Nitrogen Fixation by Diazotrophs: A Sustainable Alternative to Synthetic Fertilizers in Hydroponic Cultivation

Abstract

Sustainable agriculture and food security are challenged by the indiscriminate use of synthetic nitrogen (N₂) fertilizers, inefficient water management, and land degradation. Hydroponic cultivation uses nutrient-rich aqueous media and is a climate-resilient and resource-efficient alternative to traditional farming methods, whose dependence on synthetic N₂ fertilizers reduces their long-term sustainability. Biological nitrogen fixation (BNF), which is mediated by diazotrophs that reduce atmospheric N₂ to plant-available ammonium, has emerged as a sustainable alternative to synthetic N₂ input in hydroponic systems. This review discusses the integration of BNF into hydroponic systems by exploring the functional diversity of diazotrophs, root–microbe interactions, and environmental constraints. It further highlights recent advances in strain improvement, microbial consortia development, nitrogenase protection, and genome editing tools, novel bioformulation strategies to enhance microbial compatibility with hydroponic nutrient regimes, and omics-based tools for the real-time assessment of N₂ fixation and microbial functionality. Key challenges, such as microbial leaching, nitrate-induced inhibition of nitrogenase activity, and the absence of standardized biostimulant protocols, are discussed. Case studies on staple crops have demonstrated enhanced NUE and yield productivity following diazotroph applications. This review concludes with future perspectives on synthetic biology, regulatory policies, and omics-based tools for the real-time assessment of N₂ fixation and microbial functionality.

1. Introduction

Global agriculture is facing increasing pressure due to rapid population growth, urbanization, and climate change. According to the United Nations, the global population is projected to reach 9.7 billion by 2050, with a significant increase in food demand [1,2]. Currently, 55% of the global population resides in urban areas, which is expected to exceed 75% by 2050, indicating the need for highly efficient agricultural systems in urban and peri-urban zones [3]. Agricultural productivity is declining due to land degradation, soil infertility, water scarcity, and climate variation [4]. Abiotic stresses such as drought, salinity, nutrient imbalance, and extreme temperatures can reduce staple crop yields by up to 23% annually [3].

Controlled Environmental Agriculture, particularly hydroponic agriculture, has emerged as a sustainable alternative to conventional farming. Hydroponics is a soilless cultivation method that supplies nutrients directly to plant roots, reducing dependence on arable land and irrigation water, while enabling year-round crop production [3,5]. The global hydroponics market was valued at approximately USD 9.8 billion in 2022 and is expected to increase owing to technological development and food production demands in urban areas [6]. However, most hydroponic systems rely on synthetic N₂ fertilizers derived from the energy-intensive Haber–Bosch process, contributing to over 80% of anthropogenic reactive N₂ emissions [7]. Improper N₂ management leads to nutrient accumulation, nitrate (NO₃^–) toxicity, and environmental contamination [8].

Biological nitrogen fixation (BNF) is a sustainable alternative to synthetic N₂ fertilizers. Diazotrophic microorganisms, such as Azospirillum, Azotobacter, and Rhizobium, and N₂-fixing cyanobacteria convert atmospheric N₂ into bioavailable ammonia for plant uptake, enhancing N₂ availability and promoting nutrient use efficiency (NUE) in plants [9]. Although BNF has been studied in soil and rhizosphere systems, emerging evidence suggests that the incorporation of diazotrophic microorganisms has shown promising adaptability in hydroponic environments. They exhibit a range of nitrogen-fixing strategies, such as root colonization, biofilm formation, and nitrogenase protection mechanisms, which enhance nitrogen acquisition and plant growth in hydroponic systems [10,11,12]. However, their effectiveness is inhibited by several physiological, chemical, and ecological constraints, including microbial compatibility with host crops, nutrient solution composition, and system-specific abiotic conditions [13]. To address these limitations, recent research has focused on microbial consortium design, diazotroph strain engineering, and the use of protective bioformulations [14,15]. Ongoing advances in imaging, transcriptomics, and formulation technologies are enhancing our understanding of microbial interactions and facilitating their effective integration into precision hydroponic systems [16,17]. Moreover, regulatory and policy frameworks are being developed to define the safety and efficacy standards for microbial bioinputs in controlled environments [18,19].

This review discusses the following: (i) The potential of integrating diazotrophic microorganisms into hydroponics, focusing on their diversity, functionality, and root interactions under hydroponic conditions. (ii) Recent advancements in identifying potential diazotrophic strains, developing synthetic and native consortia, and strategies to protect nitrogenase enzymes from oxidative and NO₃^–-induced stress. (iii) Novel bioformulation strategies, such as alginate-based encapsulation, nanocellulose carriers, and co-inoculation techniques, to enhance stability and compatibility with hydroponic nutrient solutions. (iv) Omics-based tools, including metagenomics, transcriptomics, and biosensor technologies, for the real-time monitoring of N₂ fixation efficiency. (v) Key challenges, such as microbial leaching in recirculating systems, NO₃^–-induced inhibition of nitrogenase, and the absence of standardized protocols for bioinoculant applications. (vi) Case studies on the integration of diazotrophic microorganisms into hydroponic crops have shown improved NUE and crop productivity. (vii) Future research directions include synthetic biology approaches to nitrogenase engineering, development of regulatory guidelines, and the emerging role of AI-assisted nutrient management tools as potential enablers for enhancing the integration of BNF into hydroponic agriculture. These strategies aim to enhance the transition from synthetic N₂ to microbe-based nutrient solutions in hydroponic farming.

2. Nitrogen Dynamics in Hydroponic Systems

2.1. Forms and Functions of Nitrogen in Hydroponics

Nitrogen (N) is an essential macronutrient that plays a crucial role in various physiological and biochemical processes involved in plant growth and development. It constitutes the backbone of vital biomolecules, such as DNA, proteins, amino acids, chlorophyll, and phytohormones [20]. Plants absorb N from nutrient solutions in the form of NO₃⁻ and ammonium (NH₄⁺). Under certain conditions, plants absorb organic N₂ compounds, such as amino acids, peptides, and urea, especially when they are abundant in the root zone or during specific physiological or stress conditions [21]. NO₃⁻ is the preferred form because of its high mobility in nutrient solutions, low phytotoxicity, and compatibility with oxygenated root systems [13].

Under well-aerated hydroponic conditions, NO₃⁻ contributed up to 90% of the total N uptake. The uptake and utilization of NH₄⁺ depend on factors such as plant genotype, temperature, pH, and the ionic composition of the nutrient solution [13]. Although NH₄⁺ requires less energy for plant assimilation than NO₃⁻, excess NH₄⁺ can cause rhizotoxicity, disrupt ion homeostasis, and induce oxidative stress in plant tissues [22]. NH₄⁺ accumulation leads to rhizosphere acidification, which affects the uptake of essential cations such as calcium (Ca²⁺) and magnesium (Mg²⁺). Maintaining an optimal NO₃⁻:NH₄⁺ ratio is essential for plant health in hydroponics and should be optimized for specific crops and growth conditions. A 25:75 NH₄⁺:NO₃ ratio enhanced Chinese kale growth without inducing NH₄⁺ toxicity [23]. The form of N supplied influences plant traits, including root architecture, secondary metabolism, and antioxidant enzyme activity [24]. While synthetic nutrient formulations allow for precise control of nitrogen inputs, they often create conditions that suppress the functional expression of microbial nitrogenase. Comparative studies evaluating how different N sources influence diazotroph activity in hydroponics remain limited, highlighting a gap in understanding nitrogen–microbe–plant interactions in soilless environments.

2.2. N Uptake and Assimilation

In hydroponically cultivated crops, N uptake occurs through membrane-bound transporter proteins and assimilatory enzymes. Primary inorganic N sources (NO₃^– and NH₄⁺) are transported into plant cells through nitrate transporters (NRTs) and ammonium transporter protein families [13,25]. The NRT gene family includes low-affinity (NRT1) and high-affinity (NRT2) transporters that function at distinct external NO₃⁻ concentrations. NRT1 proteins function under various NO₃⁻ concentrations and are regulated by phosphorylation to adapt to nutrient availability. NRT2 is activated under NO₃⁻-limited conditions and has a high substrate specificity [25].

When NO₃⁻ enters the cell, it undergoes a two-step reduction process prior to assimilation. Nitrate reductase (NR) catalyzes the conversion of NO₃⁻ to nitrite (NO₂⁻) in the cytosol, which then moves to plastids, where nitrite reductase (NiR) reduces it to NH₄⁺ (Figure 1). NH₄⁺ or NH₄⁺ absorbed from the nutrient solution enters assimilation via the glutamine synthetase (GS)–glutamate synthase (GOGAT) pathway. GS catalyzes the ATP-dependent amination of glutamate (Glu) with NH₄⁺ to glutamine (Gln), which GOAT converts back to Glu using 2-oxoglutarate (2-OG), thereby forming the central NH₄⁺ assimilation pathway [21]. The GS–GOGAT cycle incorporates inorganic N into organic amino acids, thereby enhancing biomass accumulation and metabolic homeostasis.

Other key enzymes also regulate the N metabolism. (i) Glutamate dehydrogenase (GDH) catalyzes the reversible amination of 2-OG to Glu under stress or when NH₄⁺ is abundant. (ii) Asparagine synthetase (ASN) converts Gln to aspartate (Asp) or NH₄⁺ to asparagine (Asn) for N storage. (iii) Carbamoyl phosphate synthetase converts bicarbonate (HCO₃⁻) and NH₄⁺ to carbamoyl phosphate for pyrimidine and arginine biosynthesis [21]. These reactions facilitate N₂ assimilation and integration with metabolic pathways to regulate the growth and stress responses in hydroponic systems.

2.3. NUE in Hydroponics

NUE is an important agronomic trait in hydroponic cultivation that influences nutrient management, crop yield, and environmental sustainability. NUE indicates the plant’s absorption, assimilation, and utilization of N for biomass or yield production. Optimizing NUE is essential in hydroponic systems to reduce N loss and fertilizer costs [26].

Several strategies have been employed to enhance NUE in hydroponics, including genotype selection, nutrient optimization, and biostimulants. Studies on lettuce grown using the nutrient film technique (NFT) reported that NUE increased at NO₃^– concentrations of 100–150 mg L⁻¹, whereas higher NO₃^– supplementation decreased NUE and increased N leaching [26]. Similarly, reducing NO₃^– concentration from 7.5 mM to 1.0 mM during tuber initiation increased NUE without reducing hydroponic potato yield [27].

Genotypic variation plays a crucial role in optimizing NUE. Spinach varieties with distinct growth rates have shown differential N uptake kinetics and N₂-assimilation efficiencies, based on root morphology and associated enzyme activities [28]. Hybrid “super rice” lines bred for enhanced root architecture have shown higher NH₄⁺ acquisition and improved NUE under hydroponic conditions [29].

Physiological strategies have shown promising results in improving NUE. Grafting watermelons onto bottle gourd rootstocks enhances root system architecture, NO₃⁻ uptake, and stress tolerance under N-limited conditions [20]. Biostimulants, such as legume protein hydrolysates, effectively enhance the N₂-assimilation efficiency. Foliar application of these biostimulants to leafy vegetables enhances N metabolism enzymes and antioxidant defense mechanisms, improving NUE in hydroponically grown plants [30].

2.4. Environmental Impact of N Leaching in Hydroponic Discharge Water

Open and semi-closed hydroponic systems generate nutrient-rich discharge water that contains high concentrations of N and other macronutrients. When released untreated, they pose ecological risks, including eutrophication, groundwater contamination, and greenhouse gas emissions [3,31]. Korean hydroponic farms reported total N concentrations in discharge effluents ranging from 48 to 494 mg L⁻¹, exceeding the national safety limit of 40.0 mg L⁻¹ [32]. Similarly, effluents from acyclic hydroponic systems contain N concentrations above 400 mg L⁻¹, potentially contaminating the surface and groundwater ecosystems [33].

In addition to water pollution, NO₃⁻ and NH₄⁺ accumulation in the rhizosphere promotes anaerobic microbial activity, causing denitrification and nitrous oxide release, which is a greenhouse gas with a 300-fold greater global warming potential than carbon dioxide (CO₂) [34]. Several remediation strategies have been explored to mitigate these effects. Sequencing Batch Reactors have achieved >89.5% NO₃⁻ removal efficiency, providing effective bioremediation of nutrient-rich effluents [32]. Algal–bacterial bioreactors have emerged as promising systems for nutrient removal and value-added biomass production for feed, biofertilizer, and bioenergy applications [10].

The transition to closed-loop hydroponic systems is essential for ensuring environmental sustainability. In the Netherlands, greenhouse growers must eliminate nutrient discharge by 2027, leading to the widespread adoption of advanced real-time nutrient sensors, automated fertigation controls, and nutrient recovery platforms [35].

3. BNF: Principles and Diazotrophic Microorganisms

3.1. Overview of Nitrogenase Enzyme and Energy Requirements

BNF is an enzymatic process in which diazotrophic microorganisms convert N₂ into NH₄⁺, providing plants with bioavailable N for cellular metabolism, protein synthesis, and biomass accumulation [9]. Although lightning, fossil fuel combustion, and industrial N₂ fixation through the Haber–Bosch process contribute to the global N cycle, diazotroph-mediated BNF remains the predominant biological pathway for NH₃ production in ecosystems [36]. Living organisms depend on BNF to synthesize essential biomolecules, including nucleic acids, proteins, amino acids, and chlorophyll, which are fundamental for growth and metabolism [9].

The stoichiometric reaction catalyzed by the nitrogenase enzyme complex is as follows:

$${\mathrm{N}}_2+{8\mathrm{H}}^{+}+{8\mathrm{e}}^{-}+16\mathrm{A}\mathrm{T}\mathrm{P}\to 2\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}_2+16\mathrm{A}\mathrm{D}\mathrm{P}+16\mathrm{P}\mathrm{i}$$

Nitrogenase-mediated N reduction requires a high ATP input. Approximately 16 ATP molecules are hydrolyzed per N₂ molecule reduced to NH₃, and 12 ATP equivalents are required for NH₄⁺ assimilation, translocation, and incorporation into organic nitrogenous compounds. The total energy cost is approximately 28 ATP molecules per reduced N₂ molecule. Despite this high energy demand, BNF remains more sustainable than the Haber–Bosch process. To contextualize sustainability, we incorporated a techno-economic comparison between industrial Haber–Bosch and BNF. Industrial Haber–Bosch ammonia costs USD 160–500 t⁻¹, with smaller plants and increased energy or transport factors edging costs above USD 500 t⁻¹; energy demands are approximately 28–32 GJ t⁻¹ NH₃ (~40 MJ kg⁻¹ N⁻¹) and emissions are ~1 t CO₂ t⁻¹ NH₃ [37,38]. In contrast, BNF requires ~16 MJ kg⁻¹ N⁻¹ or ~4.1 g C g⁻¹ N⁻¹ fixed [39].

BNF is thermodynamically exothermic (ΔH° = −45.2 KJ mol⁻¹ NH₃), but kinetically limited due to its high activation energy (~420 KJ mol⁻¹). To overcome this, nitrogenase enzymes couple ATP hydrolysis with electron donation from redox carriers such as ferredoxin or flavodoxin. Studies have used chemical reductants, such as dithionite, to simulate the reaction environment [40].

The nitrogenase enzyme complex consists of two metalloproteins: dinitrogenase reductase (Fe protein) and dinitrogenase (MoFe protein). The Fe protein encoded by nifH transfers electrons to the MoFe protein encoded by nifD and nifK [9]. Nitrogenases are classified into three types based on the metal cofactors at the active site: molybdenum (Mo)-dependent (FeMo-co), vanadium (V)-dependent (FeV-co), and iron-only (FeFe-co). Each variant has unique catalytic properties, with FeMo-Co being the most common and efficient under natural conditions [36].

The major biochemical limitation of nitrogenases is their extreme sensitivity to oxygen. Molecular oxygen irreversibly oxidizes and deactivates the metal clusters at the active site. Diazotrophs possess diverse protective mechanisms against environmental stressors. Unicellular cyanobacteria, such as Cyanothece, conduct BNF exclusively at night, separating nitrogenase activity from oxygenic photosynthesis [11]. Obligate aerobes, such as Azotobacter spp., use “respiratory protection,” maintaining high respiration rates to reduce intracellular oxygen levels [41]. Filamentous heterocystous cyanobacteria, including Anabaena and Nostoc, form specialized thick-walled heterocysts that isolate nitrogenase from the oxygen produced during photosynthesis [42]. Additional mechanisms include oxygen-scavenging proteins (e.g., FeSII), extracellular polysaccharide layers as oxygen diffusion barriers, and microaerobic growth in biofilms and root nodules [43].

From an agroecological perspective, symbiotic N₂ fixation requires significant carbon input from the host plants. The microbial fixation of 1 g of N₂ requires approximately 12 g of glucose [36]. However, in hydroponic systems, BNF is a renewable, low-emission strategy for N₂ input that promotes circular bioeconomy principles and reduces dependence on synthetic N₂ fertilizers.

3.2. Plant–Microbe Symbiosis in BNF

Diazotrophic microorganisms comprise diverse prokaryotes, including taxa from the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, and the phyla Cyanobacteria, Firmicutes, and Actinobacteria [40]. Based on their interactions with host plants, diazotrophs are categorized into three functional groups: rhizosphere, endophytic, and symbiotic groups [44]. Each group exhibits distinct colonization strategies, N₂ fixation efficiencies, and host preferences. The estimated N₂ fixation rates of various diazotrophs are summarized in

Table 1. Representative diazotrophic microorganisms and their N₂ fixation rates reported under various ecological conditions. Units vary depending on the system (soil, aquatic, or symbiotic). Reported N₂ fixation rates were estimated using the acetylene reduction assay (ARA).

Microbial Species	Estimated N2 Fixation Rate	Reference
Bacillus megaterium	210.05 ± 7.0 nmol C2H4 mg−1 protein day−1	[45]
Bacillus flexus	108.76 ± 3.66 nmol C2H4 mg−1 protein day−1
Bacillus circulans	98.28 ± 4.32 nmol C2H4 mg−1 protein day−1
Rhizobium spp.	24–584 kg ha−1	[46]
Frankia spp.	2–362 kg ha−1
Anabaena spp.	45–450 kg ha−1
Anabaena spp.	0.0216–0.073 g N m−2 day−1	[47]
Nostoc spp.	0.11–0.48 N m−2 day−1
Trichodesmium spp.	92 mmol N m−2 year−1 (range: 40–150)	[48]
UCYN-A (Candidatus Atelocyanobacterium thalassa)	4.9–9.1 nmol N L−1 d−1	[49]

.

3.2.1. Rhizospheric and Endophytic Diazotrophs

Rhizospheric diazotrophs are free-living or associative N₂-fixing bacteria that inhabit the root–soil interface, also known as the rhizosphere. This microenvironment is influenced by root exudates, including sugars, amino acids, and organic acids, which are chemoattractants that provide energy for microbial colonization [40]. These microorganisms are plant growth-promoting rhizobacteria that promote plant growth through N₂ fixation, phosphate (P) solubilization, siderophore production, and phytohormone synthesis [50].

In contrast, endophytic diazotrophs colonize internal plant tissues, including cortical cells, intercellular spaces, xylem, and phloem, without causing disease symptoms [51]. Endophytes have ecological advantages, including reduced competition, access to host-derived metabolites, and enhanced N transfer, owing to their association with plant vascular tissues [44]. Several genera, including Herbaspirillum, Gluconacetobacter, Burkholderia, Azoarcus, and Azospirillum, have been isolated from cereals, such as rice, maize, sorghum, and wheat [51,52]. Herbaspirillum seropedicae and Burkholderia vietnamiensis colonize the intercellular spaces in sugarcane tissues and enhance N₂ fixation under fertilizer-limited conditions [44]. A. brasilense, a facultative endophyte, exhibits chemotaxis towards root exudates and utilizes “respiratory protection” by increasing oxygen consumption to protect nitrogenase from oxidative inactivation [53].

Transcriptomic and metabolomic studies have shown that endophytic colonization enhances the expression of host plant genes associated with N₂ assimilation, stress signaling, and root architecture [54]. Endophytes enhance tolerance to abiotic stresses, such as drought, salinity, and nutrient deficiency, by mediating signaling networks involving plant hormones (e.g., auxins and cytokinins), reactive oxygen species, and small RNAs [50].

3.2.2. Symbiotic Diazotrophs

Symbiotic N₂ fixation occurs through mutualistic interactions between host plants and diazotrophic bacteria, forming root or stem nodules, where N₂ fixation occurs under microaerobic conditions [55]. In these symbioses, the plant provides photosynthetically derived carbon compounds and a protected niche for bacterial survival and nitrogenase activity in the root nodules [10].

Symbiosis in leguminous plants occurs in the order Rhizobiales, including Rhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, and Azorhizobium [56]. Symbiotic signaling begins with root-exuded flavonoids, which act as chemoattractants and activate nod gene expression in bacteria. Nod-factor synthesis facilitates host recognition, initiates infection thread development, and promotes cortical cell division, leading to nodule organogenesis [55]. Although legumes are the main hosts of rhizobia, certain non-leguminous species, such as Parasponia andersoni, form nodules with Rhizobium spp., representing an intermediate stage between rhizobial and actinorhizal symbioses [57].

In non-leguminous plants, actinorhizal symbiosis involves filamentous actinobacteria of Frankia spp., which form N₂-fixing nodules in woody angiosperms, such as Casuarina, Alnus, Myrica, Elaeagnus, and Hippophae [58]. Frankia-induced nodules contain vesicles that protect nitrogenase from oxygen and sporangia, which promotes microbial dissemination [59]. These symbioses facilitate afforestation, land reclamation, and ecological restoration in N₂-deficient environments [60].

4. Plant Growth-Promoting Properties of Diazotrophs Beyond N₂ Fixation

Recent advancements in plant–microbe interactions have shown that diazotrophic microorganisms have diverse plant growth-promoting properties, including phytohormone synthesis, nutrient solubilization, abiotic stress tolerance, phytopathogen inhibition, and enhancement of root architecture and rhizosphere microbial dynamics [3,31,50,61] (Table 2). These traits are exhibited under both controlled and field conditions, supporting the integration of diazotrophs into hydroponic systems [3].

Diazotrophs significantly contribute to the bioavailability of essential macro- and micronutrients, including phosphorus (P), iron (Fe), zinc (Zn), and potassium (K). Strains of A. brasilense, Azotobacter chroococcum, Bacillus spp., Pseudomonas fluorescens, Anabaena variabilis, and Tolypothrix synthesize organic acids, such as gluconic, citric, and oxalic acids, which chelate mineral ions from insoluble forms and promote plant root uptake [3,31,62,63,64,65,66]. Siderophore production, particularly by Azotobacter spp. and Herbaspirillum spp., enhances Fe acquisition under Fe-limiting conditions [67,68].

Endophytic diazotrophs, such as Gluconacetobacter diazotrophicus, enhance P solubilization and K mobilization in hydroponically grown crops [69]. For example, Ramos et al. [68] reported that H. seropedicae enhances Fe and Zn uptake in rice by stimulating root exudation and modulating phytohormone signaling pathways. Cyanobacteria, such as A. variabilis and Nostoc muscorum, contribute to nutrient acquisition and rhizosphere health by producing exopolysaccharides (EPSs) and siderophores (e.g., schizokinen), which chelate Fe³⁺ [70].

In addition to nutrient solubilization, many diazotrophs produce plant growth regulators, including indole-3-acetic acid (IAA), gibberellins, cytokinins, and abscisic acid. A. brasilense has been studied for its IAA production, which stimulates lateral root formation, root hair elongation, and increased nutrient absorption, thereby enhancing root system architecture under hydroponic conditions [71]. Although these traits are commonly associated with plant growth promotion, their efficacy under hydroponic conditions can vary significantly depending on the plant species, microbial concentration, environmental conditions, and nutrient solution composition, with some studies reporting inconsistent or negligible responses to inoculation.

Diazotrophs also improve plant resilience to abiotic stress. Inoculation of hydroponic lettuce with Bacillus subtilis enhances chlorophyll content, stomatal conductance, and drought tolerance [72]. Similarly, Bacillus amyloliquefaciens enhances salt tolerance by regulating Na⁺ transport, inducing osmolyte accumulation, and activating antioxidant defense enzymes, such as superoxide dismutase and catalase [73].

Recently, the biocontrol potential of diazotrophs has gained significant attention. A microbial consortium of Trichoderma harzianum, Streptomyces albus, and Azotobacter vinelandii inhibited Fusarium oxysporum in tomatoes and increased the fruit yield [74]. Endophytic Streptomyces spp. antagonize parasitic nematodes and foliar pathogens, while improving nutrient cycling [75]. A suspension of the cyanobacterium Nostoc sp. exhibited antifungal activity against phytopathogenic fungi and oomycetes [61]. The formulated microbial consortia, with P-mobilizing and polysaccharide-degrading capabilities, restored fertility in hydroponic wastewater and in degraded soil. Genetically modified consortia in post-mining landscapes improve nutrient availability and plant establishment [14].

Table 2. Biostimulant mechanisms and observed plant benefits of diazotrophic and photosynthetic microbial inoculants.

Microbial Species	Host Plants	Biostimulant Mechanisms	Plant Benefits	References
Bacterial Inoculants
Azospirillum brasilense	Maize, lettuce, common bean	IAA, GA, CKs, P solubilization, antioxidant enzymes, and siderophore production	Improved root/shoot growth and biomass, nutrient uptake (N, P, K, Ca, Fe, Zn), NUE, photosynthesis, abiotic stress tolerance	[62,76,77]
Azotobacter chroococcum	Cotton, cucumber, tomato, cabbage, sugar beet	IAA, GA, JA, ACC deaminase, P and K solubilization, siderophore	Increased biomass, yield, leaf nutrients, photosynthesis, stress and disease resistance	[63,67,78]
Rhizobium leguminosarum	Pea, common bean, soybean, peanut, chickpea, lentil, faba bean, cowpea, lupin	IAA, CKs, P solubilization	Improved seedling growth, vigor, biomass, and yield, antioxidant activity, and secondary metabolites (phenols, flavonoids, tannins)	[79,80,81]
Gluconacetobacter diazotrophicus	Tomato, rice, Arabidopsis thaliana	Endophytic colonization, N2 fixation, modulation of growth- and defense-related gene expression	Increased plant height, fresh weight, leaf chlorophyll, yield under both N-rich and N-deficient conditions, and drought tolerance	[82,83,84]
Herbaspirillum seropedicae	Palm, rice	Endophytic colonization; stimulation of vacuolar H+-ATPase and H+-PPase activity; N2 fixation	Enhanced plant growth, nutrients (N, K, Ca, and Mg), photosynthesis	[68,85]
Pseudomonas fluorescens	Sedum alfredii, okra, tomato	IAA, ACC deaminase, P solubilization, ISR, VOC, antibiotic and siderophore production	Enhanced biomass, leaf chlorophyll, Cd uptake, essential oil biosynthesis, drought tolerance	[66,86,87]
Bacillus amyloliquefaciens	Maize, soybean, tomato	IAA, GA, ABA, antibiotics (surfactin, bacillomycin D), VOCs (e.g., 2,3-butanediol), ACC deaminase, antifungal activity	Increased plant height, dry matter, yield, Fusarium and Alternaria inhibition, stress tolerance, and photosynthesis	[64,65,88]
Cyanobacterial Inoculants
Nostoc muscorum	Basil, garlic, barley	IAA, CKs, EPS, secondary metabolite secretion, antioxidant induction	Fusarium oxysporum inhibition, increased leaf weight, essential oil yield, stress resilience, and biomass	[89,90,91]
Anabaena variabilis	Basil, rice, soybean, wheat, okra, millet, mungbean	IAA, GA, N2 fixation, P solubilization, siderophore, EPS, hydrolytic enzyme (e.g., chitosanase) production	Enhanced growth, yield, seed germination, and root architecture, and reduced Fusarium infection and oxidative stress	[89,92,93]
Tolypothrix sp.	Basil, tomato, barley	N2 fixation; phytohormone and organic acid secretion; defense enzyme and chitosanase production; antifungal activity	Improved seed germination, plant height, and soil quality, and reduced soil-borne diseases	[89,92]
Microalgal Inoculants
Chlorella vulgaris	Arabidopsis thaliana, common bean, cucumber, wheat, soybean	IAA, GA	Increased root/shoot biomass, flower number, antioxidant capacity, glucosinolate biosynthesis, drought resistance	[94,95,96]
Scenedesmus quadricauda	Lettuce, sugar beet	Activation of C and N metabolism enzymes (GOGAT, GS, CS, MDH), PAL activity, phytohormone production	Improved chlorophyll, carotenoid, proteins, biomass, nutrient uptake, root architecture	[97,98]
Arthrospira platensis	Rosemary, wheat, tomato	IAA, GA, CKs, antioxidant enzymes (SOD, CAT, GR, PPO), improved nutrient uptake	Enhanced growth parameters (height, root length, fresh/dry weight); increased essential oil content and photosynthetic pigment levels; improved stress tolerance under drought and salinity	[99,100,101]

Abbreviations: IAA, indole acetic acid; GA, gibberellin; CKs, cytokinins; ACC, 1-aminocyclopropane-1-carboxylate; JA, jasmonic acid; ISR, induced systematic resistance; VOCs, volatile compounds; ABA, abscisic acid; EPS, exopolysaccharide; GOGAT, glutamate synthase; GS, glutamine synthetase; CS, citrate synthase; MDH, malate dehydrogenase; PAL, phenylalanine ammonia lyase; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; PPO, polyphenol oxidase.

Table 2. Biostimulant mechanisms and observed plant benefits of diazotrophic and photosynthetic microbial inoculants.

Microbial Species	Host Plants	Biostimulant Mechanisms	Plant Benefits	References
Bacterial Inoculants
Azospirillum brasilense	Maize, lettuce, common bean	IAA, GA, CKs, P solubilization, antioxidant enzymes, and siderophore production	Improved root/shoot growth and biomass, nutrient uptake (N, P, K, Ca, Fe, Zn), NUE, photosynthesis, abiotic stress tolerance	[62,76,77]
Azotobacter chroococcum	Cotton, cucumber, tomato, cabbage, sugar beet	IAA, GA, JA, ACC deaminase, P and K solubilization, siderophore	Increased biomass, yield, leaf nutrients, photosynthesis, stress and disease resistance	[63,67,78]
Rhizobium leguminosarum	Pea, common bean, soybean, peanut, chickpea, lentil, faba bean, cowpea, lupin	IAA, CKs, P solubilization	Improved seedling growth, vigor, biomass, and yield, antioxidant activity, and secondary metabolites (phenols, flavonoids, tannins)	[79,80,81]
Gluconacetobacter diazotrophicus	Tomato, rice, Arabidopsis thaliana	Endophytic colonization, N2 fixation, modulation of growth- and defense-related gene expression	Increased plant height, fresh weight, leaf chlorophyll, yield under both N-rich and N-deficient conditions, and drought tolerance	[82,83,84]
Herbaspirillum seropedicae	Palm, rice	Endophytic colonization; stimulation of vacuolar H+-ATPase and H+-PPase activity; N2 fixation	Enhanced plant growth, nutrients (N, K, Ca, and Mg), photosynthesis	[68,85]
Pseudomonas fluorescens	Sedum alfredii, okra, tomato	IAA, ACC deaminase, P solubilization, ISR, VOC, antibiotic and siderophore production	Enhanced biomass, leaf chlorophyll, Cd uptake, essential oil biosynthesis, drought tolerance	[66,86,87]
Bacillus amyloliquefaciens	Maize, soybean, tomato	IAA, GA, ABA, antibiotics (surfactin, bacillomycin D), VOCs (e.g., 2,3-butanediol), ACC deaminase, antifungal activity	Increased plant height, dry matter, yield, Fusarium and Alternaria inhibition, stress tolerance, and photosynthesis	[64,65,88]
Cyanobacterial Inoculants
Nostoc muscorum	Basil, garlic, barley	IAA, CKs, EPS, secondary metabolite secretion, antioxidant induction	Fusarium oxysporum inhibition, increased leaf weight, essential oil yield, stress resilience, and biomass	[89,90,91]
Anabaena variabilis	Basil, rice, soybean, wheat, okra, millet, mungbean	IAA, GA, N2 fixation, P solubilization, siderophore, EPS, hydrolytic enzyme (e.g., chitosanase) production	Enhanced growth, yield, seed germination, and root architecture, and reduced Fusarium infection and oxidative stress	[89,92,93]
Tolypothrix sp.	Basil, tomato, barley	N2 fixation; phytohormone and organic acid secretion; defense enzyme and chitosanase production; antifungal activity	Improved seed germination, plant height, and soil quality, and reduced soil-borne diseases	[89,92]
Microalgal Inoculants
Chlorella vulgaris	Arabidopsis thaliana, common bean, cucumber, wheat, soybean	IAA, GA	Increased root/shoot biomass, flower number, antioxidant capacity, glucosinolate biosynthesis, drought resistance	[94,95,96]
Scenedesmus quadricauda	Lettuce, sugar beet	Activation of C and N metabolism enzymes (GOGAT, GS, CS, MDH), PAL activity, phytohormone production	Improved chlorophyll, carotenoid, proteins, biomass, nutrient uptake, root architecture	[97,98]
Arthrospira platensis	Rosemary, wheat, tomato	IAA, GA, CKs, antioxidant enzymes (SOD, CAT, GR, PPO), improved nutrient uptake	Enhanced growth parameters (height, root length, fresh/dry weight); increased essential oil content and photosynthetic pigment levels; improved stress tolerance under drought and salinity	[99,100,101]

Abbreviations: IAA, indole acetic acid; GA, gibberellin; CKs, cytokinins; ACC, 1-aminocyclopropane-1-carboxylate; JA, jasmonic acid; ISR, induced systematic resistance; VOCs, volatile compounds; ABA, abscisic acid; EPS, exopolysaccharide; GOGAT, glutamate synthase; GS, glutamine synthetase; CS, citrate synthase; MDH, malate dehydrogenase; PAL, phenylalanine ammonia lyase; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; PPO, polyphenol oxidase.

5. Evidence of Diazotrophs in Hydroponics: A Case Series

Recent research has demonstrated the integration of diazotrophic microorganisms into hydroponic systems, resulting in enhanced plant growth, nutrient assimilation, and reduced dependence on synthetic N₂ fertilizers. Inoculation of Phaseolus vulgaris with Rhizobium tropici under hydroponic conditions enhanced nodule formation from 4 to 80 nodules per plant, thereby increasing N₂ assimilation [102]. Co-inoculation with Azospirillum brasilense reduced fertilizer input by 25% by enhancing nutrient uptake and enzyme activity in hydroponically grown arugula [103]. Co-cultivation with cyanobacteria, such as Synechococcus sp., Anabaena sp., Nostoc sp., Calothrix sp., Scytonema sp., and Cylindrospermum sp., has been shown to improve plant growth and nutrient uptake in hydroponic crops [3]. Diazotrophic endophytes and free-living microbes can enhance N₂ acquisition in non-leguminous crops [104]. Inoculation of lettuce with A. brasilense strains AbV5 and AbV6 increased plant biomass, photosynthetic efficiency, and N₂ assimilation, reducing the requirement for external N₂ fertilizers in hydroponic systems [105]. Similarly, co-inoculation of hydroponically cultivated maize with A. brasilense and H. seropedicae enhanced N uptake, K and P accumulation, and shoot vigor [106].

Several studies have reported quantifiable yield benefits following diazotroph inoculation of hydroponic vegetable crops. Inoculation of tomatoes with endophytic bacteria, Azotobacter sp., Azospirillum sp., and phosphate-solubilizing bacteria (PSB) resulted in an 8–9% increase in fruit yield compared with uninoculated controls [107]. In cherry tomatoes grown hydroponically under substrate culture, co-inoculation with plant growth-promoting rhizobacteria (PGPR) and mycorrhizal fungi produced 29–64% higher fruit weight per plant, with the significant gains observed at full nutrient concentrations [108]. In lettuce, the application of A. brasilense strains AbV5 and AbV6 increased leaf yield, fresh and dry biomass, nutrient assimilation, and photosynthetic parameters, with chlorophyll content up by 46%, net photosynthesis by 85%, and biomass by 40–60% compared to uninoculated plants [105]. In addition, basil grown in hydroponics with PGPR and mycorrhizal treatments achieved an exceptional 214% yield increase during the second harvest compared to the controls [109]. In our previous experiment, a comparative hydroponic cultivation trial using Knop nutrient solution was used to evaluate the effect of an authentic strain of Chlorella vulgaris Beijerinck on cucumber seedlings. Supplementation with Chlorella suspension led to a 25% increase in dry root biomass, rising from 0.04 g to 0.05 g. These results suggest enhanced nutrient allocation and potential biostimulant effects under hydroponic conditions (Figure 2) [110]. These data demonstrate that diazotrophic inoculation can consistently yield ~10–200% increases in vegetable yield under hydroponic conditions.

Symbiotic diazotrophs, such as R. tropici and R. sophoriradicis, exhibit effective root nodulation and N₂ fixation in hydroponically grown common beans, resulting in increased pod numbers, tissue N content, and yield [111]. Cyanobacterial diazotrophs, such as N. muscorum and A. variabilis, have received attention for their functional roles in N₂ fixation and plant growth promotion. The incorporation of Nostoc spp. into spinach hydroponics improved ascorbic acid content by 0.37-fold, antioxidant capacity by 0.36-fold, and β-carotene levels, thereby enhancing the yield and nutritional quality [112]. Similarly, the supplementation of hydroponically grown cucumbers with Anabaena spp. improves lateral root development, increases N uptake, and enhances NUE [3].

The survival and functionality of diazotrophic microorganisms in hydroponic systems depends on their physiological and biochemical adaptations. One of the main challenges in integrating diazotrophs into hydroponic systems is the conflicting oxygen requirement; while plant roots demand high DO levels for aerobic respiration, nitrogenase is highly sensitive to oxygen and requires microaerobic conditions. Several strategies have been developed to overcome this issue. Obligate aerobes, such as A. vinelandii utilize "respiratory protection" by increasing cellular oxygen consumption to protect nitrogenase from oxidative damage [113]. Similarly, heterocyst-forming cyanobacteria, such as Anabaena and Nostoc, physically compartmentalize nitrogenase within oxygen-impermeable cells [114]. Additionally, EPS-producing strains, such as A. brasilense and H. seropedicae, form a barrier that limits oxygen diffusion and enhances biofilm-based colonization [115,116]. Spatial segregation of diazotrophs within root-interfacing microzones or physical carriers (e.g., alginate beads and porous capsules) may allow local microaerobic niches to persist, even under oxygen-rich conditions. Such strategies hold promise for optimizing plant and microbial oxygen requirements in engineered hydroponic environments [117].

Endophytic diazotrophs, such as G. diazotrophicus, colonize the root cortical tissues and are involved in nutrient exchange. In hydroponic systems, these bacteria regulate quorum sensing and upregulate stress response genes in response to electrical conductivity (EC) and pH fluctuations, thereby contributing to microbial stability and plant resilience [82]. Recent advances in confocal laser scanning microscopy and metatranscriptomics have enabled the direct visualization and gene expression profiling of root colonization, demonstrating the importance of maintaining optimal carbon/nitrogen (C:N) ratios and oxygen availability for sustained microbial function [118]. The effective application of diazotrophs in hydroponic systems depends on their nitrogenase activity, ecological compatibility with plant hosts, and nutrient requirements (

Table 3. Case studies of N₂-fixing microbial inoculants that enhance the growth, nutrition, and stress resilience of crops under hydroponic conditions.

Host Plant	N2-Fixing Microbial Species	Key Findings	Reference
Basil (Ocimum basilicum L.)	Chlorella vulgaris, Bacillus subtilis, Bacillus megaterium, and Pseudomonas fluorescens	Increased yield, leaf area, branches, phenolics, flavonoids, antioxidants, nutrients (N, P, K, Ca, Mg, Fe, Mn, Zn, Cu)	[119]
Buckwheat (Fagopyrum esculentum)	Azotobacter vinelandii	Enhanced plant growth, chlorophyll A, exometabolites, proteins, carbohydrates, phenolics	[120]
Chrysanthemum (Chrysanthemum morifolium)	Anabaena torulosa, Anabaena doliolum, Anabaena laxa	Improved IAA production, biofilm formation, PEPC, leaf chlorophyll	[118]
Common bean (Phaseolus vulgaris L.)	Rhizobium sophoriradicis, Rhizobium tropici	Increased nodulation, N2 fixation, and pod yield	[111]
Lettuce (Lactuca sativa)	Azospirillum brasilense	Increased fresh biomass, nutrient uptake (N, P, K, Ca, Zn, Cu, Mn, Fe), leaf chlorophyll content, photosynthesis	[119]
	A brasilense, Trichoderma harzianum	Increased root growth, leaf number, nutrient uptake (K, P, Ca, Mg, Fe, Mn, Cu, Zn), reduced NO3– accumulation	[76]
	Pseudomonas lundensis, Pseudomonas migulae	Improved plant growth, IAA, ISR, myo-inositol, and acetic acid	[121]
	Gluconacetobacter diazotrophicus	Increased plant biomass, NUE	[122]
Maize (Zea mays L.)	Herbaspirillum seropedicae, A. brasilense	Enhanced dry biomass, nutrients (N, P, K), NO3– assimilation, NO3− reductase activity, reduced NH4+ and sugar levels	[120]
	Bacillus amyloliquefaciens	Promoted seedling growth, leaf chlorophyll, soluble sugars, glutathione, antioxidant enzyme activity (POD, CAT), reduced Na+ accumulation and modulated stress-related genes (e.g., RBCS, RBCL, HKT1, NHX1-3)	[73]
Pac choi (Brassica rapa var. chinensis)	Bacillus amyloliquefaciens	Improved NUE	[123]
Rice (Oryza sativa L.)	Nostoc commune, Scytonema bohneri	Increased shoot and root length, biomass, and leaf area under chlorpyrifos stress	[124]
Strawberry (Fragaria vesca)	Azotobacter spp., Azospirillum spp.	Increased plant height, leaf chlorophyll, root biomass, yield, and soluble solid content	[125]
Tomato (Solanum lycopersicum L.)	Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas putida, Pseudomonas syringae	Reduced Pythium ultimum-induced root rot, increased seedling growth and fruit yield	[126]
	Arthrospira platensis	Increased plant growth, root weight, and node development	[3]

Abbreviations: IAA, indole acetic acid; PEPC, phosphoenolpyruvate carboxylase; ISR, induced systematic resistance; NUE, nitrogen use efficiency.

). Despite promising evidence of diazotroph activity in hydroponic systems, the reproducibility of these outcomes across different setups remains unclear. Most existing studies have been limited to short-term trials in controlled environments, limiting real-time validation across different crops, nutrient recipes, and climatic setups. Variations in nutrient composition, aeration, and root architecture can significantly influence colonization efficiency and nitrogen fixation rates.

6. Strategies to Improve Diazotroph Performance in Hydroponics

6.1. Microbial Strain Selection and Engineering

Recent advances in microbial biotechnology have enhanced the isolation, characterization, and engineering of efficient diazotrophic strains for use in hydroponic systems. High-throughput screening platforms, such as droplet microfluidics, fluorescence-based reporter assays, and synthetic promoter libraries, have emerged as powerful tools for the rapid identification and optimization of microbial strains with high N₂ fixation potential.

Droplet microfluidics enables the encapsulation of individual microbial cells within picoliter-scale droplets (typically 1 pL to 10 nL), allowing for the high-resolution screening of large microbial populations for nitrogenase activity. This approach enables the real-time evaluation of N₂ fixation potential, thereby reducing the time and resources required for strain selection [127]. Agresti et al. [128] demonstrated that droplet-based screening of enzyme variants led to a 10-fold increase in catalytic activity compared to the host strain, validating its utility for diazotroph selection.

Fluorescence-based reporter assays facilitate the monitoring of nif gene expression using fluorescent protein reporters regulated by N-responsive promoters. This approach involves cell encapsulation with fluorogenic substrates, enabling the visual and quantitative identification of nitrogenase activity, where fluorescence intensity correlates with enzymatic function [129].

Synthetic promoter libraries have revolutionized the precision tuning of gene expression, particularly within nif gene clusters in diazotrophic bacteria. These libraries regulate N₂ fixation activity based on host plant requirements and environmental conditions in hydroponic systems [130].

Genetic engineering approaches, including the targeted overexpression of nitrogenase structural genes (nifH, nifD, and nifK), have enhanced the N2-fixing capacity of engineered diazotrophs. The integration of nif genes from A. vinelandii into Escherichia coli resulted in a 10-fold increase in nitrogenase activity through the co-expression of electron transfer proteins, such as flavodoxin (fldA) and ferredoxin–NADP+ reductase (ydbK) [131].

To address nitrogenase oxygen sensitivity, which limits functionality in aerobic hydroponic systems, engineered strains include oxygen-scavenging enzymes, such as flavohemoglobins. These enzymes detoxify reactive oxygen and N species via NO dioxygenase activity, preserving nitrogenase activity under fluctuating oxygen levels [15].

Genome editing technologies, particularly CRISPR-Cas9, have enabled the insertion of N₂ fixation operons into non-diazotrophic plant-associated bacterial and chassis strains, thereby advancing the development of next-generation synthetic symbioses. Wang et al. [132] generated leghemoglobin mutants in Lotus japonicus using CRISPR-Cas9, which showed synergistic roles in maintaining optimal N₂ fixation in plants. Similarly, Gao et al. [133] applied cell type-specific CRISPR-Cas9 genome editing in legumes, achieving precise gene knockout within specific nodule zones, and providing insights into stage-specific gene regulation.

Recent studies have expressed multiple nitrogenase components in cereal crops, including rice, although the stability and proteolytic degradation of these components, such as NifH, remain challenging to address. Shang et al. [134] introduced a 13-gene nitrogenase biosynthesis cluster into rice plants, resulting in the cytosolic expression of 11 nitrogenase proteins and a stable NifDK tetramer formation. NifH variants that are resistant to host plant endoproteinase degradation have been engineered to overcome this instability.

6.2. Formulation and Delivery Technologies

The method of delivering microbial inoculants determines their efficiency in hydroponic systems. Conventional nutrient solutions in hydroponics lead to the rapid leaching of bioinoculants, resulting in low microbial retention and reduced colonization, which reduces the persistence and effectiveness of the inoculants. Encapsulation technologies have gained significant attention in addressing these challenges. Alginate, a naturally derived biodegradable polysaccharide, is widely used for microbial encapsulation because of its gel-forming ability, nontoxicity, and ease of use. However, alginate beads alone cannot provide sufficient protection during drying, hardening, and storage. To enhance the stability and stress tolerance of alginate-based capsules, they are combined with organic compounds, such as starch, glycerol, chitin, skim milk, and humic acids [135]. These additives improve the capsule strength and increase microbial survival under desiccation and storage conditions. The addition of starch to alginate formulations enhances protection by promoting microbial adhesion and shielding cells from physical stress and ultraviolet radiation, which maintains their activity during storage [117]. A high starch concentration improves capsule viscosity and reduces porosity, allowing for the controlled release of microorganisms. Recently, nanocellulose-based hydrogels have emerged as novel carriers for microbial encapsulation. Owing to their high surface area, mechanical stability, and water-holding capacity, nanocellulose gels effectively deliver beneficial microbes to hydroponic systems [136].

Recent advances in encapsulation technologies have improved the shelf life and storage potential of bioinoculants, while maintaining their microbial activity. Freeze-drying removes moisture by sublimation under low-temperature and vacuum conditions, preserving the physiological integrity of diazotrophic microbes for long-term storage. Studies have shown that freeze-dried formulations maintain high activity and are readily reactivated upon application [137]. Microencapsulation using biodegradable carriers, such as alginate, starch, gum arabic, or nanocellulose, provides physical protection against UV radiation, heat, and dehydration, thereby promoting gradual root colonization in hydroponic systems [117,135,136]. Spray-freeze-drying (SFD) combines atomization and freeze-drying, wherein microbial suspensions are rapidly frozen into droplets and freeze-dried, resulting in microcapsules with high structural integrity and survival rates [138]. SFD has shown promising results in preserving probiotic bacteria during drying and in enhancing their rehydration potential [139].

The efficacy of diazotrophic bioinoculants can be enhanced by incorporating bioformulation additives such as humic substances, molasses, or prebiotics. These carbon-rich compounds stimulate microbial activity and promote the colonization of hydroponic systems. Carboxymethyl cellulose (CMC) improves the microbial delivery and performance of bioformulations. Formulations containing CMC with A. chroococcum and Trichoderma afroharzianum enhanced plant growth, photosynthetic efficiency, and nutrient uptake in sweet basil [140]. Moreover, co-inoculation with compatible PGPR or mycorrhizal fungi has synergistic effects, resulting in improved rhizosphere colonization and resource utilization [141]. Furthermore, bioformulation stability and performance under fluctuating EC and pH conditions remain underexplored.

6.3. Optimization of Hydroponic Conditions

Optimizing the environmental parameters in hydroponic systems is crucial for enhancing the activity, persistence, and N₂-fixing efficiency of diazotrophic microorganisms. Among these parameters, the regulation of pH and N₂ levels in nutrient solutions is essential. A pH range of 5.5–6.5 promotes optimal nutrient availability and microbial activity [142]. The N₂ source (NO₃⁻ or NH₄⁺) can influence pH dynamics, with NO₃^– typically inducing alkalinization and NH₄⁺ inducing acidification. Studies have reported that maintaining a NO₃^–-to-NH₄⁺ ratio of approximately 3:1 stabilizes pH, which significantly enhances microbial growth and efficient N₂ fixation [143]. An adequate oxygen supply to the nutrient solution is essential for aerobic respiration in plant roots and microbial communities. Studies have reported that dissolved oxygen (DO) levels above 3.8 mg L⁻¹ in NH₄⁺-based systems and 5.3 mg L⁻¹ in NO₃^–-based systems increase photosynthetic efficiency and diazotrophic metabolism [144]. Aeration methods, such as Venturi injectors, air stones, and high-efficiency recirculating pumps, can maintain optimal DO levels in closed-loop hydroponic systems [145].

Controlling root temperature is essential for microbial growth, nitrogenase activity, and N₂ fixation. The optimal temperature in the root zone ranges from 18 to 24 °C, which increases enzymatic activity and microbial community stability, whereas deviations can inhibit nitrogenase activity through enzyme denaturation or redox balance disruption [146]. The use of real-time monitoring and automation tools, such as pH, EC, total dissolved solids, and DO sensors, enhances the regulation of physicochemical parameters. The Mamdani Fuzzy Inference System (MFIS) has been shown to be efficient in maintaining optimal conditions for stabilizing microbial activity and improving plant NUE [147].

Supplementation with organic carbon sources, particularly low-molecular-weight organic acids, such as malate and citrate, enhances microbial metabolism and promotes N transfer to host plants. These compounds act as key intermediates in the tricarboxylic acid cycle, promoting energy production and microbial growth under hydroponic conditions [148]. The exogenous application of these acids stimulates diazotroph activity, promoting N₂ fixation while regulating root exudation patterns that shape the rhizosphere microbial community structure [149]. These modifications influence microbial colonization, nutrient cycling, and host–microbe signaling pathways. Organic acid metabolism is associated with plant responses to abiotic stress, nutrient uptake regulation, and colonization, indicating the benefits of organic acid supplementation in hydroponics [149].

Adjustments to the nutrient solution composition in hydroponic systems, particularly the NO₃^–:NH₄⁺ ratio, significantly influence nif gene translational regulation in diazotrophs, such as Azospirillum and Azotobacter. High concentrations of fixed N sources, such as NH₄⁺, inhibit nif gene expression by inhibiting the transcriptional activator NifA through the action of NifL. Under N-limiting conditions, this inhibition increases, promoting nif gene expression and nitrogenase activity [150]. Maintaining a balanced NO₃^–:NH₄⁺ ratio is essential for plant nutrition and optimizing favorable conditions for diazotrophic gene expression and N₂ fixation. The NO₃^–-to-NH₄⁺ ratio influences plant N₂ assimilation pathways, which affect the rhizospheric biochemical environment and microbial colonization [151].

6.4. Omics-Based Monitoring and Precision Management

The integration of omics technologies, including genomics, transcriptomics, proteomics, metabolomics, and microbiomics, has advanced our understanding of plant–microbe interactions in hydroponic systems. These high-throughput platforms facilitate holistic system-level profiling of microbial communities, indicating their taxonomic composition and functional activity. They facilitate the systematic identification of genetic elements, regulatory networks, and metabolic pathways involved in N₂ fixation, phytohormone production, stress tolerance, and nutrient cycling. Multi-omics investigations have demonstrated how plant-associated microbiomes respond to abiotic stress factors, such as salinity, nutrient limitation, and temperature fluctuations, and the development of targeted microbial inoculants for hydroponic systems [16]. Metatranscriptomic and metaproteomic studies are valuable for monitoring nitrogenase expression and activity, providing insights into the physiological activity and functional potential of diazotrophs under hydroponic conditions.

Precision management strategies using real-time sensing technologies have emerged as critical tools for optimizing nutrient and environmental parameters in hydroponic systems. Laser-Induced Breakdown Spectroscopy (LIBS) is a rapid, nondestructive analytical method that can directly detect nutrient concentrations in aqueous solutions. LIBS platforms facilitate the real-time identification of nutrient imbalances, including deficiencies and toxicity, and enhance immediate corrective actions to maintain optimal nutrient conditions for plants and microbial populations [152]. The integration of omics datasets with AI and machine learning (ML) has advanced the field of smart agriculture. AI and ML are increasingly being integrated into hydroponic systems to enhance decision-making and nutrient regulation. For instance, CNN-based models trained on basil leaf images have achieved high accuracy in diagnosing N, P, and K deficiencies based on visual traits [153]. Explainable deep-learning frameworks have further advanced nondestructive phenotyping capabilities, facilitating the real-time monitoring of physiological responses in root systems [154]. At the Berkeley Lab, the RhizoNet platform uses deep convolutional architectures to segment and quantify root biomass dynamically in hydroponic contexts [155]. Moreover, a 2025 systematic review documented the efficacy of hybrid ML models, such as CNNs, DNNs, LSTM, fuzzy logic, and reinforcement learning, in smart AI-driven nutrient dosing systems that optimize EC, pH, and output nutrient concentrations in real time [156]. Integrating such AI-enabled systems provides the foundation for predictive diagnostics, adaptive nutrient schedules, and consistent microbial stability under fluctuating hydroponic conditions.

These approaches facilitate the interpretation of large heterogeneous datasets from omics platforms, sensor networks, and environmental control systems, thereby enabling the development of predictive models for nutrient scheduling, microbial applications, and environmental changes. AI-based systems have been effectively used to model plant nutrient uptake, detect early physiological stress, and regulate parameters, such as temperature, humidity, and pH. Such decision support systems have enhanced diazotroph colonization, N₂-fixing efficiency, crop yields, and system resilience [17].

7. Policy, Regulation, and Commercialization of BNF-Based Inputs in Hydroponics

The integration of diazotrophs into hydroponic systems is an emerging strategy to reduce dependence on synthetic N₂ fertilizers and enhance hydroponic sustainability. However, the regulatory, policy, and commercial systems governing these microbial biostimulants remain heterogeneous, underdeveloped, and dynamic, with significant regional variations. To provide a strategic context, it is noteworthy that the United States accounts for approximately 33 % of the global hydroponic crop revenue, with a large number of commercial hydroponic enterprises supported by advanced technological infrastructure [157]. Europe, led by the Netherlands and Germany, represents another key hub, while Asia–Pacific markets (especially China, India, Japan, and Singapore) are witnessing accelerated growth owing to urban agriculture strategies and investment [158]. Regarding the adoption of BNF-based biofertilizers across controlled-environment agriculture, the major biofertilizer consumers include India, China, Brazil, and the United States, mediated by policy incentives, sustainability goals, and local production targets [19].

In the European Union (EU), microbial biostimulants are regulated under Regulation (EU) 2019/1009, which classifies them under Product Function Category 6A [159]. Currently, only a limited number of microbial taxa, including Azotobacter spp., Rhizobium spp., Azospirillum spp., and mycorrhizal fungi, have been approved for agricultural use. This excludes many promising nontraditional or genetically modified diazotrophs. Researchers and agri-biotech stakeholders have advocated for periodic updates to regulatory lists based on recent advancements in microbial ecology, biotechnology, and synthetic biology [160]. The EU is developing microbe-based biostimulant products with high standards, including parameters such as viable cell count, nutrient solubilization potential, genetic stability, shelf life, and low contamination limits [161].

In the United States, the regulatory policy for microbial inoculants lacks a unified federal policy for N₂-based biofertilizers. The Plant Biostimulants Act of 2023 aims to provide consistent national guidelines; however, this system is still being developed. The United States Department of Agriculture (USDA) permits hydroponic system certification as organic, a policy that remains controversial owing to varying interpretations by certifying bodies and concerns regarding the exclusion of soil-based ecological interactions [18]. In contrast, countries such as China, India, and Brazil have structured regulatory policies for biofertilizers, administered by their respective ministries of agriculture. These systems typically require product registration, quality testing, and field trials before market approval, which enhances the product credibility in both conventional and hydroponic systems [19].

India, a pioneer in biofertilizer development, regulates biofertilizers under the Fertilizers (Control) Order of 1985, designating them as essential commodities. This policy requires labelling norms, production standards, microbial load specifications, and contamination limits. India promotes sustainable horticulture and hydroponics through subsidies ranging from 50% to 90% under the Mission for the Integrated Development of Horticulture (MIDH). The Bureau of Indian Standards (BIS) has formulated safety and efficacy benchmarks for hydroponically grown produce [162]. India’s BIS, under the Fertilizer Control Order (FCO) of 1985, revised in 2016 and 2021, has implemented formal specifications and testing protocols for biofertilizers, including Rhizobium, Azotobacter, Azospirillum, phosphate- and potassium-solubilizing bacteria, mycorrhizal fungi, and microbial consortia, as defined in IS standards IS 17134, IS 17135, IS 17136, IS 17137, IS 17672, and IS 17755 [163]. These standards set objective quality benchmarks: minimum viable cell counts (10⁷–10⁹ CFU g⁻¹), pH 6.0–8.0, moisture <10–15%, contaminant thresholds (none at 10⁵ dilution), carrier material properties, germination/nodulation efficacy, and six-month shelf life [163]. Part A of Schedule III under the FCO provides detailed guidelines for sampling, analysis, and tolerance limits, ensuring both the safety and efficacy of BNF-based inputs [164].

From a commercialization perspective, the global market for N₂-fixing biofertilizers has grown exponentially, valued at USD 800 million in 2016 and projected to exceed USD 5.2 billion by 2028 [165]. North America remains the largest market, followed by Europe and the Asia–Pacific region. Several agri-tech startups and corporations have invested in diazotrophic formulations to achieve hydroponic compatibility in their products. However, challenges related to microbial survival, colonization, and nitrogenase activity in nutrient-rich hydroponic systems remain unresolved [18].

Recent advances in microbial biotechnology and synthetic biology have led to the development of the next-generation inoculants. Modular plasmid-based gene expression systems have been engineered in A. brasilense and H. seropedicae to regulate nitrogenase activity under various conditions [166]. Similarly, engineered strains of Klebsiella variicola have shown up to a 15-fold increase in NH₄⁺ excretion, improving N bioavailability during the vegetative growth of maize [167]. Furthermore, Plasma-Activated Water (PAW) has emerged as an N-enriched carrier, enhancing root and shoot development and increasing soluble sugar and protein content in hydroponically grown radish plants compared with conventional nutrient solutions [138]. Endophytic diazotrophs have gained attention because of their ability to colonize the internal tissues of host plants. A previous study demonstrated the successful colonization of hydroponically grown tomato plants by G. diazotrophicus under N-limited conditions, colonizing root tissues and translocating them to the aerial parts [82].

EU-funded projects, such as CORDIS Projects 832574 and 961930, are investigating the use of N₂-fixing cyanobacteria for organic hydroponic fertilizer production [168,169]. The successful integration of diazotrophs into hydroponics depends on the (i) standardization of global regulatory policies, (ii) establishment of quality assurance protocols for microbial inoculants, and (iii) commercialization of robust microbial technologies. Policymakers must prioritize regulatory updates for microbial innovations, and researchers, stakeholders, and certification bodies must collaborate to develop safe and efficient microbial solutions. Investment in fundamental research, translational biotechnology, and policy coordination is crucial to realize the potential of diazotrophs in hydroponic systems for sustainable agriculture. However, standardization of microbial quality across countries remains a significant regulatory bottleneck for international commercialization.

8. Challenges and Limitations

Despite the promising results obtained by integrating diazotrophic microorganisms into hydroponic systems, this approach has several physiological, ecological, and technological limitations. However, emerging research has demonstrated targeted strategies to overcome these constraints. Unlike soil-based agroecosystems, which promote diverse microbial communities, hydroponics presents a simplified environment that can inhibit microbial colonization and BNF.

Microbial colonization is challenged by continuous nutrient flow, the absence of solid rhizospheric surfaces, and dilution effects, which reduce microbial adhesion, colonization density, and biofilm formation on the root surfaces [12]. Although endophytic colonization can partially compensate for this, many beneficial strains exhibit poor epiphytic persistence and limited endophytic competence under hydroponic conditions [44]. Biofilm development remains unstable in dynamic liquid environments because of shear stress and fluctuations in pH, EC, and DO levels, which affect the activity of diazotrophs [115,116]. Recent advances in formulation technologies, such as encapsulation in alginate–nanocellulose composites or cryoprotective matrices, have shown promise in improving microbial persistence and promoting gradual root colonization under recirculating hydroponic conditions [117,135,136].

Nitrogenase, the key enzyme in BNF, is irreversibly inactivated by oxygen, which presents a significant challenge in aerated hydroponic systems such as the NFT. This is critical for obligate diazotrophs, such as A. vinelandii and Clostridium spp., which require microaerobic or anaerobic conditions for N₂ fixation. Although some strains utilize protective mechanisms, such as increased respiration, heterocyst formation in cyanobacteria, or nocturnal N₂ fixation, these adaptations are strain-specific and insufficient for high BNF rates in hydroponic systems [41,46]. Engineered strains expressing flavohemoglobins and NO dioxygenase enzymes have improved nitrogenase stability under oxidative conditions. Synthetic biology approaches, including the insertion of N₂ fixation operons into chassis strains with oxygen-scavenging capabilities, provide promising directions for oxygen-sensitive applications [15,131,132].

The chemical composition of commercial nutrient solutions is incompatible with the sustainability of BNF. High NO₃⁻ or NH₄⁺ concentrations act as negative regulators of nitrogenase transcription and activity, causing downregulation of nif genes and a reduction in diazotrophic activity [139]. Moreover, excessive N promotes the growth of non-diazotrophic strains that replace the introduced diazotrophs. Strategies such as adjusting NO₃⁻:NH₄⁺ ratios, supplementing with low-molecular-weight organic acids (e.g., malate and citrate), and applying bioavailable carbon substrates can mitigate the inhibitory effects on nif gene expression. Optimized nutrient formulations designed for diazotroph activity can support consistent BNF activity in hydroponic systems [151].

The accurate quantification of in situ N₂ fixation remains a significant limitation. Most hydroponic studies have reported indirect indicators, such as plant biomass, total N content, and chlorophyll content, which cannot conclusively distinguish between biologically fixed and externally supplied N. Recent advances in omics-based tools, including metatranscriptomics of nif gene expression, biosensor-linked fluorescence assays, and real-time monitoring platforms using genetically encoded reporters, have provided high-resolution tools for BNF tracking. The integration of portable 15N isotope-based diagnostics into commercial workflows remains a key future direction [118].

Host specificity of diazotrophs in hydroponic systems is a key challenge. An effective diazotrophic strain that improves lettuce growth may not effectively colonize or function in cucumber or tomato because of differences in root architecture, exudate composition, and immune responses [44]. Native microbial communities in inert hydroponic substrates, such as rockwool and cocopeat, may outcompete the introduced strains, reducing the success of colonization. Precision inoculation strategies based on host genotype–microbiome compatibility, enabled by genome-based predictive models, have demonstrated improved success rates in recent studies [40,149].

Regulatory and technological gaps inhibit the commercialization of microbial inoculants in hydroponic systems. Most countries lack consistent biosafety guidelines, efficacy validation procedures, and quality control standards for bioinoculants in soilless systems [149]. Product variability in formulation types (liquid, alginate-encapsulated, freeze-dried), delivery methods (root zone, foliar spray, fertigation), and shelf-life stability limits practical applications and results in repeatability. Efforts toward integrating international quality assurance standards and large-scale multi-environmental trials are essential to promote the safe and effective use of diazotroph-based biofertilizers in hydroponics [117,135].

Thus, future research should integrate microbial ecology, plant physiology, synthetic biology, and real-time sensing to optimize the performance of diazotrophs in hydroponics. Smart bioprocessing tools, AI-assisted monitoring platforms, and standardized microbial formulations will drive the successful deployment of BNF-based fertilization in soilless agriculture.

9. Conclusions and Future Perspectives

N is a vital macronutrient that limits plant productivity in hydroponic systems. Although synthetic N fertilizers enhance crop yields, their excessive and inefficient use in hydroponics results in nutrient leaching, nitrate accumulation, and environmental degradation. BNF mediated by diazotrophic microorganisms provides a low-emission, circular, bioeconomic strategy for nitrogen input in hydroponic cultivation. This review examines the diversity, mechanisms, and applications of diazotrophs as sustainable alternatives to synthetic N in hydroponics.

The key findings of this review highlight that functional diazotrophs can enhance NUE, plant growth, and microbial stability under hydroponic conditions via nitrogenase activity, biofilm formation, EPS production, and signaling interactions. However, their successful integration is inhibited by oxygen sensitivity, microbial leaching, nitrate repression of nif genes, and limited regulatory frameworks.

Recent advances have provided innovative solutions to these limitations. For instance, microbial encapsulation using alginate–starch and nanocellulose matrices enhances microbial survival and root adhesion under hydroponic shear stress conditions, providing a slow and sustained release of diazotrophs. Freeze-dried and spray-freeze-dried formulations preserve microbial viability through moisture sublimation, enhancing shelf life and application efficiency.

Mechanistically, nitrate-induced repression of nif gene expression is mitigated by maintaining optimized NO₃⁻:NH₄⁺ ratios (~3:1) in nutrient solutions, which supports both plant uptake and diazotroph activity. Supplementation with low-molecular-weight organic acids (e.g., citrate and malate) acts as a carbon substrate for the tricarboxylic acid cycle and modulates root exudation to recruit beneficial microbes.

To address nitrogenase oxygen sensitivity, engineered diazotrophs expressing flavohemoglobins and NO dioxygenases offer protection by detoxifying reactive oxygen species. Synthetic biology tools, including nif operon expression in chassis strains and modular gene circuit design, enhance nitrogenase function in non-native oxidative environments.

Omics technologies, such as metagenomics, transcriptomics, proteomics, and biosensors, are increasingly being used to profile microbial activity, identify stress-responsive genes, and quantify in situ nitrogenase expression. When integrated with AI-driven real-time monitoring (e.g., CNN, LIBS, and RhizoNet), these systems enable dynamic nutrient scheduling, microbial diagnostics, and adaptive management in hydroponic systems.

Future research should focus on the following aspects:

• Engineering diazotrophic consortia with complementary traits such as nitrogenase activity, abiotic stress tolerance, and quorum sensing capabilities.
• Application of genome editing (e.g., CRISPR-Cas9) to enhance N fixation genes in diazotrophs and non-diazotrophic hosts, expanding microbial applicability across diverse crops.
• Development of real-time biosensors and portable diagnostics for tracking BNF efficiency, nitrogenase function, and colonization dynamics.
• Standardizing protocols for the formulation, delivery, and efficacy validation of microbial bioinputs in hydroponic environments.
• Fostering regulatory frameworks that encourage innovation while ensuring safety, reproducibility, and scale-up feasibility.
• Facilitating public–private partnerships to promote translational research and commercialization of BNF-based solutions in vertical farming and urban agriculture.

In conclusion, diazotrophic microbes are a promising solution for decarbonized, resource-efficient, and high-yield hydroponic food systems. With sustained investments in microbial engineering, sensor technology, bioformulation science, and regulatory standardization, BNF can transition from niche trials to mainstream hydroponic fertilization practices. Future studies must prioritize long-term assessments under commercial hydroponic conditions, standardized protocols for microbial application, and comparative trials across system types. Addressing these knowledge gaps is essential to translate laboratory findings into practical and scalable BNF solutions.