management strategies, such as selecting the right bioinoculants for specific soil and plant conditions. Machine learning systems can analyze historical data on the effectiveness of different microbial interventions in different environments to recommend the best practices.

Personalized Farming: In the long term, AI could enable the development of personalized farming solutions, where the microbiome and its interventions are tailored to the specific needs of each field or even each plant. This could revolutionize precision farming, minimizing fertilizer use and maximizing efficiency.

5. Conclusions

Agricultural microbiology is a breakthrough force in modern agriculture, offering irreplaceable tools for combating pathogens, increasing crop yields, and ensuring global food security. This field, utilizing microorganisms, plant extracts, and minerals, is driving a paradigm shift toward more sustainable agricultural practices. The main benefits and achievements are:

-

Microbial preparations are revolutionizing agricultural efficiency and environmental management, generating specific benefits confirmed by research, such as:

-

The use of Nod inoculants accelerates germination and increases the number of root nodules, which translates into a reduced need for synthetic nitrogen-containing fertilizers (up to 90%) and increased biomass production.

-

Mycorrhizal preparations have demonstrated the ability to increase nutrient (P, N) uptake by at least 20% and improve water uptake, resulting in reduced yield losses (up to 10%) in drought conditions.

-

Natural Plant Protection: Fungal biopreparations and other microbiological solutions effectively protect plants from pathogens. Bioinsecticides can achieve pest control efficacy of ≥70% population reduction, while simultaneously reducing yield losses by at least 10%.

-

Bioherbicides contribute to reduced chemical residues in the product (target: ≥90%) and reduced toxicity to non-target organisms (target: ≤5% mortality/damage). Bioherbicides can reduce weed mass by up to 80% and increase crop yields by ≥5%.

-

Abiotic Stress Resistance: Soil and plant microflora play a key role in increasing plant resistance to drought and salinity. Microbiological preparations can reduce stress symptoms (e.g., salinity) by at least 15%, improving plant tolerance.

-

Soil Health Regeneration: The use of effective microorganisms (EMs) can increase soil organic matter content (by at least 0.1% per year) and nutrient availability (P, K, N) by at least 10%. They also improve microbial balance and soil structure, increasing water retention.

-

Circular Economy: Microorganisms are essential for transforming organic waste into valuable resources (compost, biogas). Microorganisms in wastewater nitrification/denitrification processes can achieve total nitrogen (TN) removal efficiency of ≥90%, reducing treatment costs by at least 10% and shortening hydraulic retention time by at least 15%.

-

Precision Agriculture and Microbiome Engineering: Omics technologies (e.g., metagenomics, metatranscriptomics) enable a detailed understanding of the microbiome, enabling the design of precise, tailored biopreparations and the modification of microorganisms to enhance their effectiveness.

Challenges and Responsible Implementation: Despite their enormous benefits, there are challenges and ethical issues that must be addressed. These include:

-

Risk of Resistance: Like synthetic alternatives, microbial technologies can lead to the development of resistance in weeds, diseases, and pests. This requires continuous monitoring and product rotation.

-

Release of GMOs into the Environment: The potential release of genetically modified microorganisms (GMOs) raises ethical and regulatory concerns that require a robust legal framework and transparent dialogue.

-

Variability and Understanding Limitations: Results are sometimes inconsistent due to complex environmental interactions. For example, the effectiveness of EMs can be variable, with yield impacts ranging from −5% to +20%, depending on specific growing conditions. Continued investment in research and development (R&D) is essential to fully understand the possibilities and limitations, standardize applications, and predict outcomes.

The Way Forward: Integration and Responsible Innovation: The future of sustainable agriculture lies in integrating microbial biotechnology with other sustainable practices, such as regenerative agriculture and agroecology. By adopting a responsible approach to the development and implementation of microbial technologies, their full potential can be realized. This will significantly contribute to achieving the Sustainable Development Goals (SDGs) and building a more sustainable, equitable, and prosperous future for all.

In summary, the future of microbiology in agriculture depends on overcoming the challenges of system complexity, predictability, and acceptance, and AI and other advanced technologies will play a key role in unlocking the full potential of the microbiome soil for sustainable agriculture.