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Review

Immobilization Technology of Aerobic Denitrifying Bacteria and Its Enhanced Biological Denitrification: A Review of Recent Advances

by
Jing Li
1,
Jie Li
1,*,
Hao Mu
2,
Huina Xie
1 and
Wei Zhao
1,*
1
College of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
2
College of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710000, China
*
Authors to whom correspondence should be addressed.
Water 2025, 17(10), 1433; https://doi.org/10.3390/w17101433
Submission received: 22 April 2025 / Revised: 1 May 2025 / Accepted: 8 May 2025 / Published: 9 May 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

:
Aerobic denitrifying microorganisms, with their strong environmental adaptability, low dissolved oxygen concentration requirements, rapid growth rate, and high nitrogen removal efficiency, significantly compensate for the shortcomings of traditional aerobic chemolithoautotrophic nitrification and anaerobic heterotrophic denitrification models. The introduction of aerobic denitrifiers can effectively enhance the removal of nitrate nitrogen. However, directly inoculating aerobic denitrifiers into wastewater leads to issues such as easy loss of bacterial cells and difficulty in forming a dominant flora, thus preventing the long-term maintenance of their enhancing effect on denitrification performance. To address this problem, microbial immobilization technology has been introduced into the remediation process of nitrogen-polluted water bodies. This technology can maintain a high biomass concentration, provide a stable breeding ground for microorganisms, and effectively prevent the rapid loss of microorganisms. This article systematically reviews the current status of the isolation of aerobic denitrifying bacteria, key enzymes, and genes, as well as the application progress of aerobic denitrifying bacteria and their immobilization technology, aiming to provide solid theoretical support for the practical application of aerobic denitrification technology and promote its further development in the field of nitrogen pollution control.

1. Introduction

Excessive nitrogen emissions can lead to water eutrophication, which deteriorates aquatic ecosystems and even endangers human health [1]. Denitrification technologies are mainly divided into two categories: physicochemical methods and biological methods. Physicochemical methods include air stripping, magnesium ammonium phosphate (MAP) processes, ion exchange, etc., and are commonly used for the pretreatment of high-ammonia nitrogen wastewater. Except for some special wastewater, the traditional nitrification–denitrification biological denitrification method is often used for wastewater treatment in practice. Traditionally, it is believed that the nitrification process is completed by autotrophic nitrifying bacteria under aerobic conditions, while the denitrification process is carried out by heterotrophic denitrifying microorganisms under anoxic conditions [2]. Due to the differences in the requirements of these two types of microorganisms for oxygen concentration, carbon source concentration, and the time required for the reaction, it is necessary to design reactors separately, thus increasing the complexity of operation and space utilization, leading to an increase in treatment costs and complicating the denitrification process [3].
Aerobic denitrification refers to the process where aerobic denitrifying bacteria use both oxygen and nitrate as electron acceptors under aerobic conditions to carry out denitrification, reducing nitrate to gaseous nitrogen [4]. In 1984, the Dutch scientist Robertson discovered that the strain Thiosphaera pantotropha (later renamed Paracoccus pantotrophus) could perform denitrification under aerobic conditions, and he proposed the concept of aerobic denitrification [5]. With the isolation of a large number of aerobic denitrifying bacteria, it was found that these heterotrophic microorganisms usually possess heterotrophic nitrification performance [6,7], allowing the nitrification and denitrification processes to occur in the same reactor. In recent years, some studies have explored the bioaugmentation effects of aerobic denitrifying bacteria in reactors. However, due to their relatively poor environmental competitiveness compared to indigenous dominant flora, they are easily lost when directly added to wastewater, making it difficult to form a dominant flora and failing to effectively enhance denitrification in the long term.
Microbial immobilization is a technology that uses physical or chemical methods to retain microorganisms within a specific area. This technology originated in 1959 when Hattori and others used resins to adsorb and immobilize E. coli [8]. Compared to free microorganisms, immobilized microorganisms have many advantages: (1) They are less likely to be lost after immobilization [9]. (2) They have increased tolerance to harsh environments [10]. (3) They have an accelerated growth rate [11]. (4) They are easier to recover and reuse [12]. This technology can effectively address various issues in biological denitrification and is currently widely used in the environmental pollution control industry [13,14,15,16]. By immobilizing the selected aerobic denitrifying bacteria, the settling performance of the immobilization carrier is improved, preventing the loss of microorganisms [17]. Therefore, combining aerobic denitrification bioaugmentation technology with immobilization technology is a feasible solution to improve wastewater treatment efficiency and maintain the long-term stable operation of reactors.
Currently, most research focuses separately on the nitrogen removal performance and mechanism of aerobic denitrifiers, as well as the principles and applications of microbial immobilization technology. However, comprehensive reviews on the immobilization application of aerobic denitrifiers are rarely seen. This article summarizes the application of aerobic denitrifying microbial immobilization technology in biological nitrogen removal in recent years. It first introduces the current research status of aerobic denitrifying microorganisms, as well as traditional and novel microbial immobilization methods and their advantages and disadvantages. Secondly, it discusses the mechanism and examples of the gel entrapment method for enhancing biological nitrogen removal. Finally, it prospects the future development direction of aerobic denitrifying microbial immobilization for enhancing biological nitrogen removal.

2. Current Research Status of Aerobic Denitrifying Bacteria

2.1. Isolation and Screening of Aerobic Denitrifying Bacteria

Table 1 lists aerobic denitrifying bacteria that have been isolated and screened from environmental samples such as sewage treatment systems, soil, and activated sludge in recent years. It has been found that these bacteria are mainly distributed among the Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes phyla.
At the same time, researchers have also discovered some aerobic denitrifying bacteria with multiple special functions, such as the cold-tolerant nitrite-type aerobic denitrifying bacterium Arthrobacter nicotianae D51 [55]; the salt-tolerant aerobic denitrifying bacterium Vibrio spp. AD2 [56]; the iron-oxidizing aerobic denitrifying bacteria Arthrobacter nicotianae strain 02181 [56] and Delftia lacustris [57]; and Pseudomonas putida NP5 [35], which has the ability to simultaneously perform nitrification, denitrification, and phosphorus accumulation [58]. These aerobic denitrifying bacteria with special functions are extremely important for future applications in practical engineering.

2.2. Key Enzymes and Genes of Aerobic Denitrifying Bacteria

Denitrification refers to the process by which microorganisms reduce nitrate (NO3−-N) to nitrogen gas (N2) through a series of intermediate products, including nitrite (NO2−-N), nitric oxide (NO), and nitrous oxide (N2O). Traditional denitrification theory holds that the presence of oxygen inhibits the synthesis or activity of denitrification enzymes. During denitrification, oxygen competes with nitrate for electrons; thus, denitrification can only occur in anoxic environments. However, recent studies have found that some denitrification enzymes can be synthesized and function under aerobic conditions, and the activity of certain bacterial denitrification enzymes is not affected by dissolved oxygen, allowing them to use both oxygen and nitrate as electron acceptors. The key enzymes involved in aerobic denitrification are mainly nitrate reductase (NAR) [59], nitrite reductase (NIR) [60], nitric oxide reductase (NOR) [61], and nitrous oxide reductase (NOS) [62]. A schematic diagram of the mechanism of action of enzymes involved in the aerobic denitrification process is shown in Figure 1.
(1) Nitrate reductase (NAR): Nitrate reductase catalyzes the first step of the denitrification process, which is the reduction of NO3−-N to NO2−-N. Generally, there are two different types of nitrate reductases, namely membrane-bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap) [59], which are dominant under anaerobic and aerobic conditions, respectively. The occurrence of aerobic denitrification is related to the expression of the Nap gene [63].
(2) Nitrite reductase (NIR): Nitrite reductase is a key enzyme in the denitrification pathway, reducing NO2−-N to NO. Nitrite reductase is located in the outer membrane of the cell membrane [64]. It includes two types: the cytochrome cd1-type nitrite reductase encoded by the nirs gene, and the Cu-type nitrite reductase encoded by the nirk gene [65]. It is generally believed that these two genes do not appear simultaneously in the same strain [66].
(3) Nitric oxide reductase (NOR): Nitric oxide reductase is responsible for reducing NO to N2O. This enzyme is located on the outer membrane of the cell membrane. It was isolated from the strain P. pantotropha ATCC 35512 [67] by Fujiwara and Fukumori [68] in 1996. Due to its strong NOR activity and high affinity for NO, it can quickly reduce NO to N2O.
(4) Nitrous oxide reductase (NOS): Nitrous oxide reductase reduces N2O to N2 during the denitrification process [69]. This enzyme is located in the periplasm of the cell membrane. The NOS extracted from aerobic and anaerobic denitrifying microorganisms is similar and is commonly used as a functional marker for the identification of denitrifying bacteria [70].
For aerobic denitrifying bacteria with heterotrophic nitrification performance, Chen and Ni [71] believe that due to the different activities of hydroxylamine oxidoreductase (HAO) [72], periplasmic nitrate reductase (NAP) [73], and NIR in different bacteria, under aerobic conditions, there may be two possible pathways for the degradation of NH4+-N through nitrification and denitrification: One pathway involves the conversion through nitrate and nitrite, where ammonia monooxygenase (AMO) [74] oxidizes NH4+-N to hydroxylamine (NH2OH), and HAO further oxidizes NH2OH to NO2−-N and NO3−-N, which are then converted to N2 through denitrification (NH4+-NH2OH-NO2−-NO3−-NO2−-N2O-N2). The other pathway involves NH2OH directly, as some bacteria have lower NAP and NIR activities and cannot use nitrate or nitrite as electron acceptors to complete denitrification. Therefore, these bacteria perform denitrification through NH2OH, where NH2OH is oxidized to N2O or N2 by HAO (NH4+-NH2OH-N2O-N2). In the former pathway, due to the different expressions of denitrification enzymes in different bacteria, varying degrees of nitrite accumulation may occur. In the latter pathway, since the process of ammonia removal does not involve the conversion to nitrite or nitrate, it improves the efficiency of nitrogen removal. Chen’s [60] experimental results showed that using sodium succinate and sodium acetate as carbon sources significantly enhanced the growth of Agrobacterium sp. LAD9 strain and its nitrogen metabolism. Wan [75] compared various carbon sources and found that sodium acetate was the most suitable carbon source for Enterobacter cloacae HW-15 strain, supporting its optimal growth conditions and denitrification efficiency. In addition, Ge [27] found that Diaphorobacter sp. PD-7 could utilize phenol as the sole carbon and energy source. Under the condition of an initial phenol concentration of 1400 mg/L, Diaphorobacter sp. PD-7 almost completely removed 120.69 mg/L of ammonia nitrogen within 75 h, and the removal rate of nitrate nitrogen reached 91% within 65 h.

2.3. Current Status of Application of Aerobic Denitrification Technology

The application of aerobic denitrification technology in wastewater treatment mainly involves the addition of single strains or mixed bacterial communities to enhance the denitrification efficiency of bioreactors. Compared with control reactors without the addition of aerobic denitrifying bacteria, bioaugmentation with aerobic denitrifying bacteria can significantly improve the removal efficiency of pollutants or improve the structure of the original microbial community, thereby promoting its aerobic denitrification capacity.
Deng [76] isolated Pseudomonas sp. DM02 from an aquaculture system to enhance the treatment of aquaculture wastewater. When the carbon-to-nitrogen ratio was about 10, the total nitrogen removal rate was greater than 88.2%, and there was no accumulation of ammonia nitrogen and nitrite nitrogen during the reaction process. Chen [77] mixedly inoculated three heterotrophic nitrifying aerobic denitrifying bacteria, Agrobacterium tumefaciens LAD9, Comamonas testosteroni GAD3, and Achromobacter xylosoxidans GAD4, in equal proportions into an Sequencing Batch Reactor (SBR) for the treatment of municipal wastewater. By adding the carbon source sodium succinate to control the carbon-to-nitrogen ratio to about 8, the total nitrogen removal rate increased by 13.7% compared with the non-bioaugmented group. Although the mixed bacterial community did not become the dominant flora in the reactor, the denitrifying functional bacteria in the bioaugmented SBR reactor increased significantly. Lang [41] isolated the strain YS2, which achieved removal rates of 99.46% for NH4+-N and 80.36% for TN within 5 days. Ma [78] isolated two actinomycetes strains, M5 and M6, which achieved total nitrogen removal rates of 95.02% and 96.84%, respectively, under aerobic conditions. Zhang [79] isolated the strain Y39-6, which had an aerobic denitrification rate of 1.77 ± 0.31 mg/L·h and a nitrate removal rate of 0.324 mg/L·h under aerobic conditions.
However, the direct inoculation of aerobic denitrifying bacteria into wastewater faces challenges such as bacterial washout and difficulty in establishing a dominant population, which prevents the long-term enhancement of denitrification performance. To address this issue, microbial immobilization technology has been introduced into the remediation process of nitrogen-polluted water bodies.

3. Current Status of Immobilized Aerobic Denitrification Technology

3.1. Traditional Immobilized Microbial Technology

Immobilized microbial technology is a technique that fixes microorganisms on specific carriers, allowing them to maintain stable growth and metabolic activity in a specific environment. Currently, common microbial immobilization techniques include crosslinking, adsorption, encapsulation, and covalent binding. A diagram of common microbial immobilization techniques is shown in Figure 2.

3.1.1. Chemical Methods

Chemical methods can be divided into covalent binding and crosslinking, depending on the presence or absence of an immobilization carrier. Covalent binding involves activating the surface groups of the carrier or covalently linking bifunctional reagents to the carrier surface, followed by direct covalent coupling with active groups on the microbial surface or through bifunctional reagents [80]. Crosslinking, on the other hand, involves the formation of covalent bonds between microorganisms themselves using bifunctional reagents without a carrier; the microbial cells become interwoven, forming a network-like structure. Common bifunctional reagents include glutaraldehyde, toluene diisocyanate, etc. [81].
After chemical immobilization, microorganisms are connected to the carrier by chemical covalent bonds, resulting in strong binding and generally preventing detachment from the carrier [12]. However, chemical methods involve chemical modification of the microbial cell surface, which can be toxic to microorganisms, leading to reduced activity or even death. And the purchase cost of the crosslinking agent is relatively high. There are relatively few reports on the application of chemical methods for immobilizing denitrifying microorganisms in the field of biological denitrification, and they are generally used in combination with adsorption and crosslinking methods. Gandhi [82] used the adsorption–crosslinking method to successfully immobilize lipase by directly adding chitosan to the anion-exchange macroporous resin, achieving the highest specific activity of 24.69 U/g resin. Using this immobilized enzyme, the yield of biodiesel can reach 50.79%.

3.1.2. Adsorption Method

Unlike the strong covalent bonds, the binding forces of the adsorption method are mainly weak interactions, such as van der Waals forces, hydrogen bonds, and ionic bonds [80]. Depending on the different binding forces, adsorption can be divided into physical adsorption and ionic adsorption [83]. Common physical adsorption materials include activated carbon, polyurethane foam, montmorillonite, molecular sieves, diatomaceous earth, activated carbon, glass, ceramics, etc., while common ionic adsorption materials include ion-exchange resins and ion-exchange fibers.
The adsorption method has advantages such as low cost, convenient operation, and minimal impact on microbial activity, and it is widely used in the field of biological denitrification. An [84] used peanut-shell biochar to immobilize Pseudomonas L1 to remove Ni(II), Cr(VI), Cu(II), and nitrate from electroplating wastewater. The immobilized Pseudomonas L1 showed better pollutant removal capacity and environmental adaptability compared to the free state. However, due to the relatively weak binding force of the adsorption method, microorganisms immobilized by adsorption alone are prone to detachment from the carrier. Therefore, the adsorption method is often used in combination with chemical and encapsulation methods. Adding adsorption materials to chemical reagents or materials can better improve the immobilization effect of microorganisms. Zvulunov [85] used a composite material composed of montmorillonite and polyethyleneimine to immobilize odor-causing Pseudomonas that can degrade formaldehyde. After continuous operation for multiple cycles, it showed a stable degradation rate of formaldehyde.

3.1.3. Encapsulation Method

The encapsulation method mainly uses natural and synthetic polymer materials, while nanomaterials, magnetic materials, and inorganic porous materials are often used as additives to enhance material performance. Inorganic porous materials can significantly improve the mass transfer performance of the carrier; nanomaterials can increase the specific surface area and loading rate of the carrier, and create microenvironments suitable for the growth of different microbial groups; and magnetic materials can promote microbial growth and metabolism, and the carrier is easy to separate [86,87,88]. Commonly used embedding materials include polyvinyl alcohol (PVA), sodium alginate (SA), carrageenan, agar, and polyacrylamide (PAM), among others [89].
Depending on the different formation mechanisms, the encapsulation method can be divided into the microcapsule method and gel entrapment method [90]. The microcapsule method uses natural or artificially synthesized organic polymer materials as the capsule wall and encapsulates microorganisms in a semipermeable membrane through physical and chemical means. Most microcapsules have diameters between 1 μm and 1000 μm [91]. This technology can significantly enhance the resistance of microorganisms to harsh environments. Mollaei [92] immobilized Pseudomonas in alginate–chitosan–alginate microcapsules, which significantly improved the strain’s phenol degradation efficiency. However, the application of the microcapsule method in the field of biological denitrification is currently limited.
The gel entrapment method is a technique that intercepts and fixes microorganisms in water-insoluble gels with a three-dimensional network structure. Most gel beads have diameters between 3 and 5 mm [93]. The complex porous network structure inside the gel beads can ensure good mass transfer efficiency without leakage of microorganisms, which is conducive to maintaining good microbial activity. It is currently the most mature and widely used method for microbial immobilization. The most commonly used material for the gel method is a PVA (polyvinyl alcohol)–SA (sodium alginate) composite material [86,94,95]. Polyvinyl alcohol has high mechanical strength but low mass transfer efficiency, while sodium alginate has low mechanical strength but high mass transfer efficiency. The combination of these two materials can compensate for the above problems. Some application cases of microbial immobilization in denitrification are shown in Table 2.

3.1.4. Composite Immobilization Method

The composite immobilization method is a technique that combines two or more immobilization methods to overcome the limitations of a single method, thereby achieving higher performance or treatment efficiency than any single method and addressing the various shortcomings and deficiencies of individual immobilization methods. Common approaches in composite immobilization technology include adsorption–embedding, embedding–crosslinking, aggregation–crosslinking, and adsorption–embedding–crosslinking methods [104]. These methods integrate the advantages of different immobilization technologies to enhance the efficiency and stability of microbial immobilization.

3.2. Novel Microbial Immobilization Methods

3.2.1. Layer-by-Layer Self-Assembly Method

Layer-by-layer self-assembly is a technique that alternately deposits layers using electrostatic attraction, hydrogen bonds, covalent bonds, and other forces as driving forces to alternately attach materials to a specific template, forming thin films layer by layer [105]. Templates include but are not limited to cells, enzymes, high-molecular-weight organics, polyelectrolytes, etc., and are currently widely used in chemical, food, and other industries [106,107]. Between the layers, a molecular aggregate with an intact structure, a stable performance, and specific functions is formed [108].
The main methods to achieve layer-by-layer self-assembly are dipping, spin-coating, and spraying [109]. This technology can precisely control the structure and chemical properties of material films, and at the same time, the specific functions of the materials will be conferred to the template through this technology [110]. Its limitations lie in the long production cycle, and it mainly relies on weak interactions such as electrostatic forces and hydrogen bonds, resulting in poor stability, and the film structure is easily damaged by impact [111].
Currently, the layer-by-layer self-assembly technology has a wide range of applications in many fields, but it is mainly used for the immobilization of enzymes or single cells. For example, Wang [112] used lysozyme and SA as raw materials and employed this technology to alternately deposit cellulose acetate membranes onto the surface of nanofibers to immobilize lysozyme. The immobilized enzyme has good tolerance to harsh environments.
Because microorganisms are very sensitive to polyelectrolytes and nanoparticles, the assembly process may have adverse effects on microbial activity. At the same time, since the film formed by this technology is usually at the nanoscale level, which is much smaller than the cell diameter, the film cannot completely encapsulate the cells. Gao [113] fabricated a novel modified graphene oxide/polyvinylidene fluoride (PVDF) layered composite membrane using the layer-by-layer self-assembly technique for the treatment of aromatic compounds in petrochemical wastewater. The results demonstrated that the removal efficiency of the target pollutants reached its peak when the number of graphene oxide layers increased to 10, while the chemical oxygen demand (COD) removal rate remained consistently above 95%. Therefore, the difficulty of using this technology to immobilize activated sludge flocs containing a large number of denitrifying microorganisms will be greatly increased. Although this technology is in its infancy in the field of biological denitrification, it can confer specific material functions to microorganisms and provide corresponding “protective shells” for microorganisms against different external environmental factors, and thus it has good application prospects.

3.2.2. Biomimetic Mineralization Technology

Biomimetic mineralization technology involves introducing mineralization-related molecules onto cells that lack biomineralization ability using chemical or biological methods, thereby forming a protective shell for microorganisms [114]. This technology can enhance the microorganisms’ resistance to shock, ultraviolet radiation, and high temperatures, and can also delay the decline of microbial activity [115]. It has been widely studied in the fermentation [116] and microbiology [117] industries, but its application in environmental pollution control still needs further exploration. Lei [118] designed an innovative nanoscale biomimetic bilayer hydrogel embedded with tendon stem cells. This design significantly promotes the formation of fibrocartilage, improves motor function, and enhances biomechanical performance. Guo [114] developed a rapid biomimetic mineralization method for immobilizing laccase derived from Bacillus subtilis. The catalytic efficiency of the immobilized enzyme reached 29.844 s−1·μM−1, which is 3.5 times higher than that of the free enzyme. Han [119] employed a layer-by-layer self-assembly technique to alternately deposit polycations and polyanions onto the surface of yeast cells. They then combined this with biomimetic silicification to encapsulate individual yeast cells in silica, thereby enhancing both cell viability and stability.
The biomimetic mineralization process is simple and mild, allowing proteins to adsorb onto the interior or surface of the mineralized material’s pores through electrostatic interactions or metal chelation, preserving their structure and biological activity in the process [120]. Therefore, biomimetic mineralization technology is suitable for constructing protein immobilization carriers. Compared to traditional immobilization materials, biomimetic mineralization materials exhibit unique advantages, such as structural controllability, high stability, and abundant porosity. Hwang [121] prepared amorphous calcium phosphate–enzyme nanocomposites (ACP-NCs) using biomineralization technology and a co-precipitation method, with an enzyme loading rate as high as 70%.
However, the current application of biomimetic mineralization technology is mainly focused on the immobilization of enzymes, while its application in the immobilization of microorganisms is relatively rare.

3.2.3. Electrospinning Method

Electrospinning is a technique that processes polymer solutions or melts into nanofibers under a high-voltage electric field. The principle involves a polymer solution being pressurized through an injection pump, forming a droplet at the nozzle [122]. The high-voltage electrostatic field between the nozzle and the collector transforms the droplet into a Taylor cone, and when the voltage is sufficiently high, the polymer solution forms a jet. After a period of motion, it lands on the collector as nanofibers [123,124].
Due to the advantages of large specific surface area, high porosity, controllable fiber structure, and low cost, electrospinning has been widely applied in various industries, such as biomedicine, food, and environmental engineering [125,126,127]. This technology can immobilize microorganisms within nanofibers, accelerating their growth and reproduction rates and enhancing their tolerance to harsh environments [128]. Jayani [129] immobilized the probiotic Lactobacillus acidophilus 016 on nanofibers through electrospinning. Mechanical properties and thermal analysis indicated that these nanofibers could be used for probiotic delivery in food, and scanning electron microscopy showed that the probiotics immobilized on the nanofibers remained undamaged for 24 days. Sarioglu [130] used nanofibers made of polycaprolactone and polylactic acid to immobilize microorganisms capable of degrading textile dyes. The results showed that the immobilized microorganisms had higher pollutant degradation capabilities and greater environmental tolerance. Fan [131] immobilized yeast on polyacrylamide nanofibers, creating a fibrous material containing live yeast that can be used in biofermentation, wastewater treatment, genetic engineering, and other fields. Although electrospinning technology has wide applications in many industries, there are few reports on its use in biological denitrification. With the development of this technology, there is great potential for using it to immobilize microorganisms in the field of biological denitrification. Li [132] fabricated nanofibrous materials from a mixture of polycaprolactone and silica through electrospinning, which not only improved the stability and hydrophilicity of the carrier but also significantly enhanced the ammonia oxidation rate to 7.36 mg/L/h, with an average total nitrogen removal rate of 5.59 mg/L/h. Additionally, compared to free bacteria, the bacteria immobilized on the modified nanofibers exhibited a higher ammonia oxidation rate. Zhao [133] employed electrospinning technology to fabricate micro-nanofibers for the immobilization of aerobic denitrifying bacteria, significantly enhancing the total nitrogen removal rate and successfully immobilizing the dominant bacterial species.
Although novel microbial immobilization methods have many advantages, they still require a long development time in the field of biological denitrification. The principles (shown in Figure 3), advantages, and disadvantages of several microbial immobilization methods are shown in Table 3. Currently, the gel entrapment method remains the most widely used and mature immobilization method in the field of biological denitrification.

3.3. Mechanism of Enhanced Biological Denitrification by Gel Entrapment Method

3.3.1. Providing Protection for Microorganisms

Gel beads can provide corresponding protection for microorganisms, offering them time to adapt to the environment, while reducing the negative effects of adverse external environmental factors on microorganisms. This allows denitrifying microorganisms to grow and reproduce better, thereby improving denitrification efficiency. For example, Yu [134] used gel beads made of SA-kaolin to immobilize Pseudomonas LZ-4. In the free-strain system, the presence of Cr(VI) reduced the nitrate removal rate by 86.07%, while the immobilized strain protected the denitrification process in the system and removed 95% of the nitrate. At the same time, gel beads can also mitigate the impact of low temperatures on microbial activity and reduce the toxic effects of high salinity on microorganisms [79,135].

3.3.2. Accelerating Microbial Enrichment

The gel entrapment method can effectively enhance the growth and enrichment rate of microorganisms. This is because gel beads are easy to settle, and the immobilized microorganisms are less likely to be lost. Meanwhile, the microporous structure of the gel beads can provide favorable conditions for their growth and reproduction [136]. Extracellular polymeric substances (EPSs) released by microorganisms mainly consist of proteins and polysaccharides, and the hydrophobic interaction of proteins can cause microorganisms to form aggregates [137]. Microorganisms immobilized in gel beads are more likely to release EPSs, making them more firmly and densely combined with the network structure inside the gel beads [138]. Additionally, immobilizing microorganisms in gel beads does not alter the existing microbial community but has a positive impact on the enrichment of dominant microbial populations [139].

3.3.3. Differences in Dissolved Oxygen Concentration Inside and Outside the Gel Beads

Due to the tight structure of the gel beads and the resistance of oxygen mass transfer, the external part of the gel beads exhibits aerobic conditions, while the internal part presents anoxic conditions. The aerobic environment outside and the anoxic environment inside the gel beads can provide suitable growth conditions for traditional nitrifying/denitrifying bacteria, which is beneficial for the occurrence of simultaneous nitrification and denitrification (SND).
Aerobic denitrifiers are a type of heterotrophic bacteria that perform denitrification under aerobic conditions. Most of these bacteria also possess the function of heterotrophic nitrification, allowing aerobic denitrifiers to achieve SND from a microbial perspective [140]. Nitrite reductase (NiRs) [141] in aerobic denitrifiers can reduce NO2−-N to NO. However, NiRs are inhibited under aerobic conditions, leading to the accumulation of NO2−-N. This process is the rate-limiting step in aerobic denitrification. Through microbial immobilization technology, the accumulation of NO2−-N can be effectively reduced, and the removal efficiency of total nitrogen (TN) can be improved. Ma [142] used SA gel beads to immobilize Pseudomonas T13. The anoxic conditions inside the gel beads resulted in higher NiRs activity, with the immobilized strain achieving a maximum TN removal rate of 57.25%, while the free strain only achieved a TN removal rate of 29.7%.

3.3.4. Providing Additional Functional Microorganisms and Nutrients

The gel entrapment method can be used to immobilize functional microorganisms that are lacking in biological denitrification systems and then add them to the original system to enhance denitrification. Constructed wetlands are a new wastewater treatment technology that developed in the late 1970s. They are widely used to treat various types of wastewaters due to their multiple advantages, such as low cost, high efficiency, energy saving, and good landscape effects [143,144]. Nitrogen in constructed wetlands is removed through plant root absorption, filler matrix adsorption, ammonia volatilization, and microbial action. Zhang [145] found that microbial-driven nitrification/denitrification reactions are the main pathways for nitrogen removal in constructed wetlands, accounting for 66.9–80.5% of the total nitrogen removal. However, factors such as hydraulic flushing and predation by protozoa can lead to insufficient carbon sources and low biomass concentrations in constructed wetlands, thus greatly limiting their denitrification efficiency.
To address these two major issues, additional carbon sources and nitrifying/denitrifying bacteria can be added to the constructed wetlands. However, due to interference from external factors, the growth rate and denitrification efficiency of directly added free bacteria are low, and the bacteria are easily lost. Adding immobilized microorganisms can greatly improve the denitrification efficiency of constructed wetlands. Wang [146] immobilized nitrifying bacteria and added small-molecule organic carbon sources to enhance denitrification in constructed wetlands. However, the amount of small-molecule organic carbon sources added directly to constructed wetlands is uncertain. Insufficient addition can lead to incomplete denitrification, while excessive addition can cause secondary pollution. Yu [147] used rice hulls as a solid slow-release carbon source after treatment and co-immobilized rice hulls and denitrifying bacteria with PVA and SA to form gel beads, which were then added to a horizontal subsurface flow constructed wetland. The average TN removal rate of the constructed wetland enhanced with gel beads reached 78.4%, while the control group only had a TN removal rate of 23.69%. The sustained release of carbon sources and the increase in denitrifying bacteria abundance were the main reasons for the increase in TN removal rate. At the same time, it is also possible to immobilize some special microorganisms that are salt-tolerant and cold-tolerant to improve the denitrification efficiency of constructed wetlands under high-salinity and low-temperature conditions [147,148,149].

4. Current Status of Immobilized Aerobic Denitrifiers

Combining aerobic denitrification bioaugmentation technology with immobilization technology by immobilizing aerobic denitrifiers in carriers, on the one hand, offers a good settling performance that makes it difficult for microorganisms to be washed out of the reactor, thus increasing the retention of microorganisms in the reactor [150]; on the other hand, immobilization in carriers provides a relatively ideal living environment for microorganisms, enhancing their resistance to shock. Even under adverse environmental conditions, they can maintain high microbial activity, which is beneficial for improving the growth rate and denitrification efficiency of aerobic denitrifiers [151,152].
Tang [152] used graphene oxide-modified polyvinyl alcohol and sodium alginate gel beads as the carrier for Pseudomonas fluorescens Z03 to improve denitrification efficiency at low temperatures (6–8 °C). The results showed that the addition of graphene oxide can improve denitrification efficiency. When the dosage was 0.15 g/L, the highest denitrification efficiency was achieved, with NH4+-N and NO3−-N removal rates of 96.38~97.24% and 98.82~99.12%, respectively, and the accumulation of NO2−-N was below 0.1 mg/L. Ma [142] immobilized Pseudomonas stutzeri T13 using fungal beads and inoculated them into an SBR reactor. The results showed that the average TN removal rate of the SBR reactor could reach 77.0%; Pseudomonas stutzeri T13 formed a dominant population in the SBR reactor, providing long-term and stable bioaugmentation. Yu [153] used Pseudomonas H1 to treat domestic sewage, achieving TN and NH4+-N removal rates of 66.04% and 62.11%, respectively. To achieve solid–liquid separation and recycling of the strain, polyvinyl alcohol, sodium alginate, nano-Fe3O4, and bacterial cellulose were used to immobilize Pseudomonas H1 for domestic sewage treatment. Compared with the free bacteria, the TN removal rate of the effluent was increased from 66.04% to 77.22%. Zhao [154] used sodium alginate as the embedding agent and Ba2+ as the crosslinking agent to encapsulate P. denitrificans DYTN-1 for industrial wastewater treatment. After five cycles of use, the amount of DYTN-1 in the immobilized gel increased, and the total nitrogen removal rate was significantly accelerated, reaching 84.1% in just 1 h, which shortened the denitrification reaction time by 11 h compared to the bacteria suspension. Lin [155] utilized sodium alginate and polyvinyl alcohol to encapsulate tea-dreg powder and Marinobacter alkaliphilus strain JY28 for treating nitrogen in wastewater from a marine recirculating aquaculture system (MRAS). The highest removal rate of NO3−-N reached 99.4 ± 0.6% at a hydraulic retention time (HRT) of 10 h, while the cumulative concentration of NO2−-N remained at the lowest level (0.04 ± 0.05 mg/L). Han [156] employed PVA-SA embedding technology to immobilize the bioagent AHM M3, resulting in the complete removal of nitrate within 48 h with virtually no accumulation of NO2−-N. The MBBR system incorporating PVA/SA&AHM-M3 achieved complete nitrate removal and an 88.0% total nitrogen removal rate. The successful enrichment of Zobellella sp. MAD-44 and Halomonas alkaliphila HRL-9 within the MBBR system further demonstrated the effectiveness of PVA/SA&AHM-M3. Tian [157] immobilized the aerobic denitrifying bacterial strain PCN-1 on polyurethane biocarriers and applied it to an oxidation ditch. The results showed that N2O emissions were reduced by 49.13% and 59.70% when subjected to ammonia load shock and aeration failure shock, respectively.

5. Future Research

The future research on immobilized aerobic denitrifying bacteria can be developed from the following aspects:
(1) Research on new immobilization materials and technologies to deeply explore more materials and technologies suitable for immobilizing aerobic denitrifying bacteria, so as to improve the efficiency and service life of immobilization.
(2) Research on the long-term stability of the immobilization system to evaluate the long-term stability and effectiveness of immobilized aerobic denitrifying bacteria in practical applications, ensuring their continuous effectiveness in environmental governance.
(3) Research on large-scale application to explore the large-scale production and application technology of immobilized aerobic denitrifying bacteria, reduce costs, and improve their economic feasibility in environmental governance.
(4) Research on the structure of microbial communities to study the changes in the structure of microbial communities during the immobilization process, revealing their role in the denitrification process.
These research directions will help promote the further development and application of immobilized aerobic denitrifying bacteria technology in the field of environmental governance. Besides the aforementioned research directions, there is relatively little research on how to effectively recover immobilized carriers from wastewater treatment systems; how to clean and regenerate immobilized carriers to restore their activity and reuse them; how to safely dispose of immobilized carriers if they cannot be reused; and how to treat the microorganisms on the immobilized carriers to prevent secondary pollution. These issues also need further attention and optimization.

6. Conclusions

Aerobic denitrification technology demonstrates great potential in wastewater treatment and environmental protection. Currently, scholars mainly focus on the isolation, identification, influencing factors, enzyme activity of aerobic denitrifiers, and development of immobilized aerobic denitrification technology. Immobilizing aerobic denitrifiers using microbial immobilization technology not only reduces the negative impact of external adverse environmental factors on the bacteria but also promotes their growth rate and increases cell density. Although the combination of microbial immobilization technology and aerobic denitrification for nitrogen removal is relatively mature, further research is still needed in the following key areas:
(1) Development of new immobilization materials and processes:
Screening and synthesizing immobilization carrier materials with higher diffusion efficiency, stronger stability, and lower environmental impact.
Optimizing the immobilization process to improve efficiency and reduce costs, aiming for industrial applications.
(2) Analysis of microbial community structure and function:
Utilizing high-throughput sequencing and metagenomics to thoroughly analyze the dynamic succession patterns of microbial communities within the gel beads.
Clarifying the interaction mechanisms between denitrifying functional bacteria and other microorganisms to construct an efficient microbial community structure.
(3) Elucidation of the metabolic mechanisms of functional microorganisms:
Employing metabolomics and proteomics to deeply study the metabolic pathways and regulatory mechanisms of functional microorganisms within the gel beads.
Exploring metabolic engineering strategies to enhance the growth and enrichment of functional microorganisms, thereby improving denitrification efficiency.
(4) Optimization of practical engineering application parameters:
Conducting pilot and large-scale experiments to optimize operational parameters of the immobilized aerobic denitrification technology, such as hydraulic retention time, dissolved oxygen concentration, and organic loading.
Establishing a long-term operation monitoring system to evaluate the stability and shock load resistance of the immobilized system, ensuring long-term stable operation of the technology.
Through in-depth exploration of these research directions, the aerobic denitrification technology will be further promoted from laboratory research to practical engineering applications, providing more efficient and sustainable solutions for addressing water body nitrogen pollution.

Author Contributions

Conceptualization, J.L. (Jie Li) and J.L. (Jing Li); methodology, J.L. (Jing Li) and H.M.; software, J.L. (Jing Li) and H.M.; validation, J.L. (Jing Li), H.X. and W.Z.; formal analysis, J.L. (Jing Li) and H.M.; investigation, J.L. (Jing Li); resources, J.L. (Jing Li); data curation, J.L. (Jing Li) and H.M.; writing—original draft preparation, J.L. (Jing Li); writing—review and editing, J.L. (Jing Li); visualization, J.L. (Jing Li) and H.M.; supervision, J.L. (Jie Li) and W.Z.; project administration, J.L. (Jie Li) and J.L. (Jing Li); funding acquisition, J.L. (Jie Li) and J.L. (Jing Li). All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Gansu Provincial Department of Science and Technology Natural Science Foundation (No. 24JRRA234).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic diagram of the mechanism of action of enzymes involved in the aerobic denitrification process.
Figure 1. Schematic diagram of the mechanism of action of enzymes involved in the aerobic denitrification process.
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Figure 2. Classification and schematic diagram of traditional immobilization methods.
Figure 2. Classification and schematic diagram of traditional immobilization methods.
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Figure 3. Schematic diagram of novel immobilization methods.
Figure 3. Schematic diagram of novel immobilization methods.
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Table 1. Some aerobic denitrification strains isolated in 2011–2025.
Table 1. Some aerobic denitrification strains isolated in 2011–2025.
GenusSpeciesSourceYear
Bacillus sp.YX-6Fishery pond2011 [18]
Bacillus methylotrophicusL7Wastewater sample2012 [19]
Rhodococcus sp.CPZ24Swine wastewater2012 [20]
Halomonas campisalisha3Saline–alkali lake2013 [21]
Paracoccus versutusLYMSeabed sludge2013 [22]
Acinetobacter sp.Y16Drinking water source2013 [23]
Chryseobacterium sp.R31Slaughterhouse wastewater2014 [24]
Aeromonas sp.HN-02CASS reactor2014 [25]
Alcaligenes faecalisC16Aeration tank2015 [26]
Diaphorobacter sp.PD-7Coking-plant wastewater ponds2015 [27]
Pseudomonas aeruginosaPCN-2Landfill leachate treating reactor2015 [28]
Cupriavidus sp.S1Coking wastewater2016 [29]
Pseudomonas tolaasiiY-11Long-term flooded paddy soil2016 [30]
Raoultella sp.R11Eutrophic lake2017 [31]
Acinetobacter sp.H36Sediment2017 [32]
Pseudomonas putidaY-9Long-term flooded paddy soil2017 [33]
Pseudomonas stutzeriXL-2Secondary sedimentation tank2018 [34]
Pseudomonas putidaNP5Activated sludge2019 [35]
Acinetobacter johnsoniiWGX-9Sediment of a drinking-water reservoir2019 [36]
Acinetobacter sp.T-1Membrane bioreactor2019 [37]
Paracoccus sp.YF1Activated sludge2019 [38]
Acinetobacter sp.JR1Pharmaceutical raw water2019 [39]
Pseudomonas sp.JQ-H3Packed-bed reactor2020 [40]
Acinetobacter sp.YS2Aerobic pond2020 [41]
Bacillus thuringiensisWXN-23Piggery bran feed filtrate2021 [42]
Pseudomonas putidaW207-14Landfill leachate2022 [43]
Bacillus sp.T28Paddy soil2023 [44]
Rhodococcus sp.CPZ24Biofilm reactor2023 [45]
Rhodococcus sp.SY24Soil2023 [46]
Paracoccus sp.HY-1Landfill leachate treatment plant2024 [47]
PseudomonasXF-4Activated sludge2024 [48]
ZobellellaB307Jiaozhou Bay sediment2024 [49]
Stutzerimonas sp.os3Shrimp aquaculture sediment2025 [50]
Pseudomonas sp.WS-03Sludge of an actual wastewater treatment plant2025 [51]
Ralstonia sp.J4Piggery wastewater2025 [52]
Paracoccus binzhouensiswg1Propylene oxide saponification-activated sludge2025 [53]
Marinobacterium maritimum5-JSSea cucumber aquaculture pond2025 [54]
Table 2. Some application cases of microbial immobilization in nitrogen removal.
Table 2. Some application cases of microbial immobilization in nitrogen removal.
Material CompositionImmobilization MethodStrain NameTarget PollutantRemoval Efficiency (%)Reference
PVA-SAGel methodMixed Nitrifying BacteriaHigh-concentration ammoniacal nitrogen46[96]
PVA-SAGel methodMixed Nitrifying BacteriaDifferent concentrations of ammoniacal nitrogen48.3–100[97]
Mycelium ballsAdsorption methodPseudomonas GF3 Nitrate nitrogen95.91[98]
Sponge–walnut shell Carbon–MagnetiteAdsorption methodZoogloea L2 Nitrate nitrogen, diethyl phthalate83.97, 67.87 [99]
Mycelium balls–magnetiteAdsorption methodPseudomonas GF2Nitrate nitrogen98.14[88]
PVA-SAGel methodNitrosomonas GH22Ammoniacal nitrogen90.3[100]
Biochar adsorptionAdsorption methodPseudomonas mendocina GL6Nitrate nitrogen95.8[101]
Polyethylene suspended ballsAdsorption methodPseudomonas Y39-6Nitrate nitrogen under low temperature and low carbon–nitrogen ratio24.83[79]
PVA-SAGel methodAnammox granular sludgeNitrogen removal of different low temperatures52–72[102]
PVA-SAGel methodStutzerimonas stutzeri W-2Nitrogen-containing wastewater99.06[103]
Table 3. Principles and advantages and disadvantages of several methods for immobilization of microorganisms.
Table 3. Principles and advantages and disadvantages of several methods for immobilization of microorganisms.
Immobilization MethodPrincipleAdvantagesDisadvantagesCost
Chemical methodsMicroorganisms are connected to each other or to the carrier through chemical bondsStrong binding force, and microorganisms are highly concentrated and not easily detached from the carrierChemical reagents are toxic to microorganisms, leading to reduced microbial activityHigh
Adsorption methodMicroorganisms are connected to the carrier through weak interactions, such as van der Waals forces and ionic bondsSimple operation, non-toxic to microorganisms, and the carrier can be regeneratedThe binding force is weak, and microorganisms are easily detached from the carrierMiddle
Encapsulation methodMicroorganisms are trapped in water-insoluble gel polymersSimple operation, can immobilize specific microorganisms, and has wide applicabilityThe mass transfer resistance is large, and microorganisms are prone to leakage from the gel polymer after long-term operationLow
Layer-by-layer self-assemblySpecific materials are alternately deposited on microorganisms, layer by layer, through electrostatic forcesThe assembly process is controllable, the conditions are mild, and the properties of the materials can be imparted to the microorganismsPoor stability, long production cycle, and less application in biological nitrogen removalHigh
ElectrospinningNanofibers are formed from a mixture of microorganisms and polymer solution under a high-voltage electric fieldSimple operation, and the nanofibers produced have a large specific surface area and high porosityLow strength of nanofibers, low yield, and less application in biological nitrogen removalHigh
Biomimetic mineralizationMineralization-related molecules are introduced onto cells that lack biomineralization ability, forming a protective shell for microorganismsEnvironmentally friendly, has good biocompatibility, and is highly controllableThe synthesis process requires a lot of time, making it difficult to meet the needs of large-scale production, and it is less applied in biological nitrogen removalHigh
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Li, J.; Li, J.; Mu, H.; Xie, H.; Zhao, W. Immobilization Technology of Aerobic Denitrifying Bacteria and Its Enhanced Biological Denitrification: A Review of Recent Advances. Water 2025, 17, 1433. https://doi.org/10.3390/w17101433

AMA Style

Li J, Li J, Mu H, Xie H, Zhao W. Immobilization Technology of Aerobic Denitrifying Bacteria and Its Enhanced Biological Denitrification: A Review of Recent Advances. Water. 2025; 17(10):1433. https://doi.org/10.3390/w17101433

Chicago/Turabian Style

Li, Jing, Jie Li, Hao Mu, Huina Xie, and Wei Zhao. 2025. "Immobilization Technology of Aerobic Denitrifying Bacteria and Its Enhanced Biological Denitrification: A Review of Recent Advances" Water 17, no. 10: 1433. https://doi.org/10.3390/w17101433

APA Style

Li, J., Li, J., Mu, H., Xie, H., & Zhao, W. (2025). Immobilization Technology of Aerobic Denitrifying Bacteria and Its Enhanced Biological Denitrification: A Review of Recent Advances. Water, 17(10), 1433. https://doi.org/10.3390/w17101433

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