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# Nitrogen removal by heterotrophic nitrification-aerobic denitrification bacteria: A review

Shanshan Liª, Zhengming Heª, Cong Liª,*, Eric Lichtfouseᵇ, Chunmeng Sunª, Yunshu Zhangª, Jianping Yuª

## ABSTRACT

The rising urbanization and industrialization are increasing nitrogen pollution in water and wastewaters, calling for advanced remediation treatments. Traditional biological treatments are limited in area and efficiency. Therefore, heterotrophic nitrification-aerobic denitrification (HN-AD) in a single reactor has drawn significant attention and has shown higher efficiency. Here we review HN-AD bacteria with focus on bacterial species, nitrogen metabolism, and factors influencing denitrification. Nitrogen metabolism pathway includes nitrate-nitrite reduction denitrification, hydroxylamine reduction and assimilatory denitrification. Factors influencing denitrification comprise carbon source, C/N, temperature, pH, DO and so on. Compared to published literature reviews on HN-AD, our manuscript comprehensively summary the latest research on its nitrogen remove mechanisms and related enzymes for the first time.

**Keywords:**

HN-AD strains

Simultaneous nitrification and denitrification

Influencing factors

Nitrogen biotransformation pathway

Nitrogen removal enzymes

## 1. Introduction

The removal of nitrogen from wastewater is of paramount importance for preventing eutrophication of water bodies, improving the living-environment quality of aquatic animals and plants, and reducing their threat to human health. Numerous measures including physical, chemical, and biological, have been employed for nitrogen removal from wastewater [1–3]. Compared with many current nitrogen removal techniques, biological nitrogen removal (BNR) offers distinct advantages such as stable operation, simple operation, remarkable efficiency, and relatively cost-effective solutions [4]. Consequently, BNR has found widespread application in practical scenarios [1].

Biological denitrification exhibits a pronounced capacity for the biodegradation which can effectively remove the majority of soluble nitrogenous organic compounds [5]. However, despite of the successes of traditional biological denitrification in nitrogenous wastewater treatment, it does have certain limitations. Conventional biological denitrification processes need separate facilities, resulting in prolonged hydraulic retention times, time-consuming operations, and relatively low efficiency [6]. The discovery of heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria has introduced novel perspectives in nitrogen removal through simultaneous nitrification and denitrification

processes in a single reactor under aerobic conditions [7]. Comparing with traditional autotrophic nitrifying bacteria, these HN-AD microorganisms exhibit significant advantages, including faster growth rates, higher nitrogen removal efficiency, stronger environmental adaptability, and the better ability to utilize organic compounds [8].

A significant number of HN-AD bacteria have been isolated from various environments and applied successfully. Strain *Marinobacter* sp. F6 was isolated from an aerated biofilter system in a seawater aquaculture setup [9]. When grown in high-nitrate and nitrite environments, it achieved complete removal of nitrate and 73.10% removal of nitrite. Furthermore, a novel halophilic spore-forming bacterium, *Litoralis* sp. N31, was isolated from marine aquaculture waters [10]. After 48 h of cultivation in a nitrogen source medium with an initial nitrogen content of about 20 mg/L, N31 achieved removal rates of 86.3% for ammonium nitrogen, 89.3% for nitrite, and 89.4% for nitrate. When exposed to mixed nitrogen sources, ammonium nitrogen was preferentially removed, and there was no significant nitrite accumulation during nitrification and denitrification processes.

This article provides an overview of the nitrogen removal by HN-AD bacteria in water environments. The focus is primarily on the origins of different HN-AD bacterial strains, categorized based on their species relationships. The review summarizes the nitrogen removal pathways

<footnote>ªSchool of Environment and Architecture, University of Shanghai for Science and Technology, Shanghai 200093, China</footnote>

<footnote>ᵇState Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, 28 Xianning West Rd, Xi'an, Shaanxi 710049 PR China</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: congil@aliyun.com (C. Li).</footnote>
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and relevant enzymes of HN-AD, analyzes the favorable environmental factors for HN-AD, and elucidates the challenges and prospects in its application. Gaining a deeper understanding of these aspects will contribute to a better comprehension of their role in nitrogen removal from wastewater, as well as the interactions between bacterial strains, thereby fostering the development of sustainable wastewater nitrogen removal technologies in the future.

## 2. Source

In 1983, the HN-AD bacterium *Paracoccus pantotrophus* was first isolated from desulfurization and denitrification wastewater [11]. Subsequent research revealed that HN-AD bacteria, under aerobic conditions, rapidly convert ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and similar compounds into nitrogen-containing gases. The entire metabolic process has almost no accumulation of nitrite nitrogen [12]. Furthermore, HN-AD bacterial tolerance to oxygen is compatible with the utilization of organic substrates, realizing the vision of concurrent nitrification and denitrification within a unified system [10]. Consequently, bacteria possessing the capabilities of heterotrophic nitrification and aerobic denitrification, such as denitrifying *Paracoccus denitrificans*, *Pseudomonas stutzeri*, Thiosphaera *Pantotropha, Comamonas* sp., and *Alcaligenes faecalis*, have been extensively studied as potential microorganisms that may be used to overcome problems inherent in traditional methods [13].

About 20 genera bacteria isolated from HN-AD strains have been screened, revealing that HN-AD bacteria efficiently remove ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen, thereby enhancing nitrogen removal efficiency. Overall, the distribution of HN-AD bacteria is extensive, with findings across various environmental conditions [12] (As shown in Table 1). Moreover, they exhibit unique metabolic pathways, show a wide range of biological resources and environmental adaptability [14]. They predominantly originate from sources such as soil [15], aquatic environments [10,16], activated sludge [17], and various sediments [18].

The presence of abundant nitrogen sources in these environments is a significant factor contributing to the widespread existence of HN-AD. Despite being isolated from various environments, they all have the ability to remove nitrogen [19]. However, the nitrogen removal efficiency varies among different strains, and even within the same strain, the removal of $NH_4^+$-N, $NO_2^-$-N, and $NO_3^-$-N is different. Ren et al. successfully isolated *Acinetobacter junii* YB from a laboratory-scale Sequencing Batch Reactor(SBR) which showed the peak removal rates

observed were 8.82 mg/(L·h) for ammonium nitrogen, 8.45 mg/(L·h) for nitrite nitrogen, and 7.98 mg/(L·h) for nitrate nitrogen [20]. Moreover, the further research showed that, in the condition of simultaneous nitrification and denitrification with mixed nitrogen sources, ammonium nitrogen was preferentially removed [21]. Jin et al. isolated a strain which named *Klebsiella* sp. KSND from a lake wetland that exhibited simultaneous heterotrophic nitrification-aerobic denitrification [22]. Within 24 h, it achieved removal rates of 86.56%, 74.52%, and 100% for 160 mg/L ammonium, 105 mg/L nitrate, and 8.39 mg/L nitrite, respectively. The removal rates were calculated as 5.77 mg/(L·h) for $NH_4^+$-N, 3.26 mg/(L·h) for $NO_3^-$-N, and 0.35 mg/(L·h) for $NO_2^-$-N.

Overall, we observed that HN-AD strains have a wide range of sources, most of which are found in soil and water. Those strains are capable of simultaneous removal of ammonium, nitrate, and nitrite and with a remarkable removal effect. This not only shortens the denitrification cycle but also reduces the toxic effects of nitrite accumulation on the cells [33]. Moreover, HN-AD strains belong to the heterotrophic aerobic microorganisms, where the supply of oxygen and substrates not only accelerates cellular proliferation and differentiation, enabling rapid assimilation of ammonium into cellular components, but also maintains high nitrogen removal enzyme activity within the cells. This elevated enzymatic activity level expedites ammonium removal at the enzymatic level.

## 3. Nitrogen metabolism

Traditional biological nitrogen removal (Fig. 1a) primarily involves two processes. Firstly, the nitrification process occurs under aerobic conditions, where ammonia nitrogen in water undergoes nitrification. Ammonia is converted to nitrite through ammonia-oxidizing bacteria and further oxidized to nitrate by nitrite-oxidizing bacteria. Secondly, the nitrogen removal process utilizes organic substances as carbon sources and nitrate/nitrite as electron donors. Denitrifying bacteria reduce nitrate and nitrite to nitrogen gas via intermediates such as NO and $N_2O$ [23].

Compared with traditional nitrogen removal process, the complete nitrogen removal cycle of HN-AD can be accomplished in a single reactor. The nitrification and denitrification pathways of HN-AD are illustrated in Fig. 1b. Microorganisms convert a small portion of nitrogen from wastewater into intracellular organic nitrogen through assimilation. Simultaneously, they convert $NH_4^+$, $NO_3^-$, or $NO_2^-$ in wastewater to gaseous nitrogen through respiration and remove it from the

<table><caption>**Table 1**
Partially isolated HN-AD strains from various environments.</caption><thead><tr><th>Genus</th><th>Name</th><th>Source</th><th>Nitrogen removal efficiency</th><th>Ref.</th></tr></thead><tbody><tr><td>Alcaligenes sp.</td><td>Alcaligenes faecalis No. 4</td><td>Sewage and sludge</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 71%</td><td>[13]</td></tr><tr><td>Acinetobacter sp.</td><td>Acinetobacter junii YB</td><td>laboratory-scale SBR</td><td>The removal rates of NH<sub>4</sub><sup>+</sup>-N, NO<sub>3</sub><sup>-</sup>-N, and NO<sub>2</sub><sup>-</sup>-N are 8.82, 8.45, and 7.98 mg/(L·h), respectively</td><td>[20]</td></tr><tr><td>Alcaligenes sp.</td><td>Alcaligenes faecalis NR</td><td>Membrane bioreactor</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 26.9 mg/(L·h)</td><td>[23]</td></tr><tr><td>Pseudomonas sp.</td><td>Pseudomonas stutzeri YG-24</td><td>Sediment</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 88.1%</td><td>[24]</td></tr><tr><td>Pseudomonas sp.</td><td>Pseudomonas sp. AND-42</td><td>Pond</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 75.8%</td><td>[16]</td></tr><tr><td>Klebsiella sp.</td><td>Klebsiella pneumoniae CFS9</td><td>Domestic wastewater</td><td>85% NH<sub>4</sub><sup>+</sup>-N is removed within 24 h, and 100% NO<sub>3</sub><sup>-</sup>-N is removed within 18 h</td><td>[25]</td></tr><tr><td>Pseudomonas sp.</td><td>Pseudomonas sp. DM02</td><td>Biofloc reactor of a recirculating aquaculture system</td><td>The removal rates of total NH<sub>4</sub><sup>+</sup>-N and NO<sub>3</sub><sup>-</sup>-N are 100% and 95%, respectively</td><td>[26]</td></tr><tr><td>Golden bacillus</td><td>Golden bacillus R31</td><td>Slaughterhouse wastewater</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 95.9%</td><td>[27]</td></tr><tr><td>Coctobacillus sp.</td><td>Cupriavidus sp. S1</td><td>Coking wastewater</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 99.7%</td><td>[28]</td></tr><tr><td>Agrobacterium sp.</td><td>Agrobacterium tumefaciens LAD9</td><td>Landfill leachate</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 100%</td><td>[29]</td></tr><tr><td>Acinetobacter sp.</td><td>Sxf14</td><td>landfill</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate 94.7%</td><td>[30]</td></tr><tr><td>Acinetobacter sp.</td><td>Acinetobacter sp. ND7</td><td>Municipal wastewater</td><td>99.8% NH<sub>4</sub><sup>+</sup>-N, 96.2% NO<sub>3</sub><sup>-</sup>-N, and 97.18% NO<sub>2</sub><sup>-</sup>-N</td><td>[31]</td></tr><tr><td>Chryseobacterium sp.</td><td>Chryseobacterium sp. R31</td><td>Slaughterhouse wastewater</td><td>NH<sub>4</sub><sup>+</sup>-N removal rate within 48 h 95.87%</td><td>[32]</td></tr><tr><td>Bacillus litoralis</td><td>Bacillus sp. N31</td><td>Mariculture water</td><td>The removal efficiency of NH<sub>4</sub><sup>+</sup>-N, NO<sub>3</sub><sup>-</sup>-N and NO<sub>2</sub><sup>-</sup>-N is 86.3%, 89.3% and 89.4%.</td><td>[10]</td></tr><tr><td>Klebsiella sp.</td><td>Klebsiella sp. KSND</td><td>Lake-wetland sediment</td><td>The 24-hour removal rates for NH<sub>4</sub><sup>+</sup>-N, NO<sub>3</sub><sup>-</sup>-N, and NO<sub>2</sub><sup>-</sup>-N are 86.56%, 74.52% and 100%</td><td>[22]</td></tr></tbody></table>
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<figure><img src="image_1.png" /><figcaption>Fig. 1. a) Traditional nitrogen removal process, b) Nitrogen metabolism of HN-AD bacteria.</figcaption></figure>

sewage. Transformations among inorganic nitrogen, organic nitrogen, and gaseous nitrogen occur through oxidation, reduction, and assimilation processes. In addition, the HN-AD process exhibits minimal accumulation of nitrite and nitrate, thereby facilitating its extensive application.

Although a multitude of HN-AD bacteria have been identified, the complexity of the enzymatic systems involved in the nitration and denitration processes across different genera, coupled with the significant variations in nitrogen removal characteristics under diverse external environmental conditions, has posed challenges in comprehensively understanding the HN-AD mechanisms. As a result, the specific reaction pathways remain inadequately explored.

### 3.1. Nitrate-nitrite reduction denitrification

Currently, widely accepted is a coupled model of heterotrophic nitrification-aerobic denitrification proposed by Wehrfritz et al. in 1993 [34]. This model outlines the heterotrophic nitrification pathway as $NH_4^+ \rightarrow NH_2OH \rightarrow NO_2^- \rightarrow NO_3^-$ (as depicted in Fig. 1b). In this process, $NH_4^+$ is oxidized to hydroxylamine ($NH_2OH$) by ammonia monooxygenase (AMO). Subsequently, $NH_2OH$ is transformed to $NO_2^-$ by hydroxylamine oxidoreductase (HAO), and $NO_2^-$ is further converted to $NO_3^-$ by nitrite oxidoreductase (NXR) [18]. In addition, acetone oxime dioxygenase is another enzyme that catalyzes the production of nitrite from hydroxylamine. Hydroxylamine is converted to pyruvate oxime through a non enzymatic reaction with pyruvate, and then oxidized to nitrite and pyruvate through POD [35]. A strain of HN-AD bacteria, DN13-1, isolated by Zhang et al. in 2020 [36], was found capable of transforming hydroxylamine into nitrite. However, the activities of HAO and POD enzymes were not detected in this strain. This observation suggests the possibility of alternative nitrification pathways in the DN13-1 strain. Therefore, further studies are required to elucidate the key enzymes and metabolic pathways involved in the denitrification process of HN-AD bacteria.

### 3.2. Nitrogen removal by hydroxylamine reduction

While the coupled model nitrogen removal pathway has been widely accepted, there are certain HN-AD bacteria that do not strictly follow this pathway. Some scholars propose an alternative nitrogen removal pathway for HN-AD, involving the conversion of hydroxylamine to nitrogen as a means of ammonia nitrogen removal [37]. Research has revealed that certain bacteria exhibit heterotrophic nitrification and generate nitrate, nitrite, and $N_2$. However, when these bacteria were inoculated into culture media containing nitrite or nitrate as a nitrogen source under aerobic conditions, no reduction of nitrate or nitrite was observed [38,39]. But when hydroxylamine was the sole nitrogen source, nitrogen gas was produced during the cultivation process. The bacterium *Acinetobacter calcoaceticus* strain HNR [38] cannot utilize $NO_3^-$ and $NO_2^-$ for denitrification, yet it was able to reduce ammonia nitrogen and hydroxylamine to $N_2O$ and $N_2$ for removal. This suggests a denitrification pathway for this HN-AD strain as $NH_4^+ \rightarrow NH_2OH \rightarrow N_2O \rightarrow N_2$, which is consistent with Alcaligenes faecalis NR [23] and Alcaligenes faecalis No.4 [13].

### 3.3. Assimilatory denitrification

Nitrogen assimilation holds a pivotal role in the denitrification process within HN-AD. Several studies have indicated that nitrogen assimilation could account for up to 40% of the total nitrogen removed in the process [13]. In the denitrification process, the HN-AD strain *Pseudomonas stutzeri* T13 can assimilate nitrogen under both aerobic and anaerobic conditions [40]. Nitrogen can serve as both an electron donor ($NH_4^+$) and an electron acceptor ($NO_3^-$) for assimilation [20]. The HN-AD strain *Klebsiella* sp. lacks genes associated with nitrification (AMO and HAO genes) and denitrification (NIR and NOS genes), suggesting that the removal of $NH_4^+$ is through assimilation [37]. In the assimilation process, $NO_3^-$ is converted into nitrite by nitrate reductase, and then transformed into ammonia by nitrite reductase. Ultimately, glutamate dehydrogenase converts ammonia into glutamate for cell
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<figure><img src="image_2.png" /><figcaption>Fig. 2. Enzymes related to aerobic denitrification.</figcaption></figure>

growth. While HN-AD bacteria are typically found in soil and exhibit some capability for organic nitrogen removal, certain bacteria also display significant assimilation in inorganic nitrogen environments [41], which is beneficial for improving the adaptability of micro-organisms to high ammonia nitrogen.

### 3.4. Key enzymes

Four main enzymes are involved in HN-AD, among which Ammonia Monooxygenase and Hydroxylamine oxidase play a role in the nitrification reactions. In the aerobic denitrification process, nitrite reductase (NIR) and nitrate reductase (NAR/NAP) are essential enzymes (Fig. 2). Additionally, nitric oxide reductase (NOR) and nitrous oxide reductase (NOS) also play significant roles in aerobic denitrification [42]. The catalyzed reactions of these enzymes are shown in Table 2. Typically, research on the denitrification enzyme system of these bacteria mainly focuses on Ammonia Monooxygenase and Nitrate reductase. The presence of these enzymes serves as crucial molecular biology indicators for identifying whether a strain is an HN-AD bacterium.

#### 3.4.1. Ammonia monooxygenase

AMO is a crucial enzyme in the nitrification process, initially discovered in ammonia-oxidizing bacteria and more recently in ammonia-oxidizing archaea. It plays a key role in the oxidation of ammonia nitrogen during nitrification, making the gene encoding AMO a reliable indicator for identifying whether a strain has nitrification capability. The activity of AMO can also serve as a standard for evaluating overall nitrification activity in the environment [46]. AMO is a membrane-bound multiple subunit enzyme [47], and the AmoA, AmoB, and AmoC genes together form a functional AMO operon [48]. Difficulties in purifying AMO have limited in-depth studies on its mechanism [49]. The activity of AMO is significantly influenced by environmental factors. When the pH rises in environment, the proportion of free ammonia in ammonia

nitrogen rises, which facilitates the catalytic reaction of AMO [50]. Various metal ions have been found to influence the catalytic performance of AMO. The presence of Cu²⁺ can enhance the stability and activity of AMO, thereby improving nitrification efficiency of strains [51]. The membrane-associated AMO purified from *Paracoccus denitrificans* consists of two subunits with molecular masses of 38 kDa and 46 kDa. It belongs to quinone oxidoreductases and its activity is inhibited by light and various chelating agents. In the presence of Cu²⁺, it participates in the oxidation of NH₂OH [52], showing similarities to the AMO of Nitrosomonas europaea [53]. AMO exhibits high sensitivity to temperature. Sayavedra-Soto et al. found that as the temperature increases from 15 °C to 20 °C, the relative activity of AMO decreases from 100% to 15.1% [54]. The diversity of AMO in HN-AD bacteria contributes to the complexity of NH₄⁺ oxidation mechanisms, further research is necessary for comprehensive understanding.

#### 3.4.2. Hydroxylamine oxidase

HAO is another key enzyme in the nitrification process [12], suggests that HAO can produce different metabolic products from hydroxylamine in heterotrophic nitrifying bacteria under aerobic or anaerobic conditions. Under aerobic conditions, hydroxylamine is oxidized to NO₂⁻ by HAO, while under anaerobic conditions, hydroxylamine is oxidized to N₂O gas. Compared to the HAO in traditional autotrophic nitrifying bacteria, the structure of HAO in heterotrophic nitrifying bacteria may be simpler. The HAO in heterotrophic nitrifying bacteria is a monomeric protein that can use Fe atoms as cofactors to maintain enzyme activity. Therefore, the addition of Fe²⁺ significantly promotes the hydroxylamine oxidation process [52,55]. The diversity of heterotrophic nitrifying bacteria contributes to variations in the presence and mechanisms of HAO enzymes. Additionally, there is limited researches on the genes involved in this process. Further studies using complementary techniques are needed to provide a solid theoretical foundation for explaining the nitration mechanism in heterotrophic nitrifying bacteria that are involved in the HN-AD process.

<table><caption>**Table 2**
Nitrogen removal related enzymes and the reactions.</caption><thead><tr><td>Enzyme</td><td>Abbreviation</td><td>Reaction equation</td><td>Ref.</td></tr></thead><tbody><tr><td>Ammonia Monooxygenase</td><td>AMO</td><td>NH<sub>3</sub> + a reduced acceptor + O<sub>2</sub> ⇌ NH(2)OH + an acceptor + H<sub>2</sub>O</td><td>[43]</td></tr><tr><td>Hydroxylamine oxidase</td><td>HAO</td><td>Hydroxylamine + O<sub>2</sub> ⇌ nitrate + H<sub>2</sub>O + H<sup>+</sup></td><td>[44]</td></tr><tr><td>Nitrate reductase</td><td>NAR/NAP</td><td>Nitrate + reduced acceptor ⇌ nitrite + acceptor</td><td>[45]</td></tr><tr><td>Nitrite reductase</td><td>NIR</td><td>Nitrite + ferrocytochrome c + 2H<sup>+</sup> ⇌ Nitric oxide + H<sub>2</sub>O + ferrocytochrome c</td><td>[45]</td></tr><tr><td>Nitric oxide reductase</td><td>NOR</td><td>2 nitric oxide + NAD(P)H ⇌ Nitrous oxide + NAD(P)<sup>+</sup> + H<sub>2</sub>O</td><td>[45]</td></tr><tr><td>Nitrous oxide reductase</td><td>NOS</td><td>nitrous oxide + 2 ferrocytochrome c + 2H<sup>+</sup> ⇌ Nitrogen + H<sub>2</sub>O + 2 ferrocytochrome c</td><td>[45]</td></tr></tbody></table>
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### 3.4.3. Nitrate reductase

NAR is a crucial enzyme in the nitrate reduction pathway and is primarily located in the cytoplasm or cell membrane. In HN-AD bacteria, this enzyme exists in two types [56]: periplasmic nitrate reductase (Nap), which is freely present in the periplasmic space, and membrane-bound nitrate reductase (NarG), which is involved in the $NO_3^- \rightarrow NO_2^-$ pathway. These two types of nitrate reductase have different sensitivities to oxygen molecules. The NarG gene is expressed under anaerobic conditions, while the NapA gene is mainly expressed under aerobic conditions. For example, under aerobic conditions, the activity of Nap is significantly higher than that of Nar in *Paracoccus pantotrophus* DN13-1 [36], whereas under anaerobic conditions, the situation is reversed. Nap consists of two catalytic subunits, NapA and NapB, which form a dimer. The amplification of the NapA gene serves as a diagnostic tool to ascertain the involvement of periplasmic nitrate reductase in aerobic denitrification. Currently, the functional gene NapA is commonly used as a biomarker and for evaluating the diversity of nitrate-reducing microbial communities [57]. Additionally, there is an assimilatory nitrate reductase, Nas, in the bacterial cells. It directly converts nitrate nitrogen into intracellular nitrogen through microbial assimilation, producing a small amount of nitrite nitrogen in the reduction reaction.

### 3.4.4. Nitrite reductase

NIR catalyzes the reduction of $NO_2^-$ to NO. Depending on the reactants and cofactors, NIR can be categorized into different types: copr oper-dep endent nitrite reductase (Cu-NIR), cytochrome cd1 nitrite reductase (cd1-NIR), cytochrome c nitrite reductase (cc-NIR), and ferredoxin-dep endent nitrite reductase (Fd-NIR). Although Cu-NIR and cd1-NIR have different protein structures, they perform similar functions in the process of reducing $NO_2^-$ to NO during denitrification [58,59]. These two enzymes do not coexist in the same bacterial strain simultaneously [60]. cc-NIR consists of two nrfA subunits and one nrfH subunit [61]. These two subunits are encoded by nrfA and nrfH genes, respectively. In terms of spatial structure, there is a certain distance between the catalytic subunit nrfA and heme iron, which ensuring rapid and efficient electron transfer. cc NIR can effectively catalyze the reduction of $NO_2^-$: The nrfH subunit is a membrane-bound cytochrome containing four type C heme groups. It is responsible for transferring electrons from hydroquinone to the catalytic subunit [62]. Fd-NIR mainly exists in photosynthetic microorganisms such as algae and cyanobacteria. It is a polypeptide chain present in the light and tissues. Fd-NIR transfers six electrons to reduce $NO_2^-$ to ammonia. The majority of nitrite reductases are found in the periplasmic space of Gram-negative bacteria [63], and the presence of all four types of nitrite reductases in a single microorganism has not yet been discovered [64].

### 3.4.5. Nitrite oxide reductase

The reduction of NO to $N_2O$ is catalyzed by NOR, and this reaction occurs in the inner cell membrane [65]. Depending on the electron donor, NOR can be classified into cytochrome c-dep endent NOR (cNOR), quinol-dep endent NOR (qNOR), and copr oper-dep endent NOR (Cu ANOR). cNOR was the first NOR to be isolated and is the most extensively studied. It has been reported only in denitrifying bacteria. This enzyme is composed of an electron transfer subunit (NorC) and a catalytic subunit (NorB). qNOR is a single-subunit enzyme that lacks heme c and, as a result, cannot accept electrons from cytochromes. It accepts electrons from quinones (ubiquinol or menaquinol) for its catalytic activity. Cu ANOR has only been reported in *Bacillus* at present and consists of two subunits. The smaller subunit contains a double copper atom active center (CuA), heme b, and the binuclear catalytic center located on the larger subunit [66]. These different types of NORs play a crucial role in the denitrification process by reducing NO to $N_2O$ or other nitrogen compounds, depending on the specific enzyme and reaction conditions.

### 3.4.6. Nitrous oxide reductase

The conversion of $N_2O$ to $N_2$ is catalyzed by NOS. In the majority of HN-AD bacterial strains, NOS is a soluble periplasmic enzyme and exists as a dimeric protein [67]. The activity of NOS is reduced under low pH and high concentrations of metal ions. About one-third of denitrifying bacteria containing the genes NIR and NOR lack the NOS gene. NOS catalyzes the final step of denitrification, producing nitrogen gas ($N_2$) as the end product. As a result, the largest structural gene in the NOS gene cluster, *nosZ*, is generally considered a molecular marker for detecting whether microbes are capable of complete denitrification (with the end product being nitrogen gas) [16]. The NOS enzyme plays a critical role in the conversion of nitrous oxide, a potent greenhouse gas, into harmless nitrogen gas, thereby contributing to the reduction of greenhouse gas emissions.

## 4. Influencing factors

Indeed, environmental conditions have a significant impact on the initiation of HN-AD functions [14]. Extensive research indicates that temperature, carbon source, pH value, carbon-to-nitrogen ratio (C/N), and dissolved oxygen (DO) are the main factors influencing the growth and metabolism of HN-AD bacteria [22,29,39,68]. Therefore, further research into the effects of environmental factors is not only beneficial for optimizing denitrification performance but also provides important theoretical references for strain applications.

### 4.1. Carbon source and C/N

Carbon and nitrogen metabolism are fundamental cellular pathways in all biological water treatment techniques. Carbon sources constitute the main components of cell structures, providing energy for the life activities of HN-AD bacteria and serving as electron donors for their denitrification processes [69]. Different carbon sources occupy different positions in the carbon cycle utilization of strains, leading to varying utilization rates [70] (As given in Table 3), ultimately affecting the denitrification efficiency of the strains.

In most studies, the carbon sources used for cultivation include glucose, sodium acetate, sodium succinate, sodium citrate, trisodium citrate, and sucrose, among others. Different strains also exhibit varying adaptability to different carbon sources. When sodium acetate, glucose, sodium succinate, and sucrose are used as the sole carbon sources, the strain *Vibrio diabolicus* SF16 [18] achieves a nitrogen removal rate of over 88%. However, when sodium citrate or potassium bitartrate is used as the sole carbon source, the ammonia nitrogen removal rate is significantly lower compared to the other four carbon sources. The strain *Acinetobacter* sp. ND7 [31] demonstrates effective denitrification capabilities under aerobic conditions when sodium acetate, sodium succinate, or sodium citrate are used as the sole carbon sources, with sodium citrate yielding the best performance. However, denitrification efficiency is lower when sucrose or glucose is used as the sole carbon source. Sodium citrate, not effective for the *V. diabolicus* SF16 strain, turns out to be the most efficient carbon source for denitrification in *Acinetobacter* sp. ND7. This phenomenon might be related to strain-specific redox capabilities [24] as well as the chemical properties and molecular structures of different carbon sources [71]. Among this several carbon sources, sodium acetate exhibits the highest degree of oxidation, while glucose and sucrose are the most reduced compounds. Between sodium citrate and sodium succinate, sodium citrate has a higher degree of oxidation, which suggesting that a suitable denitrification carbon source might be a characteristic of a particular strain or species [24].

Some strains have been found to utilize both inorganic carbon for autotrophy and organic carbon for heterotrophy. *Bacillus subtilis* A1 can grow well in inorganic carbon conditions [72]. According to nitrogen balance experiments, around 10% of the ammonia nitrogen is assimilated by the cells, about 20% is transformed into nitrogen gas for
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<table><caption>**Table 3**
Nitrogen removal efficiency of HN-AD bacteria under different carbon sources and C/N conditions.</caption><thead><tr><th>Strain</th><th>Available carbon sources</th><th>Optimal carbon source</th><th>C/N range</th><th>Optimal C/N</th><th>Nitrogen removal effect</th><th>Ref.</th></tr></thead><tbody><tr><td>Alcaligenes faecalis No. 4</td><td>citrate medium, Trisodium citrate, sodium succinate, sodium acetate, sucrose, glucose</td><td>Sodium Succinate</td><td>1-20</td><td>12</td><td>nitrification ratio reached 62%</td><td>[13]</td></tr><tr><td>Pseudomonas sp. DM02</td><td>Ethanol, trisodium citrate, sodium succinate, sodium acetate, sucrose, glucose</td><td>Sucrose, glucose</td><td>0-12</td><td>12</td><td>NO<sub>3</sub>-N removal rate86%</td><td>[26]</td></tr><tr><td>Pseudomonas stutzeri YG-24</td><td>Citrate, Succinate Acetate Glucose Sucrose</td><td>Citrate</td><td>2-10</td><td>8</td><td>NH<sub>4</sub><sup>+</sup> -N remove rate 90.73%, NO<sub>3</sub>-N removal rate 94.17%</td><td>[24]</td></tr><tr><td>Pseudomonas aeruginosa SNDPR-01</td><td>Sodium citrate</td><td>Sodium citrate</td><td>2.5-20</td><td>10</td><td>ammonium and phosphate removal rate 99%</td><td>[33]</td></tr><tr><td>Rhizobium sp. W37</td><td>Sodium citrate, Glucose, Sodium acetate, Sodium succinate</td><td>sodium succinate</td><td>1-30</td><td>10</td><td>NO<sub>3</sub>-N remove rate 100%</td><td>[75]</td></tr><tr><td>Acinetobacter indicus ZJB20129</td><td>Sodium acetate, sodium citrate, sodium succinate, glucose and sucrose</td><td>Sodium acetate, sodium succinate</td><td>6-18</td><td>15</td><td>TN removal rate 77.38%</td><td>[76]</td></tr><tr><td>Brevibacillus Agri N2</td><td>Citrate, succinate, acetate, sucrose, glucose, starch, calcium carbonate</td><td>sodium succinate</td><td>3-15</td><td>12</td><td>Ammonia nitrogen removal rate 63.84%</td><td>[77]</td></tr><tr><td>Alcaligenes faecalis TF-1</td><td>Sodium citrate, glucose, sodium acetate and sodium succinate</td><td>sodium citrate</td><td>4-24</td><td>16</td><td>Ammonia nitrogen removal rate 99.98%</td><td>[78]</td></tr><tr><td>Enterobacter cloacae DK-6</td><td>Sodium acetate, sodium succinate, sodium citrate, glucose</td><td>sodium succinate</td><td>3-15</td><td>8</td><td>NO<sub>3</sub>-N removal rate 94.04</td><td>[79]</td></tr><tr><td>Paracoccus sp. HY-1</td><td>Sodium succinate, sodium citrate, glucose, sucrose, sodium acetate</td><td>sodium succinate</td><td>1-25</td><td>10</td><td>TN removal rate 92.16%</td><td>[70]</td></tr><tr><td>Acinetobacter sp. Z1</td><td>Acetate, pyruvate, succinate, citrate, glucose</td><td>Sodium acetate</td><td>4-20</td><td>12</td><td>Ammonia nitrogen removal rate 14.02 mg-N L<sup>-1</sup>h<sup>-1</sup></td><td>[80]</td></tr><tr><td>Paracoccus denitrificans AC-3</td><td>Sodium acetate, trisodium citrate, sodium succinate, sucrose, glucose and ethanol</td><td>Sodium acetate, trisodium citrate, sodium succinate</td><td>4-12</td><td>10</td><td>Ammonia nitrogen removal rate 83.3 5.2%</td><td>[81]</td></tr></tbody></table>
removal, and the COD concentration increases from 0 to 94.7 mg/L, indicating its ability to synthesize and utilize organic carbon under autotrophic conditions. When provided with organic carbon sources, strain A1 can shift to a heterotrophic nitrification mode.

Variations in the C/N directly affect the nitrogen removal efficiency of HN-AD bacteria [38]. Sufficient carbon sources can promote the expression of denitrification genes, but excessively high C/N ratios can lead to inadequate organic matter utilization, and high carbon concentrations inhibiting bacterial growth. If the carbon-nitrogen ratio is too low, bacteria may not receive sufficient nutrients for growth, and their denitrification capacity would weaken due to the lack of electron donors, thus affecting their denitrification efficiency. Therefore, exploring the suitable C/N ratio for strains is crucial. In general, the optimal C/N ratio for most HN-AD bacteria ranges from 8 to 15, within which strains can achieve a better growth rate and denitrification efficiency. For example, the strain *Bacillus m ethylotrophicus* L 7 h as excellent heterotrophic nitrification performance within a C/N range of 6-20, with the best denitrification efficiency observed at a C/N of 12 [10]. While, in nitrification media with a C/N ratio less than 10, some HN-AD bacteria have shown reduced denitrification performance [18,73].

Nevertheless, some strains can achieve good denitrification efficiency under conditions of excessively high or low C/N. Huang et al. found that the strain *Acinetobacter* sp. Y16 achieved its best denitrification performance at a C/N ratio of 2 [74].

## 4.2. Temperature

Most nitrogen transformation processes of HN-AD bacteria are sensitive to temperature changes [82], because changes in temperature can affect enzyme activity and cell membrane characteristics. The optimal growth temperature for most HN-AD bacteria is between 25 °C and 35 °C. Table 4 presents the optimal nitrogen removal temperatures for some strains. However, the actual temperature in wastewater treatment environments often exceeds the tolerance of microorganisms. When exposed to excessively high or low temperatures, the growth and enzyme activity of most thermophilic microorganisms is inhibited, leading to a sharp decline or even termination of nitrogen removal efficiency. Therefore, strains need to be screened and domesticated for specific environments. While the change of temperature significantly affects their activity, some strains can still maintain partial nitrification and nitrogen removal capabilities under excessively high or low temperatures [14]. Wei et al. found *Rhizobium* sp. WS7 as having excellent nitrogen removal performance at low temperature in winter [75]. This

strain can achieve 98.73% removal of $NH_4^+$-N, 99.98% removal of $NO_3^-$-N, 100% removal of $NO_2^-$-N, and approximately 100% removal of mixed nitrogen ($NH_4^+$-N and $NO_3^-$-N) at 15 °C. Chen et al. discovered that the strain *Anoxybacillus contaminans* HA has an optimal temperature of 55 °C and can adapt to a high-temperature up to 65 °C [83]. The discovery of HN-AD at different temperatures suggests that it plays a significant role in nitrogen cycle at diverse temperature environments.

## 4.3. pH

Changes in pH alter the cell surface charges and affect the absorption of necessary nutrients, and secondly the activity of relevant enzymes in bacteria is also influenced by changes in pH. In general, extremely acidic (pH < 5) or highly alkaline (pH > 10) conditions have negative effects on the growth of HN-AD bacteria [26]. Most of the HN-AD strains can grow in pH 4 to 10 (Table 5), thereby it exhibiting a broader pH tolerance compared with autotrophic strains. For different strains, however, have distinct pH requirements. Jin et al. found that the strain KNSD has an optimal pH range of 5 to 8 [22]. Zheng et al. discovered that the strain *Paracoccus denitrificans* AC-3 exhibits efficient nitrogen removal in pH 6 to 10 [81]. Based on the theory that ammonia monooxygenase utilizes $NH_3$ more than $NH_4$, a higher amount of free $NH_3$ in slightly alkaline culture media may be more favorable for heterotrophic nitrification [87]. Most HN-AD strains are better adapted to alkaline conditions, and few strains can grow at extreme pH values. However, the strain HN-02 exhibits excellent acid adaptability, growing and nitrogen removal occurring even at a low pH of 2.3 [88].

Most of the relevant studies exploring the effect of pH on HN-AD strains have been carried out under laboratory conditions, and may ignoring the pH changes brought about by the reaction itself, which increases with the release of $OH^-$ during the hydrolysis of organic nitrogen to $NH_4^+$. Xia et al. discovered that during the HN-AD process using $NH_4^+$-N as a nitrogen source, the pH of the strain *Acinetobacter* sp. ND7 increased from 7.0 to 8.7 within 36 h [31]. While, some HN-AD bacteria possess the ability to self-regulate pH during aerobic denitrification processes. The strain *Serratia marcescens* W5 is able to reduce pH from 10.0 to 7.2 at a C/N of 20, in order to achieve optimal growth rates [89].

## 4.4. Dissolved oxygen

Compared with traditional anaerobic denitrification, HN-AD bacteria can perform denitrification under aerobic conditions. DO concentration significantly affects the activity of enzymes (NAR, NIR, NOR

<table><caption>**Table 4**
Optimum Nitrogen Removal Temperature of HN-AD Strain.</caption><thead><tr><th>Strain</th><th>Temperature range (°C)</th><th>Optimum temperature (°C)</th><th>Nitrogen removal effect</th><th>Ref.</th></tr></thead><tbody><tr><td>Alcaligenes faecalis No. 4</td><td>20-37</td><td>30</td><td>The maximum removal rate of ammonia is 26.4 mg/L/h</td><td>[13]</td></tr><tr><td>Bacillus strain N31</td><td>15-40</td><td>30</td><td>Ammonia nitrogen removal rate 85.80 ± 5.34%</td><td>[10]</td></tr><tr><td>KNSD</td><td>5-40</td><td>35</td><td>Ammonia nitrogen removal rate 98%</td><td>[22]</td></tr><tr><td>Pseudomonas stutzeri YG-24</td><td>20-40</td><td>35</td><td>99.89%</td><td>[24]</td></tr><tr><td>Pseudomonas aeruginosa SNDPR-01</td><td>20-40</td><td>30</td><td>Ammonia nitrogen removal rate 65%</td><td>[33]</td></tr><tr><td>Pseudomonas sp. N31942</td><td>20-40</td><td>30</td><td>Ammonia nitrogen removal rate 86%</td><td>[84]</td></tr><tr><td>Rhizobium W37</td><td>15-30</td><td>30</td><td>NO<sub>3</sub>-N removal rate 100%</td><td>[75]</td></tr><tr><td>Acinetobacter indicus ZJB20129</td><td>20-40</td><td>35</td><td>Ammonia nitrogen removal rate 98.94%</td><td>[76]</td></tr><tr><td>Brevibacillus Agri N2</td><td>45-60</td><td>50</td><td>Ammonia nitrogen removal rate 62.17%</td><td>[77]</td></tr><tr><td>Alcaligenes faecalis TF-1</td><td>6-10</td><td>7</td><td>Ammonia nitrogen removal rate 73.45%</td><td>[78]</td></tr><tr><td>Enterobacter cloacae DK-6</td><td>20-40</td><td>30</td><td>NO<sub>3</sub>-N removal rate 98.74%</td><td>[79]</td></tr><tr><td>Paracoccus HY-1</td><td>25-40</td><td>35</td><td>N removal rate 15.48 mg-N L<sup>-1</sup> h<sup>-1</sup></td><td>[70]</td></tr><tr><td>Acinetobacter sp. Z1</td><td>15-40</td><td>30</td><td>Ammonia nitrogen removal rate 98.3 ± 0.4%</td><td>[80]</td></tr><tr><td>Paracoccus pantotrophus MA3</td><td>26-40</td><td>37</td><td>NO<sub>3</sub>-N removal rate 96.5 ± 3.5%</td><td>[85]</td></tr><tr><td>Acinetobacter pittii sp. SY9</td><td>10-35</td><td>30</td><td>Ammonia nitrogen removal rate 99%</td><td>[86]</td></tr></tbody></table>
<table><caption>**Table 5**
Optimum nitrogen removal pH of HN-AD strains.</caption><thead><tr><th>Strain</th><th>pH range</th><th>Optimum pH</th><th>Nitrogen removal effect</th><th>Ref.</th></tr></thead><tbody><tr><td>Bacillus strain N31</td><td>6.5-9</td><td>8</td><td>Ammonia nitrogen removal rate 86%</td><td>[10]</td></tr><tr><td>KNSD</td><td>4-10</td><td>5-8</td><td>Ammonia nitrogen removal rate 91-92%</td><td>[22]</td></tr><tr><td>HN-02</td><td>4-10</td><td>10</td><td>Ammonia nitrogen removal rate 95.95%</td><td>[88]</td></tr><tr><td>Pseudomonas sp. DM02</td><td>5-9</td><td>6</td><td>NO<sub>3</sub>-N removal rate 86%</td><td>[26]</td></tr><tr><td>Pseudomonas sp. N31942</td><td>6-8</td><td>7</td><td>Ammonia nitrogen removal rate 84%</td><td>[84]</td></tr><tr><td>Rhizobium W37</td><td>6-10</td><td>7</td><td>NO<sub>3</sub>-N removal rate 100%<br>TN removal rate 78.65%</td><td>[75]</td></tr><tr><td>Acinetobacter indicus ZJB20129</td><td>5-9</td><td>8</td><td>Ammonia nitrogen removal rate 99.27%</td><td>[76]</td></tr><tr><td>Brevibacillus Agri N2</td><td>7-9</td><td>8</td><td>Ammonia nitrogen removal rate 74.42%</td><td>[77]</td></tr><tr><td>Enterobacter cloacae DK-6</td><td>5-9</td><td>6</td><td>NO<sub>3</sub>-N removal rate 98.03%</td><td>[79]</td></tr><tr><td>Paracoccus HY-1</td><td>6-9</td><td>7</td><td>Ammonia nitrogen removal rate 13.77 mg-N L<sup>-1</sup> h<sup>-1</sup></td><td>[70]</td></tr><tr><td>Acinetobacter sp. Z1</td><td>7-11</td><td>8</td><td>Ammonia nitrogen removal rate 97.6 ± 0.3%</td><td>[80]</td></tr><tr><td>Paracoccus denitrificans AC-3</td><td>6-10</td><td>6-10</td><td>Ammonia nitrogen removal rate 99%</td><td>[81]</td></tr><tr><td>Acinetobacter pittii sp. SY9</td><td>5-9</td><td>7</td><td>TN removal rate 92%</td><td>[86]</td></tr></tbody></table>

<table><caption>**Table 6**
Optimum DO concentration of HN-AD strains.</caption><thead><tr><th>Strain</th><th>Shaking speed (rpm)</th><th>Optimum Shaking speed (rpm)</th><th>Nitrogen removal effect</th><th>Ref.</th></tr></thead><tbody><tr><td>Marinobacter sp. F6</td><td>0-200</td><td>150</td><td>TN removal rate > 80%,NO<sub>2</sub>-N minimal accumulation</td><td>[9]</td></tr><tr><td>Rhizobium sp. W37</td><td>90-150</td><td>150</td><td>NO<sub>3</sub>-N removal rate 100%<br>TN removal rate 75.74-78.19%</td><td>[75]</td></tr><tr><td>Brevibacillus Agri N2</td><td>50-200</td><td>200</td><td>Ammonia nitrogen removal rate 79.15%</td><td>[77]</td></tr><tr><td>Pseudomonas putida strain NP5</td><td>80-200</td><td>200</td><td>Ammonia nitrogen removal rate > 95%</td><td>[8]</td></tr><tr><td>Enterobacter cloacae DK-6</td><td>3.6-5.6 mg/L</td><td>5.2 mg/L</td><td>NO<sub>3</sub>-N removal rate 99.06%</td><td>[79]</td></tr><tr><td>Cupriavidus sp. S1</td><td>0-180</td><td>120</td><td>NO<sub>3</sub>-N removal rate 100%</td><td>[28]</td></tr><tr><td>Paracoccus HY-1</td><td>80-240</td><td>240</td><td>Ammonia nitrogen removal rate 17.47 mg-N L<sup>-1</sup> h<sup>-1</sup></td><td>[70]</td></tr><tr><td>Acinetobacter sp. Z1</td><td>50-200</td><td>150</td><td>NH<sub>4</sub><sup>+</sup>-N and NO<sub>3</sub>-N removal rate is 92.3 ± 1.1% and 87.6 ± 3.2% respectively</td><td>[80]</td></tr><tr><td>Acinetobacter haemolyticus ZYL</td><td>0-200</td><td>120</td><td>NO<sub>3</sub>-N removal rate 100%</td><td>[92]</td></tr><tr><td>Paracoccus pantotrophus MA3</td><td>60-180</td><td>180</td><td>Ammonia nitrogen removal rate 100%</td><td>[85]</td></tr><tr><td>Acinetobacter pittii sp. SY9</td><td>40-180</td><td>120</td><td>Ammonia nitrogen removal rate 100%<br>TN removal rate 90.13%</td><td>[86]</td></tr><tr><td>Pseudomonas putida strain NP5</td><td>0-200</td><td>150</td><td>Ammonia nitrogen removal rate 96.5%</td><td>[93]</td></tr></tbody></table>

and NOS), modifying microbial community, bacterial co-occurrence [90]. Once the DO concentration deviates from the optimal range, the growth and function of microorganisms will be inhibited [75]. Researchers often adjust the shaking speed of the incubator to control DO concentrations and explore the optimal DO range for HN-AD bacteria. Different strains exhibit varying tolerance to DO levels. Most HN-AD bacteria can remove nitrogen in the system when the shake rate keeping under 200 rpm (Table 6). Generally, within the DO tolerance range of a strain, increasing DO concentration does not significantly affect its nitrogen removal ability. However, when DO concentration exceeds a threshold, the nitrogen removal capacity of the strain will be inhibited by DO concentration. Zheng et al. found that the strain *Marinobacter* sp. exhibited enhanced denitrification within a certain range of DO concentrations, but its nitrogen removal efficiency significantly decreased once DO exceeded a certain threshold [9]. Moreover, some strains have been found to tolerate higher DO concentrations. Ouyang et al. isolated *Acinetobacter tandoii* MZ-5 from a polluted river, and even when the DO concentration increased to 7.66 mg/L, its ability to remove ammonia nitrogen remained at over 99% [91]. A similar phenomenon was observed in the strain *Pseudomonas putida* strain NP5 [8].

## 5. Conclusion

Among the remediation techniques for nitrogen-contaminated water bodies, microbial methods offer a relatively cost-effective and environmentally friendly approach compared to other chemical and physical techniques. HN-AD bacteria proliferate rapidly, have a wide range of applicability, and are diverse. They can simultaneously eliminate ammonia, nitrate, and nitrite, key enzymes such as AMO, HAO,

NAR, NIR play crucial roles in this process. In the HN-AD process, temperature, carbon source, pH, C/N, and DO are the primary factors influencing the growth and metabolism of HN-AD bacteria. Different strains showing varying adaptability to specific environment. The optimal range of C/N typically falling between 8 and 15. Temperature influences enzyme activity and cell membrane characteristics, with most HN-AD bacteria thriving between 25 °C and 35 °C. The value of pH affects cell surface charges and enzyme activity, with most strains growing in pH 5 to 10.

Although these isolated and identified HN-ADs have been found in various environments, this does not mean that they have HN-AD functions in some environments. In order to determine whether the function can proceed, the expression of relevant functional genes and the contaminant removal performance need to be determined. So far, there are no HN-AD bacterial models that can decipher general molecular information through modern omics approaches (genomics, transcriptomics, and proteomics). Further exploration is warranted to uncover the potential functions of HN-AD bacteria. Reports indicate that some genera of HN-AD can cause diseases such as Klebsiella pneumoniae, while others can prevent diseases and improve water quality. The classification and functions of HN-AD are still evolving, requiring ongoing efforts to deepen our understanding of these microorganisms. The growth and metabolic characteristics of different HN-AD bacteria are obviously different, and the coordination and interaction with other microorganisms need to be further explored.

Moreover, the practical application of HN-AD strains primarily remains in the laboratory stage, with only a few being employed in large-scale actual wastewater degradation processes. In addition, such bacteria have a low survival rate and vulnerable metabolic activity in
wastewater. Consequently, researching the role of HN-AD bacteria in various real wastewaters, analyzing their status in nitrogen cycling processes, and optimizing processes and conditional parameters to enhance their prominence as denitrifying agents are critical areas for future research. At present, domestic and international research on HN-AD focuses mostly on the isolation of strain, with limited exploration of reactors or other practical integrated systems.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationship s that could have app earedto influ-ence the work reported in this paper.

## Acknowledgements

This work was supported by the Natural Science Foundation of Shanghai Municipality, grant number No. 20ZR1438200 and the National Natural Science Foundation of China, grant number No. 51778565.

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