Heterotrophic Nitrification-aerobic Denitrification Research Articles

Activation of Algicidal Bacteria and Nitrogen-Phosphorus Removal Bacteria During Controlling Cyanobacteria Bloom in Taihu Lake by Artemisinin Algaecide

Cyanobacterial harmful algal blooms (CyanoHABs) in Taihu Lake pose a persistent environmental challenge. This study investigated the inhibitory effects of artemisinin algaecide (AMA) on cyanobacteria in Taihu Lake and assessed its impact on nutrients, as well as the structures of particle-attached (PA) and free-living (FL) bacterial communities and potential ecological mechanisms. The results indicated that A-3 (0.8g artemisinin/L) effectively inhibited CyanoHABs (inhibition rate = 93%) and significantly increased the alpha diversity of PA and FL bacterial communities during the stationary phase， thereby promoting the proliferation of algicidal bacteria (AB) (e.g., Acinetobacter, Stenotrophomonas, and Exiguobacterium) and heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria (e.g., Acinetobacter, Stenotrophomonas, and Bacillus) through the utilization of dissolved organic carbon (DOC) from the dead cyanobacteria. This proliferation enhanced nitrogen metabolism and increased the abundance of nitrogen-cycling functional genes, improving nutrient cycling and enhancing system stability. The increased abundance of AB continuously suppressed cyanobacteria, while the proliferation of HN-AD bacteria removed nitrogen and phosphorus from the water, thus limiting nutrients available for cyanobacterial growth. Our findings demonstrate that AMA effectively inhibits CyanoHABs and prevents secondary blooms, providing a scientific foundation for the widespread application in cyanobacterial management, enhancing the effectiveness and sustainability of CyanoHAB control efforts.

A novel Zobellella endophytica W14 strain for nitrogen removal from hypersaline wastewater through simultaneous nitrification and denitrification.

To address the challenges associated with biological treatment of high-salinity wastewater, a novel salt-tolerant strain, Zobellella endophytica W14, was isolated. This strain exhibited heterotrophic nitrification-aerobic denitrification (HN-AD) capabilities. Strain W14 could grow and remove ammonium in high-salinity environments with salinity levels ranging from 0 to 11% (w/v). At 5% salinity, strain W14 demonstrated high removal efficiencies for nitrite, ammonium, and nitrate (100%, 99.58%, and 98.85%, respectively), when these compounds were provided as the single source of nitrogen. In cases of mixed nitrogen sources, total nitrogen removal efficiency of strain reached 95.22%. The nitrogen balance analysis confirmed the utilization of nitrogen sources by strain W14 through both assimilation and dissimilation. Through the amplification of functional genes involved in nitrogen metabolism (i.e., hao, napA, nirS, and nosZ), the nitrogen metabolism pathway of strain W14 was predicted to be: NH₄⁺ → NH₂OH → NO₂⁻ → NO₃⁻ → NO₂⁻ → NO → N₂O → N₂. The study reveals that the novel W14 strain can efficiently remove total nitrogen from high-salinity wastewater and has significant potential for biological treatment of such wastewater.

Will the removal of carbon, nitrogen and mixed disinfectants occur simultaneously: The key role of heterotrophic nitrification-aerobic denitrification strain

The capacity and mechanism of heterotrophic nitrification-aerobic denitrification (HNAD) strain (H1) to remove carbon, nitrogen, disinfectants chloroxylenol (PCMX) and benzethonium chloride (BEC) were investigated in this study. PCMX was removed via metabolism and chemical oxygen demand co-metabolism process. BEC was eliminated through bacterial adsorption, which greatly inhibited the removal of other pollutants. Carbon source optimization tests revealed that glucose was the optimal carbon source for co-removal of pollutants under mixed disinfectants circumstances (PCMX + BEC). Comparing the groups without (G1) and with disinfectants (G2), the content of extracellular polymeric substances was higher, and hydrophobicity was enhanced under the hazardous conditions of G2. All the nitrogen metabolism functional genes in G2 were up-regulated, and the electron transport system activity was also improved. At the same time, G2 had lower reactive oxygen species content, which reduced the probability of resistance genes dissemination, but the abundance of most quantified resistance genes was elevated in G2. Toxicity assessment assays found a dramatic reduction in the virulence of G2’s effluent compared with the mixed disinfentants. The results confirmed that H1 strain could be used to treat the disinfectant-containing wastewater, which may aid in the application of HNAD process.

Genomic insights into heterotrophic nitrifying-aerobic denitrifying bacteria from petroleum terminal effluents

Heterotrophic nitrification-aerobic denitrification (HN/AD) is a single-organism process that converts ammonia into nitrogen gas under strictly aerobic conditions, playing a crucial role in biological ammonia removal from industrial wastewater. Despite several studies, significant knowledge gaps remain about the genes involved in the process. This study aimed to characterize the genomes of four HN/AD bacterial strains, Pseudomonas stutzeri UFV5, Pseudomonas balearica UFV3, Rhodococcus ruber UFV2, and Gordonia amicalis UFV4, and identify potential genes involved in the HN/AD process. Results revealed that shared genes of these strains were primarily involved in amino acid and protein biosynthesis. The two Pseudomonas strains had more genes linked to nitrogen metabolism than the others. Additionally, four strains showed a significant number of hypothetical proteins and genes related to oxidative stress. Notably, no common nitrogen metabolism genes were found among the strains, indicating a lack of a shared HN/AD pathway. However, comparing these genomes with previous transcriptomic data of the P. stutzeri UFV5 identified nine shared proteins as potential HN/AD pathway candidates. This study enhances our understanding of the genomes of these HN/AD-capable bacterial strains and identifies nine candidate genes as markers for the HN/AD process.

Characteristics of a heavy metal resistant heterotrophic nitrification–aerobic denitrification bacterium isolated from municipal activated sludge

The heterotrophic nitrification-aerobic denitrification (HNAD) is a new biological denitrification technology, the present study isolated a new HNAD strain named Cupriavidus metallidurans TX6 with heavy metal resistance. The gene expression, electron transport, enzyme activity and nitrogen removal property of strain TX6 were studied with different influencing factors. Strain TX6 has five nitrogen metabolism pathways (NH4+ → NH2OH → NO → NO2− → NH4+ → GOGAT/GDH; NH4+-N → NH2OH → NO → N2O → N2; NH4+ → NH2OH → NO → NO2− → NO3−; NO3− → NO2− → NH4+ → GOGAT/GDH; NO3−→ NO2− → NH4+ → GOGAT/GDH). Nitrogen balance analysis shows that 29 ± 4 mg/L of N was converted to intracellular nitrogen by assimilation and 50 ± 3 mg/L N loss may be attributed to aerobic denitrification. The results provide a theoretical basis for the HAND bacteria application in nitrogen removal from wastewaters containing heavy metals.

Transcriptome analysis expands underlying mechanisms of quorum sensing mediating heterotrophic nitrification-aerobic denitrification process at low temperature

Quorum sensing (QS) could regulate the behavior of microbial communities and help them resist adverse low-temperature environments. A newly isolated heterotrophic nitrification-aerobic denitrification (HN-AD) bacterium, strain YB1107, exhibited strong tolerance to harsh cold environments, removing 93.5 % of ammonia within 36 h and achieving a maximum specific growth rate of 0.28 h−1 at 10 °C. Strain YB1107 secreted large amounts of N-butanoyl-L-homoserine or N-octanoyl-L-homoserine lactones in response to cold stimuli. The add-back experiments indicated that these two signaling molecules jointly manipulated microbial physiological behavior by improving ammonia oxidation and biofilm formation, while inhibiting aerobic denitrification. The transcriptome analysis revealed that QS systems enhanced the cold resistance of HN-AD bacteria by promoting nitrogen assimilation and reducing dissimilation through regulating related genes. This study provided new molecular insights into how QS mediated HN-AD at low temperatures and laid the foundation for the potential applications of psychrophilic HN-AD bacteria in wastewater treatment.

Fulvic acid mediated highly efficient heterotrophic nitrification-aerobic denitrification by Paracoccus denitrificans XW11 with reduced C/N ratio

Reducing the C/N ratio requirements for heterotrophic nitrification-aerobic denitrification (HNAD) is crucial for its practical application; however, it remains underexplored. In this study, a highly efficient HNAD bacterium, Paracoccus denitrificans XW11, was isolated. The HNAD characteristics of XW11 were studied, and the redox mediator fulvic acid (FA) was used to reduce the C/N requirements. Whole-genome sequencing revealed multiple denitrification genes in XW11; however, nitrification genes were not identified, because heterotrophic nitrification-related gene sequences were not included in the database. However, the nitrogen removal related enzyme activity test revealed complete nitrification and denitrification pathways. Reverse transcription PCR showed that the membrane-bound nitrate reductase (NarG), rather than the periplasmic nitrate reductase, was responsible for aerobic denitrification. The conventional nitrite reductase (NirS) also does not mediate nitrite denitrification. When the C/N ratio was 10, the ammonia removal efficiency of the Control was 71.71 % and the addition of FA increased it to 86.12 %. Transcriptomic analysis indicated electron flow from the carbon source to FA without proton transmembrane transport, and the presence of FA constructs another electron transfer system. The redox potential of oxidized FA/reduced FA is 0.3679 V, avoiding competition for electrons from Complex III. Thus, ammonia monooxygenase obtains electrons more easily, thereby promoting nitrification. The enzyme activity test of the nitrification process confirmed this view. In addition, NarG expression increased, and the denitrification process was enhanced. Overall, FA improved HNAD efficiency by facilitating electron transfer to the nitrogen dissimilation process, offering a novel approach to reduce the C/N requirement of HNAD.

Calcium self-release bioremediation system combined with microbially induced calcium precipitation for the removal of ammonium nitrogen, phosphorus and heavy metals

Natural landscape water and substrate were taken to simulate the release of substrate pollutants in micropolluted water bodies, and a calcium silicate- sodium alginate (CS-SA) bioremediation system was constructed. The removal of ammonium nitrogen (NH4+-N), total nitrogen (TN) and Phosphate (PO43--P) were 99.14 %, 98.90 % and 84.44 %, respectively. The Ca2+ continuously released by CS-SA provided good microbial-induced calcium precipitation (MICP) conditions for Acinetobacter calcoaceticus strain HM12, and the removal efficiency of both Zn2+ and Cd2+ was 100 %, and the pH after remediation was consistent with that of natural water bodies. NH4+-N was removed by heterotrophic nitrification- aerobic denitrification (HN-AD) of strain HM12, and heavy metals and phosphorus were removed by co-precipitation and adsorption by MICP. High-throughput sequencing results showed that strain HM12 was effective as a bioinoculant for the remediation of the aquatic environment. The construction and operation of this bioremediation system provided a method for micropolluted water treatment and recovery of phosphorus and heavy metals.

Research on the application of heterotrophic nitrification-aerobic denitrification bacteria in membrane bioreactor (MBR).

Inoculating heterotrophic nitrification-aerobic denitrification bacteria (HN-AD) to enhance membrane bioreactor (MBR) efficiency may result in the loss of functional bacteria. Therefore, this study compares the application results of enhancing MBR with a self-designed biological amplifier coupled with HN-AD against the performance of conventional MBR. After enhancement, the MBR achieved a removal efficiency of 96.7% for NH4+-N (100mg/L) and 96.4% for COD (400mg/L) in synthetic wastewater. There was a 33% increase in TN (100mg/L) removal efficiency. The dominant bacteria in the MBR were Alcaligenes (48.4%) and Thauera (15.2%). Additionally, the abundance of denitrification genes (nirK, norB, nosZ) increased in the enhanced MBR, contributing to improved TN removal efficiency. The use of a biological amplifier effectively solved the problem of HN-AD loss in sewage treatment.

Promotion of heterotrophic nitrification-aerobic denitrification by nitrite and efficient removal of total nitrogen of strain EN-F2

The inhibition of heterotrophic nitrification-aerobic denitrification (HN-AD) process and low efficiency of total nitrogen conversion under nitrite stress were overcome by strain EN-F2. Results demonstrated that nitrite addition increased total nitrogen conversion to 91.36% and 87.02% for ammonium and nitrate systems, respectively, representing improvements of 5.61% and 15.41%. This enhancement is likely due to the simultaneous acceleration of cell growth, and consumption of ammonium and nitrate. Furthermore, 10 mg/L of hydroxylamine could be almost completely oxidized in a wide range of environmental conditions in the presence of 50 mg/L nitrite, and 100% and 89.82% of nitrite and total nitrogen could be degraded under the conditions of 25 °C, sodium succinate, 7.40 mg/L of dissolved oxygen, C/N ratio 20, initial pH 7.40–7.80 and inoculation quantity of 0.5 × 108 CFU/mL. Altogether, the HN-AD performance of strain EN-F2 can be promoted by nitrite, and no nitrate and hydroxylamine accumulation were found.

The impact of aeration rate on the denitrification performance and microbial community characteristics of the HN-AD bacteria-inoculated MBBR system

This study investigated the influence of aeration rates on the denitrification performance and microbial community of heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria-inoculated MBBR systems treating nitrate wastewater. The results demonstrated high removal efficiencies for COD (93.8 %) and TN (86.2 %) at an optimal aeration rate of 1 mL/min, indicating enhanced TN removal with reduced energy consumption. High-throughput sequencing results revealed that Proteobacteria, Bacteroidota, and Chloroflexi were key players in denitrification, with notable genera including Stappia, Thauera, norank_f__A4b, SM1A02, Pseudomonas, and Flavobacteria. HN-AD bacteria Stappia and Thauera played the key denitrification role at 1 mL/min and lower aeration rate was more conducive to the enrichment of Stappia and Thauera. Functional gene analysis further highlighted the importance of narG, narH and narI in denitrification processes.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

Outdoor tubular photobioreactor microalgae-microorganisms biofilm treatment of municipal wastewater: Enhanced heterotrophic assimilation and synergistic aerobic denitrogenation

This research evaluated a microalgae consortium (MC) in a pilot-scale tubular photobioreactor for municipal wastewater (MWW) treatment, compared with an aeration column photobioreactor. Transitioning from suspended MC to a microalgae-microbial biofilm (MMBF) maintained treatment performance despite increasing influent from 50 L to 150 L in a 260 L system. Carbon and nitrogen removal were effective, but phosphorus removal varied due to biofilm shading and the absence of phosphorus-accumulating organisms. High influent flow caused MMBF detachment due to shear stress. Stabilizing and re-establishing the MMBF showed that a stable phycosphere influenced microbial diversity and interactions, potentially destabilizing the MMBF. Heterotrophic nitrification-aerobic denitrification bacteria were crucial for MC equilibrium. Elevated gene expression related to nitrogen fixation, organic nitrogen metabolism, and nitrate reduction confirmed strong microalgal symbiosis, highlighting MMBF’s treatment potential. This study supports the practical application of microalgae in wastewater treatment.

A sustainable, zero-waste approach for production of biohydrogen from chicken manure slurry by hybrid recycling of digestate

This study provides a sustainable approach to produce biohydrogen through the long-term hydrogenic fermentation of chicken manure (CM) in a semi-continuous stirred tank reactor (CSTR) under different organic loading rates (OLRs). To ensure the zero-waste route and sustainability, the total digestate was mechanically separated into liquid and solid fractions. The solid digestate fraction was thermally treated to produce biochar, while the liquid digestate fraction was biologically treated through bioaugmentation with a novel heterotrophic nitrification-aerobic denitrification (HD-AN (bacterial strain (Alcaligenes sp. ME-1). The results showed that the high concentration of free ammonia nitrogen (FAN) contents of CM could inhibit methanogenesis and induce hydrogen production. The ORLs of 2.5, 3.5, 4.5, and 5.5 gVS/L.d imposed to the CSTR resulted in volumetric hydrogen production (VHP) of 643.3 ± 37, 908.8 ± 64.5, 1077.8 ± 95.17, and 1383.3 ± 156.8 mL/L.d, respectively. The microbial community structures indicated the long-term adaptation of hydrogen-producing bacteria and methanogenesis inhibition. The bioaugmentation process removed 83.3 % of total ammonia and 66.2 % of the chemical oxygen demand (COD) from the liquid digestate fraction. The characteristics of the biochar derived solid digestate is suitable for land applications as fertilizer. The hybrid approach combining biohydrogenation, pyrolysis, and bioaugmentation, maximizes the utilization of chicken manure slurry while conforming to the sustainable development goals.

Transcriptome analysis of the degradation process of organic nitrogen by two heterotrophic nitrifying and aerobic denitrifying bacteria.

The heterotrophic nitrification aerobic denitrification bacteria (HNDS) can perform nitrification and denitrification at the same time. Two HNDS strains, Achromobacter sp. HNDS-1 and Enterobacter sp. HNDS-6 which exhibited an amazing ability to solution nitrogen (N) removal have been successfully isolated from paddy soil in our lab. When peptone or ammonium sulfate as sole N source, no significant difference in gene expression related to nitrification and denitrification of the strains was found according to the transcriptome analysis. The expression of phosphomethylpyrimidine synthase (thiC), ABC transporter substrate-binding protein, branched-chain amino acid ABC transporter substrate-binding protein, and RNA polymerase (rpoE) in HNDS-1 were significantly upregulated when used peptone as N source, while the expression of exopolysaccharide production protein (yjbE), RNA polymerase (rpoC), glutamate synthase (gltD) and ABC-type branched-chain amino acid transport systems in HNDS-6 were significantly upregulated. This indicated that these two strains are capable of using organic N and converting it into NH4+-N, then utilizing NH4+-N to synthesize amino acids and proteins for their own growth, and strain HNDS-6 can also remove NH4+-N through nitrification and denitrification.

Characteristics of Novel Heterotrophic Nitrification–Aerobic Denitrification Bacteria Bacillus subtilis F4 and Alcaligenes faecalis P4 Isolated from Landfill Leachate Biochemical Treatment System

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are the key functional microorganisms needed to achieve simultaneous nitrification and denitrification (SND). In this study, 25 strains of HN-AD bacteria were successfully isolated from a stable landfill leachate biochemical treatment system, of which 10 strains belonged to Firmicutes and 15 strains belonged to Proteobacteria. Bacillus subtilis F4 and Alcaligenes faecalis P4 displayed good tolerance at a wide range of ammonia nitrogen (NH4+-N) concentrations. When the C/N ratio was 20, the removal rates of ammonia nitrogen were 90.1% and 89.5%, and the chemical oxygen demand (COD) removal rates were 92.4% and 93.9%, respectively. The napA gene encoding periplasmic nitrate reductase (Nap) and the nirS gene encoding nitrite reductase (Nir) were detected, and nitrogen balance showed assimilation and HN-AD was the main nitrogen metabolism mode in both strains. The use of immobilization materials could increase removal rate of ammonia nitrogen by 21.1% and 29.6%, respectively. The research results of this work can provide theoretical basis and technical support for the practical application of HN-AD bacteria to enhance the treatment of high ammonia nitrogen wastewater with high efficiency and low consumption.

Ammonium nitrogen and phosphorus removal by bacterial-algal symbiotic dynamic sponge bioremediation system in micropolluted water: Operational mechanism and transformation pathways

Construct a bacteria-algae symbiotic dynamic sponge bioremediation system to simultaneously remove multiple pollutants under micro-pollution conditions. The average removal efficiencies of NH4+-N, PO43−-P, total nitrogen (TN), and Ca2+ were 98.35, 78.74, 95.64, and 84.92 %, respectively. Comparative studies with Auxenochlorella sp. sponge and bacterial sponge bioremediation system confirmed that NH4+-N and TN were mainly removed by bacterial heterotrophic nitrification - aerobic denitrification (HN-AD). PO43−-P was removed by algal assimilation and the generation of Ca3(PO4)2 and Ca5(PO4)3OH, and Ca2+ was removed by algal electron transfer formation of precipitates and microbially induced calcium precipitation (MICP) by bacteria. Algae provided an aerobic environment for the bacterial HN-AD process through photosynthesis, while respiration produced CO2 and adsorbed Ca2+ to promote the formation of calcium precipitates. Immobilization of Ca2+ with microalgae via bacterial MICP helped to lift microalgal photoinhibition. The bioremediation system provides theoretical support for research on micropolluted water treatment while increasing phosphorus recovery pathways.

Hormesis and synergistic effects of disinfectants chloroxylenol and benzethonium chloride on highly efficient heterotrophic nitrification-aerobic denitrification functional strain: From performance to mechanism

The heterotrophic nitrification-aerobic denitrification (HNAD) strain Exiguobacterium H1 (H1) was isolated in this study. The changes in nitrogen metabolism functions of H1 strain were discussed in presence of disinfectants chloroxylenol (PCMX) and benzethonium chloride (BEC) alone and combined pollution (PCMX+BEC). The H1 strain could use NH4+-N, NO2--N and NO3--N as nitrogen sources and had good nitrogen removal performance under conditions of C/N ratio 25, pH 5–8, 25–35 oC and sodium acetate as carbon. PCMX and BEC alone exhibited hormesis effects on H1 strain which promoted the growth of H1 strain at low concentrations but inhibited it at high concentrations, and combined pollution showed synergistic inhibitory on H1 strain. H1 strain owned a full nitrogen metabolic pathway according to functional genes quantification. PCMX encouraged nitrification process of H1, while BEC and combined pollution mostly blocked nitrogen removal. PCMX, but not BEC, mainly led to the enrichment of resistance genes. These findings will aid in systematic assessment of contaminant tolerance characteristics of HNAD strain and its application prospects.

Characteristics of Nitrogen Removal and Functional Gene Transcription of Heterotrophic Nitrification-Aerobic Denitrification Strain, Acinetobacter sp. JQ1004

In this study, the heterotrophic nitrification–aerobic denitrification strain JQ1004 was investigated in terms of its nitrogen removal mechanism and kinetic properties, laying the foundation for its application in the field of wastewater treatment. Nitrogen balance analysis revealed that the final metabolic product was N2, and approximately 54.61% of N was converted into cellular structure through assimilation. According to the fitting of the Compertz model, the maximum degradation rates of ammonia and nitrate were 7.93 mg/(L·h) and 4.08 mg/(L·h), respectively. A weakly alkaline environment was conducive to N removal, and the sensitivity of functional genes to acidic environments was amoA > nirS > narG. An appropriate increase in dissolved oxygen significantly enhanced heterotrophic nitrification activity, and notably, the denitrification-related functional gene narG exhibited greater tolerance to dissolved oxygen compared to nirS. The transcription level of amoA was significantly higher than that of narG or nirS, confirming that there might have been direct ammonia oxidation metabolic pathways (NH4+→NH2OH→N2) besides the complete nitrification and denitrification pathway. The annotation of nitrogen assimilation-related functional genes (including gltB, gltD, glnA, nasA, nirB, narK, nrtP, cynT, and gdhA genes) in the whole-genome sequencing analysis further confirmed the high assimilation nitrogen activity of the HN-AD strain.

Influence and mechanism of typical transition metal ions on the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria

To investigate the inhibitory effects of various transition metal ions on nitrogen removal and their underlying mechanisms, the single and combined effects of Cu2+ Ni2+ and Zn2+ on Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria Acinetobacter sp. TAC-1 were studied in a batch experiment system. The results revealed that increasing concentrations of Cu2+ and Ni2+ had a detrimental effect on the removal of ammonium nitrogen (NH4+-N) and total nitrogen (TN). Specifically, Cu2+ concentration of 10 mg/L, the TN degradation rate was 55.09%, compared to 77.60% in the control group. Cu2+ exhibited a pronounced inhibitory effect. In contrast, Zn2+ showed no apparent inhibitory effect on NH4+-N removal and even enhanced TN removal at lower concentrations. However, when the mixed ion concentration of Zn2++Ni2+ exceeded 5 mg/L, the removal rates of NH4+-N and TN were significantly reduced. Moreover, transition metal ions did not significantly impact the removal rates of chemical oxygen demand (COD). The inhibition model fitting results indicated that the inhibition sequence was Cu2+ > Zn2+ > Ni2+. Transcriptome analysis demonstrated that metal ions influence TAC-1 activity by modulating the expression of pivotal genes, including zinc ABC transporter substrate binding protein (znuA), ribosomal protein (rpsM), and chromosome replication initiation protein (dnaA) and DNA replication of TAC-1 under metal ion stress, leading to disruptions in transcription, translation, and cell membrane structure. Finally, a conceptual model was proposed by us to summarize the inhibition mechanism and possible response strategies of TAC-1 bacteria under metal ion stress, and to address the lack of understanding regarding the influence mechanism of TAC-1 on nitrogen removal in wastewater co-polluted by metal and ammonia nitrogen. The results provided practical guidance for the management of transition metal and ammonia nitrogen co-polluted water bodies, as well as the removal of high nitrogen.