Harnessing Nitrogen-Fixing Cyanobacteria for Sustainable Agriculture: Opportunities, Challenges, and Implications for Food Security

Abstract

Nitrogen, an essential element for plant growth and food production, presents significant challenges in agriculture due to the environmental consequences of synthetic nitrogen fertilizers. This review explores the potential of nitrogen-fixing cyanobacteria as a sustainable alternative for agricultural nitrogen fertilization. The molecular mechanisms underlying nitrogen fixation in cyanobacteria, including key genes such as nif and related biochemical pathways, are examined in detail. Biotechnological approaches for utilizing nitrogen-fixing cyanobacteria as biofertilizers are discussed, alongside strategies for genetic engineering to improve nitrogen fixation efficiency. The review further evaluates the impact of cyanobacteria on soil health and environmental sustainability, emphasizing their role in mitigating the detrimental effects of synthetic fertilizers. While promising, challenges such as oxygen sensitivity during nitrogen fixation and competition with native microorganisms are critically analyzed. Finally, future directions are proposed, including advancements in synthetic biology, integration with conventional agricultural practices, and scalable implementation strategies. This review underscores the transformative potential of nitrogen-fixing cyanobacteria in promoting sustainable agriculture and enhancing global food security.

1. Introduction

The global challenge of sustaining agricultural productivity to meet the demands of a burgeoning population hinges significantly on the availability of essential nutrients, with nitrogen playing a pivotal role [1,2]. Nitrogen is a cornerstone element for plant growth, influencing crop yields and nutritional quality. As the backbone of amino acids, proteins, and chlorophyll, nitrogen is indispensable for the development of healthy and robust crops [3,4,5]. However, the conventional reliance on synthetic nitrogen fertilizers to meet agricultural nitrogen demands has ushered in a host of challenges, ranging from economic burdens to environmental degradation [6,7]. Increasing global demand for food production has led to widespread use of nitrogen fertilizers, raising concerns related to soil and water pollution, greenhouse gas emissions, and energy-intensive manufacturing processes [8]. The over-reliance on such fertilizers has raised concerns about their sustainability and long-term environmental impact. Therefore, there is an urgent need to explore alternative approaches that meet the nitrogen requirements of crops while mitigating the adverse effects of traditional fertilization practices [9,10]. This review article embarks on a journey that examines the importance of nitrogen in agriculture and its close connection to global food security. Furthermore, the production and application of nitrogen fertilizers are energy-intensive processes, primarily reliant on fossil fuels. The carbon footprint associated with fertilizer manufacturing contributes to greenhouse gas emissions, exacerbating climate change [11,12,13]. The nitrogen cycle is also intricately linked to the emission of nitrous oxide, a potent greenhouse gas with implications for both climate change and stratospheric ozone depletion. The overuse and mismanagement of nitrogen fertilizers can lead to soil degradation and long-term environmental consequences [14,15]. Types of nitrogen-fixing plants in terrestrial ecosystems can be categorized into four types of symbiotic nitrogen fixation: legumes with rhizobial bacteria, actinorhizal plants with Frankia bacteria, elm plants with rhizobial bacteria (e.g., Parasponia with rhizobial bacteria), and certain plants such as cryptogams and Gunnera with cyanobacteria [16,17,18]. The nitrogen fixation rates, the number of species, and the main distribution areas of these various nitrogen-fixing plants vary, as summarized in Table 1.

Considering these challenges, the exploration of nitrogen-fixing cyanobacteria presents a compelling alternative. By mitigating the adverse environmental impacts associated with synthetic fertilizers, the utilization of cyanobacteria for nitrogen fixation holds the potential to address the drawbacks of conventional nitrogen fertilization practices [19]. Nitrogen-fixing cyanobacteria, renowned for their ability to convert atmospheric nitrogen into forms usable by plants, emerge as natural biofertilizers that hold great promise for sustainable agriculture. These cyanobacteria, recognized for their capacity to fix atmospheric nitrogen into bioavailable forms, are generally free-living organisms and do not form a symbiotic relationship with plants as rhizobia do. Although they do not transfer nitrogen directly to crops, their nitrogen-fixing activity enriches the environment and makes nitrogen available for plant uptake [20,21]. This biological nitrogen fixation not only reduces the reliance on synthetic fertilizers but also contributes to soil fertility and health. Additionally, cyanobacteria have inherent nutrient cycling and biofertilization capabilities, further strengthening their role in sustainable agriculture. The implications of incorporating nitrogen-fixing cyanobacteria extend beyond immediate agricultural concerns. As the global population continues to grow, ensuring food security becomes an increasingly critical challenge. The adoption of innovative and sustainable agricultural practices, such as harnessing cyanobacteria, becomes imperative for meeting the rising demand for food while minimizing environmental degradation [22,23,24,25].

Table 1. The types of N-fixing symbioses and the ecological characteristics of nitrogen-fixers in terrestrial ecosystems.

N-Fixing Symbioses	Nitrogen Fixing Rate (kg N·ha1·year−1)	Nitrogen-Fixer and Total Number	Main Distribution Area	References
Mesorhizobium ephedrae	850	Ephedra (5 species)	Tropical low pH environments in Malaysia and the Western Pacific	[26,27]
Legumes/rhizobia	300~400	Brassinae (550~660)	Humid tropics	[28,29]
		Mimosineae (88~102)	Tropical subtropics (Asia, Africa, Australia, and North America)
		Papilioninae (921~973)	Woody individuals are mainly found in tropical subtropics, while herbs are mostly found in temperate and boreal forests
Actinomycorrhizal plants	15~90	Betulaceae, Casuarinaceae and Myriceaceae Rosaceae, rhamnaceae and Elaeagniaceae Masanaceae and four trees (200)	Temperate and high northern latitudes	[30,31]
Plants/cyanobacteria	2~41	Lichen (moss), fern	Extreme environments (deserts, grasslands, and frozen soils)	[32,33]
	—	Cycads alone (more than 250)	Arid woodland of Australia and South Africa	[34]
	72	Rhizome or rhizomes, petiole only (about 50)	Found naturally in the tropical humid mountains of the Southern Hemisphere (Hawaii to the central and southern United States, New Zealand, Southeast Asia, and the southernmost parts of South America)	[33]

Table 1. The types of N-fixing symbioses and the ecological characteristics of nitrogen-fixers in terrestrial ecosystems.

N-Fixing Symbioses	Nitrogen Fixing Rate (kg N·ha1·year−1)	Nitrogen-Fixer and Total Number	Main Distribution Area	References
Mesorhizobium ephedrae	850	Ephedra (5 species)	Tropical low pH environments in Malaysia and the Western Pacific	[26,27]
Legumes/rhizobia	300~400	Brassinae (550~660)	Humid tropics	[28,29]
		Mimosineae (88~102)	Tropical subtropics (Asia, Africa, Australia, and North America)
		Papilioninae (921~973)	Woody individuals are mainly found in tropical subtropics, while herbs are mostly found in temperate and boreal forests
Actinomycorrhizal plants	15~90	Betulaceae, Casuarinaceae and Myriceaceae Rosaceae, rhamnaceae and Elaeagniaceae Masanaceae and four trees (200)	Temperate and high northern latitudes	[30,31]
Plants/cyanobacteria	2~41	Lichen (moss), fern	Extreme environments (deserts, grasslands, and frozen soils)	[32,33]
	—	Cycads alone (more than 250)	Arid woodland of Australia and South Africa	[34]
	72	Rhizome or rhizomes, petiole only (about 50)	Found naturally in the tropical humid mountains of the Southern Hemisphere (Hawaii to the central and southern United States, New Zealand, Southeast Asia, and the southernmost parts of South America)	[33]

The subsequent sections of this review paper will unravel the biological intricacies that underpin the nitrogen-fixing capabilities of cyanobacteria and explore their potential application in addressing the limitations of conventional nitrogen fertilizers. The ensuing segments of this comprehensive review will intricately explore the biological nuances and pragmatic implementations associated with the utilization of nitrogen-fixing cyanobacteria. This paper aims to provide an exhaustive perspective elucidating the manifold ways in which these microorganisms can substantively enhance sustainable agricultural practices while concurrently mitigating the ecological apprehensions inherent in conventional nitrogen fertilization methodologies. Through this exploration, we aim to shed light on how these microorganisms can revolutionize agricultural practices, offering a pathway towards enhanced soil fertility, reduced environmental impact, and ultimately, greater food security for a growing global population.

2. Nitrogen Fixation Mechanisms

Nitrogen fixation in cyanobacteria is a complex and finely regulated process controlled by specific molecular mechanisms that allow them to convert atmospheric nitrogen into a form usable by plants. The use of nitrogen-fixing cyanobacteria as a replacement for nitrogen fertilizer in agriculture requires large-scale production. Traditional methods include outdoor cultivation and photobioreactor systems. In [35], the authors highlighted the potential of nitrogen-fixing cyanobacteria found in ditches near fields as a biological nitrogen fertilizer for organic farms. Similarly, ref. [36] showed that photobioreactors can achieve higher production efficiency for the cultivation of nitrogen-fixing cyanobacteria. Their study also found that lighting conditions in laboratory-scale photobioreactors have a significant impact on Tolypothrix tenuis biomass production. The results showed that algal cell density and volumetric productivity were higher in reactors with short light path and high light intensity. However, at normal light intensity, the total area productivity is greater in reactors with a 5 cm light path. The use of halogen lamps and ion flocculation increased the viability of the cyanobacteria after drying, and grinding is significantly stronger than when drying with fluorescent tubes [36]. Since the cost of using chemical culture media is too high and it is difficult to apply on a large scale, it is necessary to find a more cost-effective growth substrate [37,38]. Understanding these intricate processes provides essential insights into the biological foundation that makes cyanobacteria promising candidates for sustainable agricultural practices [39,40,41].

2.1. Advances in Agriculture with Cyanobacteria Nitrogen Fixation Techniques

There are generally two ways to apply nitrogen-fixing cyanobacteria on agricultural land: large-scale cultivation to obtain a large amount of algal biomass, and inoculation of a small amount of algal species to provide on-site sources of nitrogen through growth and fixation of nitrogen on agricultural land. Both approaches have notable drawbacks. The first method requires extensive equipment, designated facilities for cultivation, harvesting, drying, and preservation, and significant investments in water sources and culture media, making it costly. In contrast, the second method is heavily influenced by ecological and environmental factors, leading to low survival rates, poor growth, slow nitrogen fixation, and inefficient nitrogen fixation. In practical field applications, the use of nitrogen-fixing cyanobacteria faces many challenges, including their inability to compete effectively with native plants, difficulties in maintaining a prolonged growth period, and reduced nitrogen fixation efficiency in the presence of chemical nitrogen fertilizers [42]. Field and large-area trials based on pot trials have been widely reported in recent years [43]. The development direction includes the complete replacement of chemical fertilizers in organic farming and the production model of partial replacement of chemical fertilizers, reduction in quantity, efficiency, and environmental protection [44].

2.2. Molecular Mechanisms of Nitrogen Fixation in Cyanobacteria

The nitrogenase enzyme complex is central to all forms of biological nitrogen fixation, including in cyanobacteria, where it facilitates the conversion of atmospheric dinitrogen (N₂) to ammonia (NH₃). This ammonia serves as a vital source of nitrogen for plant assimilation and supports ecosystems that depend on nitrogen availability [45]. This enzymatic machinery consists of two main components, the Fe protein (dinitrogenase reductase) and the MoFe protein (dinitrogenase), and represents a finely tuned process that is essential for a sustainable nitrogen cycle [46]. The Fe protein, functioning as an electron carrier, plays a pivotal role in the nitrogenase complex. It receives electrons from donor molecules such as ferredoxin or flavodoxin, acting as a conduit for the transfer of these electrons to the MoFe protein. The MoFe protein, housing the iron-molybdenum cofactor (FeMo-co) within its catalytic site, serves as a hub for the reduction of N₂ to NH₃ and catalyzes the multi-step process that transforms inert atmospheric nitrogen into a biologically accessible and valuable nutrient [47,48]. In Figure 1, inorganic nitrogen mainly includes dissolved nitrogen (N₂), ammonium nitrogen (NH₄⁺), nitrite nitrogen (NO₂⁻), and nitrate nitrogen (NO₃⁻). Molecular nitrogen dissolved in water is only absorbed by nitrogen-fixing bacteria and nitrogen-fixing cyanobacteria in the water. Nitrogen can be converted into a form that plants can use through nitrogen fixation. Generally, phytoplankton utilize ammonium nitrogen first, followed by nitrate nitrogen, and finally nitrite nitrogen. Nitrogen in the triple-state refers to the triple bond in dinitrogen gas (N₂). In contrast, the three inorganic forms of nitrogen, ammonium (NH₄⁺), nitrate (NO₃⁻), and nitrite (NO₂⁻), are commonly referred to as available nitrogen.

This reduction process occurs within the intricacies of the FeMo-co active site, where nitrogen molecules are successively modified through the addition of electrons and protons. The exquisite chemistry of this process exemplifies the evolutionary adaptations that cyanobacteria have undergone to thrive in nitrogen-poor environments, showcasing their ability to extract essential nutrients from the atmosphere [49,50]. A notable challenge in nitrogen fixation is the vulnerability of the nitrogenase complex to oxygen [51]. In some species, cyanobacteria overcome this challenge through specialized cells known as heterocysts. These cells form a microaerobic niche with modified cell envelopes that protect the nitrogenase complex from the inhibitory effects of oxygen. In filamentous cyanobacteria, this spatial separation ensures an optimal environment for efficient nitrogen fixation [52].

In essence, the molecular mechanisms of nitrogen fixation in cyanobacteria reflect a remarkable interplay between the Fe and MoFe proteins within the nitrogenase complex. Understanding these mechanisms not only unveils the biological intricacies of cyanobacteria but also highlights their potential application in sustainable agriculture, where harnessing these nitrogen-fixing capabilities could revolutionize current nitrogen fertilization practices, mitigating environmental concerns associated with traditional methods [53,54].

2.3. Nitrogenase Enzyme Complex

The nitrogenase enzyme complex is highly sensitive to oxygen, posing a challenge for nitrogen-fixing cyanobacteria that inhabit oxygen-producing environments. To resolve this conflict, cyanobacteria employ distinct strategies depending on their lineage. Certain filamentous species produce specialized cells called heterocysts [39], which create an anaerobic microenvironment by restricting oxygen diffusion through a thickened cell envelope. These cells spatially separate nitrogen fixation from oxygenic photosynthesis in neighboring vegetative cells, enabling simultaneous metabolic processes [40]. However, not all cyanobacteria support themselves on heterocysts; Instead, non-heterocyst species separate nitrogen fixation from photosynthesis, which often fixes nitrogen in times of production of low oxygen (e.g., at night or under microoxic conditions). This time decoupling avoids the need for physical differentiation and shows the evolutionary diversity of adaptations to the oxygen sensitivity of cyanobacteria.

2.4. Nitrogenase Genes and Regulation

The genetic foundation of nitrogen fixation in cyanobacteria hinges on a specialized group of nitrogenase genes tasked with encoding the constituents of the nitrogenase enzyme complex. This genetic arrangement is finely regulated to ensure that nitrogenase genes are expressed judiciously, responding to environmental signals and creating optimal conditions for nitrogen fixation [55,56]. The nitrogenase gene cluster comprises various genes that collectively give rise to the nitrogenase enzyme complex. This complex is vital for converting atmospheric dinitrogen into ammonia, a process integral to the biologically meaningful assimilation of nitrogen by plants [57]. Regulation of nitrogenase genes is a nuanced process, highly responsive to environmental cues [58]. Cyanobacteria have evolved intricate signaling pathways and engage multiple transcription factors to modulate the expression of nitrogenase genes. These regulatory mechanisms are finely tuned to the availability of nitrogen sources and other environmental factors [59]. Within this regulatory framework, cyanobacteria demonstrate a remarkable adaptability to diverse ecological niches [60]. They adjust nitrogenase gene expression to prevailing environmental conditions and utilize heterocysts to create microaerobic environments essential for efficient nitrogen fixation and survival [61]. In heterocystous cyanobacteria, an important aspect of nitrogen regulation is the differentiation of specialized cells, called heterocysts, under nitrogen-limiting conditions. Heterocysts create a microaerobic environment, protect the oxygen-sensitive nitrogenase complex, and demonstrate the close coordination between genetic regulation and cellular specialization [62,63]. Additionally, nitrogenase activity is subject to feedback inhibition and metabolic control. Excessive ammonia production acts as a feedback signal, downregulating nitrogenase gene expression to prevent unnecessary energy expenditure. This regulatory feature allows cyanobacteria to fine-tune nitrogen fixation rates based on the actual nitrogen requirements of the cell [64,65].

Fundamentally, the genetic regulation of nitrogenase genes in cyanobacteria is a dynamic and intricate process. This regulation, involving signaling pathways, transcription factors, and cellular differentiation, ensures precise control over nitrogen fixation, reflecting the adaptability and resilience of cyanobacteria in diverse environmental contexts. Understanding these genetic mechanisms holds significance for leveraging the nitrogen-fixing capabilities of cyanobacteria in sustainable agriculture and addressing challenges associated with traditional nitrogen fertilization practices.

2.5. Adaptations for Nitrogen Fixation in Cyanobacteria

Cyanobacteria are versatile microorganisms that exhibit a number of structural and physiological adaptations specifically designed to facilitate nitrogen fixation [49]. An important adaptation is the development of heterocysts—specialized cells that provide a microaerobic environment essential for the activity of nitrogenase, the enzyme that catalyzes the conversion of atmospheric nitrogen to ammonia. These adaptations underscore the efficiency and specialization of cyanobacteria in nitrogen fixation. The formation of heterocysts is a critical adaptation unique to filamentous and heterocystous cyanobacteria. These specialized cells effectively isolate the oxygen-sensitive nitrogenase enzyme from the oxygen-generating photosynthetic apparatus. This segregation enables efficient nitrogen fixation by maintaining the microaerobic conditions required for nitrogenase activity [66,67]. Beyond heterocyst differentiation, cyanobacteria showcase another noteworthy adaptation through the development of specialized cells designated as akinetes. Akinetes, which are found only in certain filamentous heterocytic cyanobacteria, act as specialized dormant cells to help them survive in adverse environmental conditions. In addition to their role as nitrogen reservoirs by storing excess nitrogen during the nitrogen-rich season [68], akinetes also have an increased resistance to drying out, temperature changes, and other environmental stresses. When nitrogen availability is limited or conditions are unfavorable, akinetes allow a long-term retention by entering a dormant state until conditions improve. Once germinating, they release stored nitrogen, which promotes the recovery of cyanobacterial populations and contributes to the resilience of the ecosystem [69,70]. This adaptive strategy increases the stability of cyanobacterial communities and their role in nitrogen cycling in a variety of environments [71]. In addition, cyanobacteria exhibit a remarkable capacity to modulate their metabolic pathways in response to changes in nitrogen availability. This metabolic flexibility allows these microorganisms to fine-tune their physiological responses, optimizing resource utilization and ensuring efficient nitrogen fixation under diverse environmental circumstances. Cyanobacteria can dynamically regulate the expression of genes associated with nitrogen metabolism, enabling rapid and precise adjustments to the cellular machinery in the face of changing nitrogen levels [72,73,74].

The Azolla–Anabaena relationship is a striking example of cyanobacterial symbiosis with plants, characterized by a mutualistic association rather than root nodule formation. In this partnership, the nitrogen-fixing cyanobacterium Anabaena azollae resides within specialized leaf cavities of the water fern Azolla, where it provides a continuous supply of bioavailable nitrogen to the plant [75,76]. In return, Azolla offers a protected environment and sustains the cyanobacterium through carbon and nutrient exchange. This efficient, self-sustaining symbiosis plays a crucial role in nitrogen cycling and has been widely utilized in sustainable agriculture, particularly in rice paddy systems, as a natural biofertilizer [77,78]. This mutualistic collaboration allows cyanobacteria to provide plants with a direct source of fixed nitrogen, while plants reciprocate by offering a protected environment and essential nutrients to the cyanobacteria. Such symbiotic associations underscore the versatility of cyanobacteria in establishing complex relationships that contribute to nitrogen cycling in terrestrial ecosystems [79,80]. Essentially, the adaptations employed by cyanobacteria for nitrogen fixation extend beyond the well-studied heterocyst formation [21]. The integration of akinetes, metabolic flexibility, and symbiotic relationships with plants showcases the sophisticated strategies cyanobacteria employ to thrive in diverse ecological niches [81]. Understanding these adaptations at the molecular and physiological levels not only unravels the intricacies of cyanobacterial nitrogen fixation but also holds significance for harnessing their potential in various environmental and agricultural applications [82].

In essence, the molecular mechanisms of nitrogen fixation in cyanobacteria are a testament to the evolutionary adaptations that enable these microorganisms to thrive in diverse environments. The understanding of these mechanisms provides a foundation for the practical utilization of nitrogen-fixing cyanobacteria in agriculture, emphasizing their potential to contribute to sustainable farming practices by addressing the challenges associated with traditional nitrogen fertilization methods

3. Genetic and Biochemical Factors

The genetic and biochemical intricacies underlying the nitrogen-fixing abilities of cyanobacteria constitute a fascinating realm of study, providing essential insights into the molecular mechanisms that govern this crucial biological process [83]. At the forefront of nitrogen fixation are key genes that encode proteins forming the nitrogenase complex, the enzymatic machinery responsible for converting atmospheric nitrogen (N₂) into ammonia (NH₃), a form readily assimilable by plants [84]. The primary players in nitrogen fixation are the nifH, nifD, and nifK genes, which collectively encode the three essential subunits of the nitrogenase enzyme. In Figure 2, the nifH gene encodes the iron protein (Fe protein), while nifD and nifK encode the molybdenum-iron protein (MoFe protein). These proteins collaborate to catalyze the intricate series of reactions involved in the reduction of atmospheric nitrogen [57,85,86]. The nitrogenase complex facilitates the biochemical process of nitrogen fixation through a series of tightly regulated steps. Initially, the Fe protein, encoded by nifH, provides electrons to the MoFe protein (nifD and nifK), triggering the reduction of atmospheric nitrogen [87]. This reduction involves a complex cascade of electron transfer and redox reactions, ultimately incorporating nitrogen into ammonia. The efficiency and precision of these biochemical processes are crucial for the overall success of nitrogen fixation in cyanobacteria [88,89].

Electron transfer is a critical and intricately regulated aspect of nitrogenase activity, constituting a fundamental step in the complex process of biological nitrogen fixation. The nitrogenase complex, composed of the Fe protein (encoded by the nifH gene) and the MoFe protein (encoded by the nifD and nifK genes), catalyzes the reduction of atmospheric nitrogen (N₂) into ammonia (NH₃) through a series of redox reactions.

The process of electron transfer within the nitrogenase complex begins with the Fe protein, which acts as a carrier of electrons. These electrons are donated by various electron donors, with ferredoxin and flavodoxin being prominent examples [90]. Ferredoxin and flavodoxin are small iron-sulfur proteins that serve as electron shuttles, conveying electrons to the nitrogenase complex during different stages of the catalytic cycle [91]. The redox reactions within the nitrogenase complex are tightly regulated to ensure the efficient and precise conversion of nitrogen to ammonia while minimizing undesirable side reactions [92]. The active site of the nitrogenase enzyme, located on the MoFe protein, is a complex arrangement of metal cofactors, including iron and molybdenum, that facilitates the intricate dance of electrons [93]. During the catalytic cycle, the Fe protein transfers electrons to the MoFe protein, initiating the reduction of atmospheric nitrogen. The transfer of electrons is facilitated by a finely tuned interplay of conformational changes and chemical reactions within the nitrogenase complex [49]. As electrons move through the complex, they traverse a series of iron-sulfur clusters and other cofactors, each playing a specific role in the electron transfer process [94]. The precision required for electron transfer within the nitrogenase active site is a testament to the biological sophistication of this process [95]. The tightly choreographed movement of electrons is crucial for preventing wasteful side reactions and ensuring that the reduction of nitrogen is carried out with maximum efficiency [96]. This precision is achieved through the coordinated action of the nitrogenase proteins and the specific arrangement of metal cofactors, highlighting the remarkable adaptation of cyanobacteria to perform this biologically demanding task [97].

Understanding the genetic architecture and biochemical intricacies of nitrogen fixation in cyanobacteria is essential for harnessing their potential in agriculture [98]. Researchers are actively exploring genetic manipulation techniques to enhance nitrogen-fixing efficiency in these microorganisms, with a focus on optimizing the expression and regulation of key nitrogenase-related genes.

4. Biotechnological Applications

4.1. Nitrogen-Fixing Cyanobacteria as Biofertilizers

Nitrogen-fixing cyanobacteria play a crucial role in enriching agricultural soils by converting atmospheric nitrogen into ammonia, an essential nutrient for plant growth [99]. Unlike synthetic nitrogen fertilizers, which require energy-intensive production and contribute to greenhouse gas emissions [100], cyanobacteria offer a sustainable, eco-friendly alternative by naturally fixing nitrogen and enhancing soil fertility [101]. Their presence in agricultural systems supports nutrient cycling, reducing reliance on synthetic inputs and mitigating environmental risks such as chemical runoff and ecosystem disruption [102].

Through their activity in the root zones of plants, nitrogen-fixing cyanobacteria release bioavailable nitrogen into the soil, creating a more favorable environment for crop growth and improving overall soil health [103]. This continuous nitrogen enrichment benefits both current and subsequent crops, fostering long-term soil fertility and sustainable agricultural productivity [104]. In regions with nitrogen-deficient soils, the introduction of cyanobacteria provides an effective, low-cost solution for enhancing nutrient availability and optimizing yields where conventional fertilizers may be limited or economically unfeasible [105].

Harnessing nitrogen-fixing cyanobacteria as biofertilizers represents a promising biotechnological strategy that not only improves nutrient availability and soil fertility but also contributes to resilient and environmentally sustainable agricultural practices [106].

4.2. Improving Crop Yield and Quality

The utilization of nitrogen-fixing cyanobacteria as biofertilizers translates into tangible benefits for crop yield and quality [107]. The availability of a natural and renewable nitrogen source positively influences the growth and development of plants, leading to increased yields. Additionally, the symbiotic relationship between cyanobacteria and plants can contribute to improved nutrient uptake, stress tolerance, and overall crop health [108,109]. These combined effects not only enhance the quantity of agricultural produce but also hold promise for improving the nutritional quality of crops, addressing global challenges related to food security and malnutrition [110].

4.3. Genetic Engineering Approaches for Enhanced Nitrogen Fixation

Biotechnology has opened new frontiers in optimizing the nitrogen fixation capabilities of cyanobacteria through genetic engineering [111]. Researchers are actively identifying and manipulating key genes within nitrogen fixation pathways with the aim of increasing the efficiency and stability of converting atmospheric nitrogen into biologically accessible forms [112]. Recent advances include the integration of regulatory networks to fine-tune nitrogenase activity, reduce energy loss, and improve overall metabolic efficiency. In addition, genetic engineering is being used to introduce new traits such as improved photosynthesis efficiency and resistance to environmental stresses, thereby expanding the applicability of cyanobacteria strains. These tailored modifications enable the development of biofertilizers specifically optimized for different agricultural conditions, offering unparalleled precision and effectiveness. By combining cutting-edge molecular biology with practical agronomic requirements, this approach has the potential to revolutionize nitrogen management in sustainable agricultural practices [113,114].

4.4. Strain Improvement Strategies

While genetic engineering has attracted considerable attention, complementary strain improvement strategies are equally important for increasing the agricultural utility of nitrogen-fixing cyanobacteria [115]. Classical breeding techniques, mutagenesis, and advanced selection methods are used to isolate strains with desirable traits such as superior nitrogen fixation efficiency, environmental robustness, and compatibility with certain crops [116]. Additionally, researchers are integrating systems biology and omics technologies to accelerate the identification of genetic markers associated with these traits. These efforts also extend to optimizing cyanobacteria interactions within diverse agroecosystems, including symbiotic relationships with plant roots. By holistically improving strain performance, this approach ensures the development of adaptable biofertilizers that can thrive under different climate and soil conditions. Taken together, these strategies complement genetic engineering to maximize the practical potential of nitrogen-fixing cyanobacteria, thereby contributing to global agricultural sustainability [101,113,115]. While genetic engineering has garnered significant attention, complementary strain improvement strategies are equally vital for enhancing the agricultural utility of nitrogen-fixing cyanobacteria [117]. Classical breeding techniques, mutagenesis, and advanced selection methods are being employed to isolate strains with desirable traits, such as superior nitrogen-fixing efficiency, environmental robustness, and compatibility with specific crops [118]. Moreover, researchers are integrating systems biology and omics technologies to accelerate the identification of genetic markers associated with these traits. These efforts also extend to optimizing cyanobacterial interactions within diverse agroecosystems, including symbiotic relationships with plant roots. By holistically improving strain performance, this approach ensures the development of adaptable biofertilizers capable of thriving under varying climatic and soil conditions [119]. Collectively, these strategies complement genetic engineering to maximize the practical potential of nitrogen-fixing cyanobacteria, contributing to global agricultural sustainability.

4.5. The Role of Mixed Inoculation with Other Beneficial Organisms

Mixed inoculation of nitrogen-fixing cyanobacteria with other beneficial microorganisms may have advantages over inoculation of nitrogen-fixing cyanobacteria alone. For example, [120] examined the combination of nitrogen-fixing cyanobacteria, green algae and nitrogen-fixing organisms; ZOB-1 (Anabaena variabilis, Chlorella vulgaris and Azotobacter sp.) and ZOB-2 (N. calsicola, Chlorella vulgaris and Azotobacter) showed high levels of microalgae growth rates and photosynthetic activity. ZOB-1 promotes rice seed germination and plant growth and can be used as a plant growth stimulant and biofertilizer, as shown in Figure 3. Its advantages include that plants can withstand extreme conditions, have extremely high ecological value, have an extremely high growth rate, and are immune to extremely low environmental conditions.

In field research [121], it was shown that the application of a mixed biofertilizer based on Brachyphyllum or Anabaena improved the germination rate and fresh weight of cotton (Gossypium hirsutum) “PKV081” and increased the available nitrogen in the soil by 20–50%, and microbial activity increased by 10–15%. Research by [122] showed that in two consecutive rice trials, rice grain yield in rice fields in Bangladesh treated with Azolla imbircata and nitrogen-fixing cyanobacteria increased by 10% compared to the control. Mixed application of inorganic fertilizers, organic, and biological fertilizers can increase organic carbon, total nitrogen, microbial biocarbon, and cation exchange capacity of soil in rice fields.

5. Impact on Soil Health

5.1. Influence of Nitrogen-Fixing Cyanobacteria on Soil Microbial Communities

Nitrogen-fixing cyanobacteria play a pivotal role in shaping the microbial communities within the soil environment [123]. As these cyanobacteria engage in the process of nitrogen fixation, they release bioavailable nitrogen compounds into the soil. This nitrogen influx not only serves as a direct nutrient source for plants but also acts as a stimulus for other soil microbes [103,124]. The increased availability of nitrogen alters the microbial composition, fostering a more diverse and dynamic soil microbiome. This diversification is essential for promoting symbiotic relationships among various microorganisms, contributing to overall soil health. They promote the decomposition of soil organic matter and the transformation of nutrients through some oxidation, nitrification, nitrogen fixation, etc. The types of microorganisms in the soil are extremely rich, mainly including bacteria, archaea, fungi, viruses, protozoa, etc. They have a major impact on soil formation and development, material circulation, and fertility evolution. Moreover, as illustrated in Figure 4, nitrogen-fixing cyanobacteria contribute significantly to soil health by enhancing microbial interactions and nutrient availability. Their ability to fix atmospheric nitrogen creates a self-sustaining nutrient cycle, improving soil fertility and promoting ecological balance. Additionally, the presence of cyanobacteria introduces a unique set of interactions, such as cross-feeding and competition, further influencing the intricate web of soil microbial communities [125,126,127].

5.2. Soil Fertility Enhancement and Nutrient Cycling

Cyanobacteria play a crucial role in enhancing soil fertility by fixing atmospheric nitrogen into bioavailable forms, reducing the need for synthetic fertilizers and their associated environmental risks [103]. Their continuous nitrogen input supports efficient nutrient cycling, minimizing losses and promoting long-term soil health [128]. While organic fertilizers are widely used to improve soil fertility, their composition and effects vary, requiring careful management. This article reviews the impacts of organic fertilizers on soil properties, nutrient availability, microbial communities, heavy metal accumulation, and greenhouse gas emissions (

Table 2. The influence of organic fertilizers on soil health and environmental balance.

Soil Characteristics	Mechanism	Types of Organic Fertilizers	References
Soil physical properties	Reduce soil bulk density, increase total porosity, increase the number and stability of soil aggregates, enhance the ability of soil to retain water and fertilizer, and alleviate soil acidification.	Livestock manure, farm manure, crop straw, biological waste, green fertilizer, commercial organic fertilizer	[130,131,132,133]
Soil nutrient	Improve the capacity of soil fertilizer supply; Accelerate the activation rate of humic acid to soil nutrients, improve the activities of microorganisms and enzymes related to nutrient conversion; maintain the balance of available nutrient supply and improve fertilizer utilization efficiency; increase the availability of trace elements.	Livestock manure, farm manure, crop straw, biological waste, sludge, green manure, commercial organic fertilizer	[134,135,136]
Soil microorganism	Increase organic matter and soil fertility, provide carbon source, nitrogen source, energy and binding site for soil microorganisms and enzymes; improve the soil microecological environment and promote the growth and reproduction of microorganisms.	Livestock manure, farm manure, crop straw, biological waste, sludge, green manure, commercial organic fertilizer	[137,138]
Soil heavy metals	They carry high levels of heavy metals; the availability of heavy metals was affected by changing the physical and chemical properties of soil such as pH, SOM, and Eh. The availability of heavy metals depends on the adsorption and desorption processes.	Livestock manure, sludge, commercial organic fertilizer	[139,140]
Soil greenhouse gas	Release more CO2 by increasing soil organic matter and total porosity and soil respiration; provide an abundant methanogenic matrix and suitable environment for methanogenic bacteria to grow and release more CH4; by changing soil C/N, the formation and emission of nitrification and denitrification reaction products of N2O are affected, and different organic fertilizers show uncertainty.	Livestock manure, farm manure, crop straw, biological waste, commercial organic fertilizer	[141,142]

) and provides an overview of their benefits and limitations in comparison with chemical fertilizers, with a view to guiding sustainable farming practices [129].

5.3. Effects on Soil Structure and Water Retention

The influence of nitrogen-fixing cyanobacteria on the soil goes beyond nutrient supply and influences crucial physical properties [143]. These microorganisms produce extracellular polymeric substances (EPSs) that act as binding agents and promote the formation of stable soil aggregates. This aggregation improves soil structure by preventing erosion, increasing porosity and enabling improved root penetration [144,145]. The production of EPSs by cyanobacteria plays a fundamental role in soil aggregation [146]. These substances act as cohesive forces, binding soil particles into aggregates that resist erosion by water runoff and wind. This stability is critical for maintaining topsoil integrity, particularly on farms where soil erosion poses a threat to productivity and environmental sustainability [147]. Stable soil aggregates, reinforced by EPSs produced by cyanobacteria, contribute to erosion protection. By firmly binding soil particles, these aggregates minimize the effects of water runoff and wind, thereby protecting against the loss of fertile topsoil [148]. This erosion protection is crucial for maintaining the productive capacity of agricultural land and preventing environmental damage [149]. The aggregation process also leads to increased porosity of the soil. The gaps and channels between soil particles provide a more ventilated environment and facilitate gas exchange and nutrient diffusion. Improved porosity benefits the microbial community and improves conditions for root growth and proliferation, thereby promoting overall soil health [150]. Numerous studies have shown that the occurrence of soil-borne diseases, such as bacterial wilt, is closely related to the physical, chemical, and biological properties of the soil, including soil enzyme activity, pH, nutrients, root exudates, soil microbial diversity, etc., among other influencing factors [151]. Pathogen bacteria exhibit pathogenic properties under certain environmental conditions due to sudden changes in these key indicators and thereby infect host plants (Figure 5).

Cyanobacteria play a crucial role in improving soil structure by forming stable aggregates and increasing porosity, facilitating root penetration [103]. This structural enhancement enables plant roots to penetrate the soil more effectively, allowing them to reach deeper layers for better access to water and nutrients [152]. The symbiotic relationship between cyanobacteria, soil structure improvement, and root development highlight the interconnectedness of biological and physical aspects in agricultural ecosystems [153]. In addition, cyanobacteria activities significantly contribute to the formation of stable soil aggregates, prevention of erosion, and increase of porosity, which overall increases the water retention capacity of the soil. This ability to store water is particularly valuable in regions with water scarcity or erratic rainfall, as it ensures a consistent and reliable water supply for plants, thereby reducing their vulnerability to drought stress [154,155].

In summary, the impact of nitrogen-fixing cyanobacteria on soil health is multifaceted. From influencing the composition of microbial communities to enriching soil fertility and altering physical soil properties, these microorganisms emerge as key players in promoting sustainable agriculture [156]. Their ability to foster a balanced soil ecosystem, reduce reliance on synthetic fertilizers, and enhance water retention underscores their potential to address critical challenges in modern agricultural practices while contributing to the long-term health and productivity of the soil [103,157].

6. Environmental Sustainability

6.1. Reduction of Synthetic Nitrogen Fertilizer Usage

Nitrogen-fixing cyanobacteria offer an eco-friendly alternative to synthetic nitrogen fertilizers, addressing both environmental and agricultural challenges [99]. Unlike traditional fertilizers, which contribute to greenhouse gas emissions, such as nitrous oxide, and can cause soil acidification, cyanobacteria naturally convert atmospheric nitrogen into bioavailable forms for plants [101]. This process reduces reliance on synthetic fertilizers, lowering agricultural practices’ carbon footprint and mitigating their associated environmental impacts [158].

6.2. Mitigation of Nitrogen Runoff and Environmental Pollution

Nitrogen runoff from synthetic fertilizers is a significant environmental issue, as it contributes to eutrophication and contamination of water systems. This process occurs when excessive nitrate and ammonium, derived from synthetic fertilizers, leach into the soil and eventually reach water bodies. Cyanobacterial biofertilizers, however, have been shown to reduce nitrogen runoff and leaching due to their unique mechanism of nitrogen fixation and gradual nutrient release.

Cyanobacteria contribute to a more controlled nitrogen release compared to synthetic fertilizers. Through biological nitrogen fixation, cyanobacteria convert atmospheric nitrogen into forms that can be utilized by plants, but at a slower rate than the immediate availability of nitrogen in synthetic fertilizers. Several studies have investigated the impact of cyanobacterial biofertilizers on nitrogen loss from agricultural systems, demonstrating reduced nitrogen leaching when compared to synthetic fertilizers. For example, research by [159] found that cyanobacterial biofertilizers resulted in lower nitrate leaching in comparison to synthetic fertilizers, likely due to the gradual release of nitrogen, which aligns better with plant uptake rates and reduces the potential for nitrogen to leach below the root zone. Similarly, ref. [160] observed that cyanobacterial application significantly decreased nitrate runoff in field trials, likely because cyanobacteria enhance soil aggregation and stability, reducing surface runoff and improving nitrogen retention in the soil.

Moreover, cyanobacteria’s ability to produce extracellular polymeric substances (EPSs) further contributes to nitrogen retention. EPSs help stabilize soil structure by improving soil aggregation, thus decreasing surface water runoff and promoting better nitrogen retention in the soil matrix. This characteristic has been highlighted by ref. [161], who noted that soil treated with cyanobacteria exhibited improved soil texture and reduced surface erosion, leading to lower nitrogen losses.

However, it is important to acknowledge that once nitrogen from cyanobacterial biofertilizers is mineralized into inorganic forms (nitrate or ammonium), its movement in the environment follows similar pathways to that of synthetic fertilizers. This means that while cyanobacteria reduce the risk of immediate nitrogen losses, the long-term environmental fate of the nitrogen will be influenced by soil conditions, water availability, and crop uptake rates. The advantage of cyanobacterial fertilizers lies in their more synchronized release, which helps minimize excessive nitrogen leaching, especially in systems where traditional fertilizers are used in excess. Overall, the use of cyanobacteria as biofertilizers presents a promising strategy for reducing nitrogen pollution in agricultural systems. By improving nitrogen use efficiency and minimizing the environmental impact of fertilization practices, cyanobacteria contribute to more sustainable agricultural practices. Further studies are needed to quantify the specific conditions under which cyanobacteria offer the greatest reduction in nitrogen runoff and to assess their long-term efficacy across different agricultural systems.

6.3. Carbon Sequestration and Soil Health

Cultivation of nitrogen-fixing cyanobacteria improves soil health by increasing nitrogen availability, which stimulates plant growth and organic matter accumulation [162]. This process contributes to improved soil structure and fertility, supporting sustainable agriculture. In addition, as plant biomass and soil organic matter increase, carbon sequestration is promoted, helping to mitigate climate change. By improving soil health and promoting regenerative agricultural practices, cyanobacteria play a critical role in both environmental sustainability and climate change adaptation [163,164].

7. Challenges and Limitations

7.1. Oxygen Sensitivity of Nitrogenase and Strategies to Overcome It

The efficiency of nitrogen fixation by cyanobacteria is significantly affected by the oxygen sensitivity of the nitrogenase enzyme, a crucial component responsible for catalyzing the conversion of atmospheric nitrogen (N₂) into biologically useful forms [165]. The paradox arises from the fact that cyanobacteria, as oxygen-containing photosynthesizers, produce oxygen during photosynthesis, a process that occurs simultaneously with nitrogen fixation [166]. This poses a significant challenge as the oxygen produced during photosynthesis can deactivate and damage the nitrogenase enzyme [61]. To overcome this fundamental hurdle, researchers are developing innovative strategies to create oxygen-protected microenvironments within cyanobacteria [167]. One approach involves the formation of specialized structures, such as heterocysts, within cyanobacteria filaments. These unique cells provide an oxygen-depleted environment, protecting the nitrogenase enzyme from the inhibitory effects of oxygen and enabling sustained nitrogen fixation [168].

Another avenue of exploration involves genetic engineering efforts to enhance the oxygen tolerance of the nitrogenase enzyme itself [167]. By modifying the genetic makeup of cyanobacteria, researchers aim to create strains with nitrogenase enzymes that are more resilient to the presence of oxygen. This could involve introducing mutations or incorporating elements that protect the enzyme’s activity in oxygen-rich environments [169,170]. The goal is to enable cyanobacteria to carry out nitrogen fixation efficiently even in the presence of oxygen, overcoming a significant barrier to their practical application in aerobic environments [66]. Additionally, advances in synthetic biology and metabolic engineering are contributing to the design of cyanobacterial strains with improved strategies for managing oxygen during nitrogen fixation. These strategies may include optimizing the timing of nitrogenase activity relative to the photosynthetic cycle or developing regulatory mechanisms that respond dynamically to oxygen concentrations [171,172].

As researchers continue to unravel the complexities of the nitrogenase–oxygen interaction, a multifaceted approach combining biological insights and engineering solutions is emerging. The development of cyanobacterial strains with enhanced oxygen tolerance and the creation of microenvironments conducive to nitrogen fixation represent pivotal steps toward harnessing these microorganisms for sustainable agriculture [173,174]. Overcoming the oxygen sensitivity of nitrogenase is crucial not only for maximizing nitrogen fixation efficiency but also for realizing the full potential of nitrogen-fixing cyanobacteria as a transformative tool in the quest for global food security and environmentally conscious agriculture [175].

7.2. Competition with Native Microorganisms

The introduction of nitrogen-fixing cyanobacteria into agricultural ecosystems raises concerns about potential competition with native microorganisms [176]. Soil, a complex and thriving ecosystem, hosts diverse microbial communities, including bacteria, fungi, and archaea, each contributing to essential ecological functions [177]. The influx of cyanobacteria introduces a new player into this intricate system, leading to competition for crucial resources such as nitrogen, phosphorus, and micronutrients [178]. The established microbial communities, already adapted to prevailing soil conditions, may outcompete cyanobacteria, limiting their ability to establish and contribute to enhanced soil fertility [156]. The potential consequences of this competition extend beyond resource limitation, encompassing disruptions to established ecological balances [179]. Native microbial communities, finely tuned to specific niches, may experience shifts in structure and function, impacting overall soil health and ecosystem dynamics [180]. Recognizing the need for optimization, researchers are actively exploring ways to enhance the compatibility between nitrogen-fixing cyanobacteria and native soil microorganisms [181]. This involves studying ecological niches, resource utilization patterns, and potential symbiotic relationships to facilitate the coexistence of introduced cyanobacteria with the existing soil ecosystem [182].

Understanding the intricacies of microbial interactions and identifying synergistic partnerships are essential steps toward addressing competition challenges [183]. Some studies suggest that establishing mutualistic relationships between nitrogen-fixing cyanobacteria and specific native microorganisms can enhance overall nitrogen fixation effectiveness [115]. Additionally, soil management practices play a crucial role in mediating these interactions. Sustainable agricultural practices that prioritize soil health, organic matter content, and microbial diversity can create conditions conducive to the coexistence of diverse microbial communities [184]. Researchers are working to address the challenges posed by competition with native microorganisms, focusing on strategies that facilitate the successful integration of nitrogen-fixing cyanobacteria into agricultural systems. These efforts aim to promote the establishment of balanced and sustainable ecosystems.

7.3. Field Implementation Challenges and Risks

The transition from laboratory-scale experiments to large-scale field implementation poses a set of challenges and risks [185]. Optimizing cultivation practices, ensuring effective inoculation, and maintaining the stability of cyanobacterial populations in diverse environmental conditions are among the key challenges [186]. Unforeseen ecological consequences and potential environmental risks associated with introducing genetically modified cyanobacteria into ecosystems also demand careful consideration [187]. Researchers are actively investigating these challenges through field trials, environmental impact assessments, and the optimization of cultivation techniques. Their efforts aim to mitigate potential risks while enhancing the benefits of nitrogen-fixing cyanobacteria in practical agricultural applications [188].

Addressing these challenges necessitates an interdisciplinary approach integrating molecular biology, microbiology, agronomy, and environmental science. Current research efforts focus on understanding the oxygen sensitivity of nitrogenase, optimizing cyanobacteria–microbe interactions, and refining field application strategies. Overcoming these hurdles will be key to harnessing the full potential of nitrogen-fixing cyanobacteria as a sustainable, environmentally friendly means of improving nitrogen availability in agricultural soils, ultimately supporting global food security in the face of evolving environmental conditions.

8. Future Directions

8.1. Advances in Synthetic Biology for Cyanobacterial Nitrogen Fixation

The future of sustainable agriculture hinges on the transformative capabilities of synthetic biology applied to nitrogen-fixing cyanobacteria [189]. Researchers are actively engaged in deploying advanced genetic manipulation techniques to enhance these microorganisms’ inherent nitrogen-fixing abilities. Through precise engineering, strains with optimized nitrogenase activity and increased tolerance to diverse environmental conditions are being developed. This involves the introduction of novel genes or modifications to existing ones, all aimed at boosting the efficiency of nitrogen fixation [190,191]. One important research avenue is using CRISPR-based gene editing tools, which have recently shown remarkable success in improving cyanobacteria strains for industrial and agricultural applications [192]. For example, CRISPR/Cas9 has improved nitrogen fixation efficiency, while CRISPR/Cas12a has enabled precise genome modifications to improve stress tolerance [193]. Furthermore, transposon-based CRISPR systems such as CAST have enabled large-scale genomic insertions and expanded the scope of metabolic engineering [194]. These advances allow scientists to tailor cyanobacteria traits with exceptional specificity and develop strains that not only efficiently fix nitrogen but also withstand environmental stressors such as temperature fluctuations, pH fluctuations, and changes in salinity [195,196].

Genetic engineers are incorporating genes from diverse sources, including other nitrogen-fixing organisms, to enhance cyanobacteria’s overall nitrogenase performance [197]. This approach allows for the creation of strains with superior nitrogen-fixing capabilities, contributing to increased bioavailability of nitrogen for surrounding crops. Such optimization strategies aim to make nitrogen fixation by cyanobacteria a more potent and adaptable solution for agricultural challenges [198]. The advent of synthetic biology presents an opportunity to tailor cyanobacterial strains for specific agricultural environments [199]. By addressing the unique conditions of different regions, such as nutrient-poor soils or arid climates, researchers aim to develop strains that can efficiently fix nitrogen across a variety of global agricultural contexts [200]. This adaptability ensures that the potential benefits of cyanobacterial nitrogen fixation can be realized on a broad scale, addressing diverse challenges in agriculture [201].

As synthetic biology continues to evolve, its potential for unprecedented advancements becomes increasingly apparent. The integration of genetic engineering, systems biology, and computational modeling promises to unveil novel pathways, regulatory mechanisms, and biological strategies. These discoveries hold the key to optimizing and customizing cyanobacterial nitrogen fixation, offering innovative solutions for sustainable and resilient agriculture in the face of global food security challenges.

8.2. Integration with Other Agricultural Practices for Maximum Impact

The evolution of sustainable agriculture is intrinsically tied to the harmonious integration of nitrogen-fixing cyanobacteria with established farming practices. This integration represents a paradigm shift towards holistic and resilient agricultural systems, where the synergistic interplay between cyanobacteria and other sustainable techniques forms the cornerstone of enhanced productivity, environmental stewardship, and soil health [19,202]. Researchers are actively exploring the dynamic relationships between nitrogen-fixing cyanobacteria and various existing agricultural practices. Organic farming, which is characterized by the avoidance of synthetic fertilizers and pesticides, aligns seamlessly with the ethos of sustainable agriculture [203]. Studies are underway to assess how cyanobacteria can augment organic farming methods by providing a natural and renewable source of nitrogen [204]. However, the ban on genetically modified organisms (GMOs) in organic farming poses a significant challenge to the use of genetically modified cyanobacteria in these systems. Despite this hurdle, research into non-genetically modified cyanobacteria strains and their symbiotic relationships with certain crops could help reduce the dependence on external nitrogen inputs and promote a more self-sufficient and environmentally friendly agricultural approach [205,206].

The integration of nitrogen-fixing cyanobacteria into agricultural systems is being explored as a strategy to enhance soil fertility and sustainability [103]. Researchers are investigating the potential of cyanobacteria to contribute natural nitrogen inputs while also improving soil structure and reducing erosion [207]. By colonizing the soil surface, cyanobacteria can form biological soil crusts that help stabilize the soil, suppress weeds, and promote soil moisture retention, thereby supporting sustainable agricultural practices [208]. To achieve the full potential of these integrated systems, it is imperative to unravel the complexities of interactions between cyanobacteria and different crops, as well as their compatibility with other beneficial microorganisms [209]. This necessitates in-depth research into the microbial ecology of agricultural soils, aiming to decipher the intricate web of relationships that contribute to overall ecosystem health [210]. Future research in this field will aim to further refine and optimize these synergies. Collaborative efforts between scientists and agricultural practitioners will be essential in harnessing the full potential of cyanobacterial nitrogen fixation across diverse agricultural systems. The overarching objective is to develop integrated and sustainable agricultural models that enhance the benefits of cyanobacteria while addressing potential limitations. By doing so, these advancements will contribute to a more resilient, efficient, and environmentally sustainable future for global agriculture.

8.3. Scaling-Up Strategies for Large-Scale Agriculture

As the agricultural landscape continues to evolve, the scalability of cyanobacterial nitrogen fixation becomes a central concern [211]. Researchers are exploring strategies to expand the cultivation of cyanobacteria from laboratory settings to large-scale agricultural fields [186]. This includes the development of cost-effective and efficient farming systems such as photobioreactors and open pond systems that can accommodate the increased biomass production required for widespread agricultural use [212]. Furthermore, advances in harvesting and processing technologies are being driven to optimize the extraction of nitrogen-rich biomass from cyanobacteria [213].

Collaboration among scientists, engineers, and policymakers is crucial for overcoming logistical and regulatory challenges associated with large-scale implementation. Effective scaling strategies will be essential to fully realize the potential of cyanobacterial nitrogen fixation as a sustainable and globally viable approach to enhancing agricultural productivity. Future advancements in harnessing nitrogen-fixing cyanobacteria require a multidisciplinary approach, integrating synthetic biology for genetic optimization, incorporation into diverse agricultural systems, and the development of scalable solutions for widespread adoption. These efforts hold the promise of transforming cyanobacteria from a subject of scientific interest into a practical and impactful tool for sustainable agriculture, addressing critical challenges related to nitrogen availability and environmental sustainability in global food production.

9. Conclusions

The application of nitrogen-fixing cyanobacteria offers a practical and sustainable solution to the challenges associated with nitrogen fertilization in agriculture. Advances in understanding the molecular mechanisms of nitrogen fixation, including the role of key genes like nifH and associated pathways, have opened up new possibilities for utilizing cyanobacteria such as Anabaena and Nostoc. These organisms contribute to improving soil health and reducing dependency on synthetic fertilizers, which are known to have significant environmental impacts. Despite these promising developments, the practical application of nitrogen-fixing cyanobacteria is not without its limitations. Challenges such as the oxygen sensitivity of nitrogenase, competition with native microbial communities, and the need for stable integration into diverse soil environments remain significant obstacles. Addressing these issues requires targeted genetic modifications, enhanced delivery systems for biofertilizers, and a deeper understanding of the ecological interactions that shape their performance in agricultural settings. Future research into synthetic biology and advanced genetic engineering holds great potential to optimize the nitrogen fixation efficiency of cyanobacteria under real-world agricultural conditions. Equally important is the development of scalable systems that enable the practical integration of these microbes into current farming practices without disrupting existing ecosystems. These efforts could help redefine the role of biological nitrogen fixation in supporting sustainable food production.

Nitrogen-fixing cyanobacteria represent more than just an alternative to synthetic fertilizers; they embody a broader shift toward environmentally responsible agriculture. By addressing the remaining challenges through rigorous scientific research and technological innovation, their potential to contribute to global food security and environmental sustainability can be fully realized.