Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1 under aerobic and anaerobic conditions

Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1 under aerobic and anaerobic conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1 under aerobic and anaerobic conditions Yiguo Hong, Wei Sun, Chunchen Hu, Jiapeng Wu, Mingken Wei, Jih-Gaw Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3890763/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although multiple microorganisms can remove ammonium and nitrate simultaneously, their metabolic mechanisms are not well understood. Strain R-1, isolated from the activated sludge of a sewage treatment plant, was identified as Paracoccus denitrificans , and was found to efficiently remove ammonium and nitrate under anaerobic and aerobic conditions. The maximal NH 4 + removal rate (RR = 9.94 mg·L − 1 ·h − 1 ) was significantly higher under aerobic conditions than under anaerobic conditions (RR = 2.91 mg·L − 1 ·h − 1 ). Analysis of the nitrogen balance and isotope tracers indicated that NH 4 + was consumed through assimilation, but not nitrification. The maximal NO 3 − RR of strain R-1 was 18.05 and 19.76 mg·L − 1 ·h − 1 under aerobic and anaerobic conditions, respectively, and NO 3 − reduction was able to support the growth of R-1 under anaerobic conditions. The stoichiometric consumption ratios of acetate and lactate to nitrate were 0.902 and 0.691, respectively. The 15 NO 3 − isotopic tracer experiment demonstrated that NO 3 − was reduced to N 2 by aerobic and anaerobic denitrification. These results indicated that the NO 3 − reduction by strain R-1 was a respiratory process coupled with the oxidation of electron donors. Genomic analysis showed that strain R-1 contained complete genes for the nitrogen metabolism pathways of ammonium assimilation and denitrification, but not for nitrification, which is consistent with the physiological process of inorganic nitrogen metabolism in strain R-1. Moreover, we found that ammonium assimilation and nitrate denitrification effectively promoted each other. Our findings demonstrate that the mechanism of the simultaneous removal of NH 4 + and NO 3 − by strain R-1 involves ammonium assimilation and denitrification under aerobic and anaerobic conditions. These findings provide new insights into microbial nitrogen transformation and facilitate the simultaneous removal of NH 4 + and NO 3 − in a single reaction system. Biological sciences/Biological techniques/Microbiology techniques Biological sciences/Biochemistry/Metabolomics Simultaneous removal of ammonia and nitrate Ammonium assimilation Dentrification Paracoccus denitrificans R-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nitrogen pollution is becoming increasingly severe in aquatic environments due to improper treatment of industrial wastewater, excessive discharge of domestic sewage, agricultural contamination, animal husbandry, and the metabolism of aquatic organisms [ 1 – 3 ] . Nitrogen overload leads to water eutrophication [ 4 ] , destroys ecosystem function, and threatens human health [ 5 ] . Microbiological nitrogen removal (MNR) is an economical and sustainable approach that causes no secondary pollution [ 6 ] . However, traditional nitrogen removal approaches involve two sequential reactions of aerobic nitrification coupled with anaerobic denitrification, which must be conducted in two separate pieces of equipment with independent conditions. This results in deficient performance when dealing with pollution with high concentrations of inorganic and organic nitrogen [ 7 ] . Thus, the high treatment costs have limited the development of MNR technology. Fortunately, with the continuous development of MNR technology, diverse microorganisms have been isolated and identified for nitrogen removal from both engineered and natural ecosystems. Aerobic denitrifying bacteria (ADB) show higher growth rates and nitrogen removal efficiencies than autotrophic, traditional denitrifying bacteria [ 8 , 9 ] , and can simultaneously utilize various organic substrates [ 7 ] ; therefore, aerobic denitrification technology overcomes the disadvantages of traditional nitrogen removal methods and exhibits prominent application value in the engineering process of wastewater treatment [ 10 ] . To improve nitrogen removal efficiency, microbial strains that can simultaneously remove ammonium and nitrate have been isolated and identified from various environments. Most of these strains are ADB. The nitrogen removal mechanisms of these strains are often defined as heterotrophic nitrification and aerobic denitrification [ 11 ] ; however, the evidence for this definition is insufficient. The nitrogen metabolic processes and mechanisms of ADB have not yet been clarified [ 12 ] , especially in regard to ammonium removal. Although ammonium is an important and preferable nitrogen source for the growth of most prokaryotes [ 13 , 14 ] , its removal is generally attributed to heterotrophic nitrification, and little attention has been paid to the role of microbial ammonium assimilation [ 15 , 16 ] . Cell growth and biomass accumulation are directly correlated with ammonium assimilation, which significantly affects nitrogen removal efficiency during aerobic denitrification [ 16 ] . Ammonium assimilation is an important driving force of aerobic denitrification [ 6 ] . Based on the current research in this field, the mechanism responsible for the simultaneous removal of ammonium and nitrate by ADB remains unclear. Thus, the nitrogen metabolic mechanism of ADB warrants exploration as it will be helpful for improving the nitrogen removal efficiency and application of ADB in wastewater treatment. In a previous study, we reported the genome sequence of Paracoccus denitrificans R-1 isolated from the activated sludge of a sewage treatment plant in Taiwan [ 15 ] . This strain was show to be an efficient ADB. In the present study, we investigated the performance and metabolic mechanisms of nitrogen removal by Paracoccus denitrificans R-1. The specific objectives were to: (1) examine the nitrogen removal performance of strain R-1 using growth characteristics and various forms of inorganic nitrogen under aerobic and anaerobic conditions and (2) clarify the metabolic pathways involved in the simultaneous removal of ammonium and nitrate by strain R-1 based on the production of metabolic intermediates, nitrogen balance analysis, stoichiometry between the carbon and nitrogen sources, thermodynamic analysis, 15 N-metabolic flux analysis, and metagenome analysis. A series of combined methodologies was used to elucidate the bacteria-mediated mechanism of simultaneous ammonium and nitrate removal, which will provide a deeper understanding of the nitrogen removal mechanism in ADB and promote its application in wastewater treatment. 2. Material and methods 2.1 Strain, medium, and cultivation Paracoccus denitrificans strain R-1 was isolated from the activated sludge originating from the Xinfeng Sewage Treatment Plant in Taiwan [ 15 ] . The denitrification medium (DM) used in this study comprised1.2 g·L − 1 KNO 3 , 7.9 g·L − 1 Na 2 HPO 4 ·7H 2 O, 1.5 g·L − 1 KH 2 PO 4 , 4.7 g·L − 1 CH 3 COONa·7H 2 O, and 2 mL·L − 1 trace element solution, as reported previously [ 17 ] . The above medium supplemented with only 0.3 g·L − 1 NH 4 Cl (AM) or 0.3 g·L − 1 NH 4 Cl + 1.2 g·L − 1 KNO 3 (ADM) as replacement for KNO 3 in DM was used to test the capability of nitrogen removal by strain R-1. The R-1 bacterial solution was added to the DM and cultured for 12 h until the logarithmic phase of bacterial growth was attained. The culture was centrifuged, the supernatant was removed, and the bacterial precipitate was washed three times with sterile water. Then, 1 mL of the DM solution was added to form a bacterial suspension, which was used as the seed liquid. The equation OD600 (seed liquid) × V1 = 100 mL * 0.05 was used to calculate the inoculation volume, V 1 , which ensured that the optical density at 600 nm (OD 600 ) of strain R-1 was consistently 0.05 in the culture media for the experimental groups at the initial cultivation stage. The prepared seed culture was added to a 250 mL conical flask with 100 mL sterile medium and incubated for 24 h at 30°C with a rotational speed of 150 rpm for aerobic cultivation (Fig. S1 -A). The conical flasks were replaced with anaerobic bottles to ensure an anaerobic culture system (Fig. S1 -B). Anaerobic culture technologies were based on those reported in a previous study [ 18 ] . High-purity helium (He) was subjected to sterile filtration before use. The prepared medium was split into 100 mL anaerobic bottles sealed with septa and flushed with sterilized high-purity He for 10 min. After inoculation with seed liquid, the bottles were cultured at 30°C. The cultures were sampled at different time points (2 and 4 h or 0, 6, 12, 18, and 24 h). The biomass was determined by measuring the OD 600 , and the concentrations of NH 4 + , NO 2 − , and NO 3 − were determined using a rapid spectrophotometry method [ 19 , 20 ] . 2.2 Effect of different electron donors on strain R-1 growth and nitrogen removal under aerobic and anaerobic conditions To explore the coupling of the growth and nitrogen transformation of strain R-1 with the oxidation of electron donors, R-1 cells were cultured in AM, DM, and ADM containing different electron donors (formate, acetate, succinate, pyruvate, lactate, and glucose) with an initial C/N ratio of 9.65. The concentrations of electron donors were determined by ion chromatography, as described in a previous report [ 21 ] . The stoichiometric equation for denitrification with the selected carbon sources was deduced by measuring the content of electron donors and nitrates as acceptors in the determination system. 2.3 Nitrogen removal processes of R-1 under aerobic and anaerobic conditions The nitrogen removal kinetics of R-1 were explored in AM, DM, and ADM using sodium acetate as an electron donor under aerobic and anaerobic conditions. The nitrogen removal efficiency (RE) and rate (RR) were calculated according to the following formulae: \(\text{R}\text{E} \left(\text{%}\right)=\frac{\left(\text{A}-\text{B}\right)}{\text{A}}\times 100\) and \(\text{R}\text{R} \left(\text{m}\text{g}·\text{L}\text{-}\text{1}·\text{h}\text{-}\text{1}\right)=\frac{\left(\text{C}-\text{D}\right)}{\text{T}},\) where A is the initial ammonium or nitrate concentration, B is the observed ammonium or nitrate concentration, C and D are the observed ammonium or nitrate concentrations between adjacent sampling times, and T is the time phase between adjacent samplings. All analyses were performed in duplicate. 2.4 Nitrogen balance analysis of ammonium removal Nitrogen balance analysis was used to identify the NH 4 + removal pathway. The seed liquid of strain R-1 was inoculated into 250 mL triangular flasks containing 100 mL of AM and grown for 28 h at 30°C with a shaking speed of 150 rpm. Bacterial samples were collected every 4 h to determine the biomass concentration, OD 600 , and total nitrogen (TN) concentration of the culture. The concentrations of NH 4 + , NO 2 − , NO 3 − , and TN´ in the liquid supernatant were determined after centrifugation (8,000 × g, 10 min), and the dissolved organic nitrogen (DON) concentration was calculated as the TN concentration minus the sum of the inorganic nitrogen concentrations. Biomass N was calculated as TN minus TN´ concentrations [ 22 ] . Nitrogen loss (N-loss) was calculated as the initial TN concentration minus the final TN concentration after cultivation in non-centrifuged medium. 2.5 Ammonium removal based on isotope tracing analysis The seed liquid of strain R-1 was inoculated into 250 mL triangular flasks containing 100 mL of AM, with NH 4 + replaced with the stable isotope 15 NH 4 + . After 24 h of culture, R-1 cells and supernatants were collected by centrifugation. The R-1 cells were washed three times with ultrapure water and transferred to a clean colorimetric tube. After the addition of an alkaline potassium persulfate solution, the cells were digested at 121℃ for 30 min. If 15 NH 4 + -N assimilation occurred during AM cultivation, the organic nitrogen in the R-1 cells was labeled with 15 N ( 15 N-ON) and released as 15 N-labeled inorganic nitrogen after digestion. The digested solutions were adjusted to pH 7 and then transferred to a 60 mL serum bottle, which was then used as the nitrogen source in DM and inoculated with R-1 seed liquid for 24 h of cultivation (Fig. S2 A). Meanwhile, the AM supernatants obtained after centrifugation were also used as a nitrogen source in DM. They were directly added to the serum bottle, and then inoculated with the seed liquid of strain R-1. After 24 h of cultivation, they were referred to as 15 N-IN (Fig. S2 B). Subsequently, a membrane injection mass spectrometer (MIMS) was used to determine the generation of 29 N 2 and 30 N 2 in the culture medium [ 20 ] . The variation in 30 N 2 concentration after cultivation in these two groups of experiments was used to determine whether NH 4 + -N was assimilated into the organic nitrogen of R-1 cells or nitrified to NO 2 − -N and NO 3 − -N in the supernatants. 2.6 Analysis of nitrate and ammonium removal mechanism based on isotope tracing 15 NH 4 + -N (200 µmol·L − 1 ), 15 NO 3 − -N (200 µmol·L − 1 ), or both were added into the AM, DM, and ADM, respectively, as substitute nitrogen sources under aerobic and anaerobic conditions (Fig. S3). Sterilized media were injected into 60 mL serum bottles with no headspace and aerated with high-purity He for 10 min. The seed liquid of R-1 was inoculated into serum bottles sealed with butyl rubber stoppers and cultured at 30°C. The culture systems used for the aerobic experiments comprised 30 mL of culture medium and 30 mL of headspace to ensure dissolved oxygen in the media. The experimental settings were the same as those used for the anaerobic experiments described above. Blank control experiments were included to test the effects of the medium in the absence of 15 NH 4 + -N and 15 NO 3 − -N. At 0 and 8 h, 2 mL of 50% ZnCl 2 solution was injected using a syringe to inactivate the bacterial cells in the serum bottles. The serum bottles were then transferred to a dark environment for 24 h of precipitation. A MIMS was used to determine the contents of 29 N 2 and 30 N 2 produced in the culture systems, as previously described [ 23 ] . 2.7 Genome sequencing and gene annotations to nitrogen transformation pathways Total genomic DNA was extracted from strain R-1 using a HiPure soil DNA kit (Magen, Guangzhou China). DNA was fragmented into 300–350 bp segments to construct a paired-end library. The end repair of sequences (including phosphorylation of the 5′ end and the addition of A to the 3′ end) was carried out using End Prep Enzyme Mix(NGS Fast DNA Library Prep Set for Illumina, Baiolaibo, Beijing China), and sequencing adapters were added to both ends. Magnetic beads were used to purify the fragments, and 341F/805R primers were used to amplify the V3-V4 variable region of the 16S rRNA gene [ 24 ] . The NovaSeq PE150 platform (Illumina, San Diego, CA, USA) was used to conduct paired-ended sequencing (Azenta Biotech, Suzhou, China) after mixing the DNA libraries labeled with different indices. Raw data processing was performed as described in detail in a previous report [ 15 ] . Genomic DNA (5 ~ 10 µg) was used to construct a PacBio sequencing library. An Agilent 2100 Biological Analyzer (Agilent Technologies, Palo Alto, CA, USA) was used to determine the library quality and a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA) was used to determine the concentration of the library. Sequencing was performed using a PacBio Sequel instrument (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) combined the SMRTbell library with the sequencing primers and enzymes. Sequencing data were analyzed by Azenta Biotech. The raw genome reads were deposited in GenBank under accession numbers CP087986, CP087987, and CP087988 [ 15 ] . The key genes encoding important enzymes closely related to bacterial nitrogen metabolism were identified by annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/ ) database. 3. Results and discussion 3.1 The simultaneous removal of ammonium and nitrate by strain R-1 under aerobic and anaerobic conditions 3.1.1 Effective electron donors for the removal of ammonium and nitrate P. denitrificans R-1 strain was cultured in AM, DM, and ADM supplemented with six different carbohydrates (formate, acetate, pyruvate, succinate, lactate, and glucose) under anaerobic conditions (Fig. S4). The results showed that strain R-1 could use the selected carbohydrates to remove NH 4 + -N from AM with an RE of 17.73–52.42%. RE values greater than 50% were achieved with formate, acetate, and glucose as the carbon sources, and the growth curves of R-1 in AM were all linear with OD 600 values of 0.28–0.62 (Fig. S4A-1–A-6). Comparatively, NO 3 − was almost completely reduced (with an RE of almost 100%) within 12 ~ 18 h in DM with acetate, pyruvate, succinate, lactate, and glucose, but not sodium formate, and the OD 600 values of 0.62–1.67 were higher in DM than that in AM under the same conditions (Fig. S4B-1–B-6). The RE values of NH 4 + and NO 3 − were 80–99% and almost 100%, respectively, within 12 ~ 18 h in ADM with acetate, pyruvate, succinate, lactate, and glucose, but not sodium formate, and the OD 600 values of 1.02–1.68 were higher than those for AM and DM under the same conditions (Fig.S4 C-1–C-6). Moreover, the NH 4 + -N and NO 3 − -N removal curves were consistent with the changing trend with culture time, suggesting that NH 4 + and NO 3 − were simultaneously removed from ADM. Therefore, acetate, pyruvate, succinate, lactate, and glucose could be used as carbon sources to support the growth and nitrogen metabolism of strain R-1. 3.1.2 The NH 4 + removal performance and growth of strain R-1 under aerobic and anaerobic conditions The removal of NH 4 + and the growth of strain R-1 were analyzed in AM for 24 h under aerobic conditions, and the results are shown in Fig. 1 (A-1). The concentration of NH 4 + in AM decreased from 70.56 to 64.25 mg·L − 1 during 0–4 h, with an RR of 1.79 mg·L − 1 ·h − 1 for NH 4 + - N. The concentration of NH 4 + in AM decreased from 64.25 to 0 mg·L − 1 and the average RR of NH 4 + -N by R-1 was 5.35 mg·L − 1 ·h − 1 during 4–16 h. The highest RR was 9.94 mg·L − 1 ·h − 1 at 16 h, which was close to the RR of Rhodococcus erythropolis strain Y10 (9.69 mg·L − 1 ·h − 1 ) [ 6 ] and higher than the RR of Vibrio sp. Y1-5 (2.65 mg·L − 1 ·h − 1 ) [ 25 ] , Rhodococcus sp. CPZ24 (3.1 mg·L − 1 ·h − 1 ) [ 26 ] , and Pseudomonas mendocina TJPU04 (4.69 mg·L − 1 ·h − 1 ) [ 27 ] . These results indicated that strain R-1 has a strong ammonium conversion ability. The growth curve with the OD 600 values was consistent with a decrease in NH 4 + . In contrast, the removal of NH 4 + by strain R-1 and its growth in AM were obviously different under anaerobic conditions (Fig. 1A-2). The RE of NH 4 + reached 75.09% at 24 h, with the highest RR of 2.91 mg·L − 1 ·h − 1 at 20 h. This was lower than the RR under aerobic conditions. This may be due to the lack of available electron acceptors in AM under anaerobic conditions [ 16 ] . Energy derived only from the oxidation of organic carbon may be inefficient for ammonium assimilation during heterotrophic growth or respiration. A similar result was reported in a previous study, in which the removed NH 4 + was mainly transformed into biological nitrogen through ammonium assimilation, with cell synthesis rates greatly inhibited under anaerobic conditions [ 22 ] . These results are not consistent with those of Pseudomonas stutzeri T13, which cannot utilize NH 4 + as the sole nitrogen source to grow under anaerobic conditions [ 16 ] . In addition, the accumulation of NO 2 − and NO 3 − was not observed in the AM of strain R-1 under aerobic or anaerobic conditions (Fig. 1A-1 and A-2), suggesting that NH 4 + was used as a nitrogen source for cell synthesis in the AM of strain R-1. This result is in accordance with those of previous studies of Pseudomonas stutzeri T13 [ 16 ] , Ochrobactrum anthropic LJ81 [ 11 ] , and Rhodococcus erythropolis Y10 [ 6 ] . These results suggest that NH 4 + was possibly removed through the assimilation pathway, but not through heterotrophic nitrification. 3.1.3 The NO 3 − removal performance and growth of strain R-1 under aerobic and anaerobic conditions The kinetics of nitrate removal and growth of strain R-1 were analyzed in DM for 24 h under aerobic conditions, as shown in Fig. 1 (B-1). The OD 600 value of strain R-1 increased slowly during 0–4 h, and the NO 3 − RR of strain R-1 reached 6.17 mg·L − 1 ·h − 1 at 4 h. The OD 600 value in DM then increased exponentially during 4–16 h, and the highest NO 3 − RR was 18.05 mg·L − 1 ·h − 1 at 12 h. During 16–20 h, the OD 600 value remained stable, and the NO 3 − RR decreased from 11.30 to 0.74 mg·L − 1 ·h − 1 . Finally, the OD 600 value decreased in the period 20–24 h, and the NO 3 − RR of strain R-1 decreased to the lowest value of 0.74 mg·L − 1 ·h − 1 , and the NO 3 − RE reached 95.04% at 24 h. The nitrate removal performance and bacterial growth in DM under anaerobic conditions are shown in Fig. 1 (B-2). The NO 3 − concentration decreased from 171.23 to 132.45 mg·L − 1 , and the NO 3 − RR reached 5.59 mg·L − 1 ·h − 1 at 12 h. The NO 3 − concentration decreased from 132.45 to 12.45 mg ·L − 1 during 12–20 h and the highest NO 3 − RR was 19.76 mg·L − 1 ·h − 1 at 20 h. The RE of NO 3 − reached 94.90% at 24 h under anaerobic conditions. The OD 600 was 0.97 in the stable period (20–24 h), which was lower than the OD 600 value observed under aerobic conditions (16 h, OD 600 = 1.82). Similarly, Pseudomonas stutzeri T13 and Paracoccus denitrificans HY-1 have been reported to assimilate less nitrate into their biomass under anaerobic conditions than under aerobic conditions [ 7 , 16 ] . These results showed that strain R-1 could adopt NO 3 − as both a nitrogen source and an electron acceptor when it was supplied as the sole source under aerobic and anaerobic conditions. The maximal NO 3 − RRs of strain R-1 were 18.05 and 19.76 mg·L − 1 ·h − 1 under aerobic and aerobic conditions, respectively, in DM, which were higher than those of Bacillus cereus GS-5 (2.70 mg·L − 1 ·h − 1 ) [ 28 ] , Paracoccus denitrificans HY1 (14.56 mg·L − 1 ·h − 1 ) [ 7 ] , and Rhodococcus erythropolis strain Y10 (5.43 mg·L − 1 ·h − 1 ) [ 6 ] , which suggested that strain R-1 has a strong ability for NO 3 − conversion. During the whole NO 3 − removal process, NH 4 + and NO 2 − were almost undetected, which was in accordance with the previously reported results for Pseudomonas mendocina X49 without NO 2 − accumulation [ 29 ] . Pseudomonas stutzeri T13 [ 16 ] and Rhodococcus erythropolis strain Y10 [ 6 ] accumulate NO 2 − during NO 3 − removal. These differences may be due to the different RRs among different ADB, different initial NO 3 − concentrations, or different detection times for the immediate products. 3.1.4 The simultaneous removal of NH 4 + and NO 3 − and the growth of strain R-1 under aerobic and anaerobic conditions The growth curve and simultaneous ammonium and nitrate removal by strain R-1 were determined in ADM cultured for 24 h under aerobic conditions, as shown in Fig. 1 (C-1). R-1 was in the lag phase in the first 4 h, with a slow increase in the OD 600 from 0.05 to 0.07, which was accompanied by an NH 4 + RE of 15.20% and no decrease in NO 3 − concentration at 4 h. The NH 4 + concentration then decreased rapidly from 65.33 to 2.15 mg·L − 1 in the following 12 h, with the maximum RR of 9.72 mg·L − 1 ·h − 1 between 12 and 16 h, and the OD 600 of strain R-1 rapidly increasing from 0.07 to 1.66, which suggested that R-1 was in the logarithmic phase. At the same time, a rapid decline in the NO 3 − concentration was observed from 149.49 to 9.23 mg·L − 1 between 12 and 16 h, with a maximum RR of 34.89 mg·L − 1 ·h − 1 , and the NO 2 − content rapidly increased from 0.23 to 85.65 mg·L − 1 . In the final 8 h, the growth of strain R-1 reached the stationary phase, with a slow increase in OD 600 from 1.66 to 1.97. Moreover, the RE of NH 4 + and NO 3 − were 98.37% and 100%, respectively, at 24 h and the NO 2 − concentration rapidly decreased from 85.65 to 0.24 mg·L − 1 during 16–20 h. The detection of NO 2 − at the logarithmic phase of R-1 growth may be due to the accumulation of intermediate products produced during denitrification, while the small amount of NO 2 − regenerated after 20 h was mainly due to microbial autolysis when infertile conditions occurred after nutrient depletion [ 16 , 30 ] . The NH 4 + concentration decreased slowly from 72.25 to 63.70 mg·L − 1 as it was utilized by the limited biomass increase from 0.02 to 0.04 in the initial 4 h under anaerobic conditions, as shown in Fig. 1 (C-2). The NH 4 + concentration then rapidly decreased from 63.70 to 26.89 mg·L − 1 between 4 and 16 h, with an RR of 3.98 mg·L − 1 ·h − 1 from 8 to 12 h, and the OD 600 increased from 0.04 to 0.61. Meanwhile, the NO 3 − concentration sharply decreased from 179.29 to 3.95 mg·L − 1 , with a maximum RR of 31.16 mg·L − 1 ·h − 1 from 12 to 16 h, while NO 2 − began to be detected from 8 h and reached a peak value of 74.52 mg·L − 1 at 16 h, with an average rate of increase of 9.32 mg·L − 1 ·h − 1 . In the final 8 h, strain R-1 reached the stationary phase, with a slow increase in the OD 600 value from 0.61 to 0.88, and a sharp reduction in the NO 2 − concentration from 74.52 to 0 mg·L − 1 . At the same time, the concentrations of NH 4 + and NO 3 − decreased, with an RE of 100% at 24 h. The maximal NO 3 − RRs of strain R-1 were 34.89 and 31.16 mg·L − 1 ·h − 1 under aerobic and aerobic conditions, respectively, in ADM (Fig. 1C-1, C-2), which were obviously higher than that RRs in DM with NO 3 − as the sole nitrogen source, suggesting that the addition of NH 4 + increased the denitrification rate and N 2 production for strain R-1 under both aerobic and anaerobic conditions. Similar results were found in studies of Paracoccus denitrificans HY-1 and Rhodococcus erythropolis strain Y10, which reported that the presence of NH 4 + increased the NO 3 − denitrification rates [ 6 , 7 ] . NH 4 + , a superior nitrogen source for most prokaryotes, is easily absorbed by cells to participate in the synthesis of biological macromolecules [ 14 ] . Thus, bacterial growth and reproduction are directly correlated with ammonium assimilation [ 31 ] . Increasing the initial NH 4 + concentration supplied more nitrogen for cell synthesis during ammonium assimilation and promoted the production of biomass nitrogen [ 16 ] , which provided sufficient material for NO 3 − denitrification. Although oxygen is absent under anaerobic conditions, NH 4 + could also be effectively adopted as a nitrogen source through assimilation with NO 3 − as an electron acceptor, along with nitrate respiration to provide energy for assimilation [ 16 ] . Moreover, the aerobic and anaerobic denitrification efficiency enhanced by ammonium assimilation may be related to genes associated with the nitrogen metabolism pathway and transporters and other metabolism-related genes [ 32 ] . The maximal NH 4 + RR of strain R-1 was 5.92 mg·L − 1 ·h − 1 under anaerobic conditions in ADM, which was two times higher than the RR with NH 4 + as the sole nitrogen source under anaerobic conditions (Fig. 1A-2 and C-2), suggesting that the existence of NO 3 − promotes the removal of NH 4 + to biomass nitrogen. Similarly, the NH 4 + RR increased from 3.75 to 5.70 mg·L − 1 ·h − 1 when adding NO 3 − to the novel denitrification bacteria Ochrobactrum Anthropic LJ81 [ 11 ] . Because cell synthesis is a heterotrophic growth process, organic carbon can serve as an electron donor to provide electrons for NO 3 − reduction and produce energy support for ammonium assimilation. Moreover, NO 3 − must be converted into available nitrogen sources via assimilative NO 3 − reduction for cell synthesis [ 33 ] . However, such a process may occur when NH 4 + is exhausted, because strain R-1 prefers NH 4 + over NO 3 − as a nitrogen source, similar to P. stutzeri T13, Rhodococcus erythropolis Y10, Acinetobacter haemolyticus ZYL, and Pseudomonas aeruginosa P-1 [ 6 , 16 , 33 , 34 ] . 3.2 The mechanism of ammonium removal by strain R-1 based on analysis of nitrogen balance and isotope tracing Although heterotrophic nitrification coupled with aerobic denitrification is widely accepted for ADB, some ADB may not follow this pathway. The nitrogen metabolic pathways for ADB are often deduced by conducting nitrogen intermediate product measurements corresponding to the key functional genes, as well as detection of the expressed enzyme activity and nitrogen balance calculations [ 10 , 35 ] . Due to the complex biological process of nitrogen transformation and the lack of reference genomes, the metabolic mechanisms whereby ADB utilize only one of the conventional methods remain elusive [ 35 ] . Therefore, it was necessary to integrate various methods to investigate the exact pathway of ammonium transformation by strain R-1. The NH 4 + metabolic pathway of strain R-1 was analyzed using a nitrogen balance experiment. Figure 2A presents the changes in different nitrogen species with time during the NH 4 + removal process. Within 0–8 h, the inorganic nitrogen concentration decreased slowly, and the biomass-N increased from 3.33 to 8.16%. During 8–24 h, the inorganic nitrogen concentration decreased rapidly from 91.84 to 18.17%, and the biomass-N increased from 8.17 to 65.25%. During the final stage of the incubation, the NH 4 + concentration decreased to 0.32 mg·L − 1 , with no accumulation of NO 2 − or NO 3 − , but a slight accumulation of DON (Table S2 ). The variations in TN concentration in the entire incubation system showed that 77.06% NH 4 + was transformed into biomass-N, 8.10% NH 4 + was transformed into DON, and nitrogen loss was only 14.42% through gaseous or other forms (Table S2 ). These results suggest that the removal of ammonium by strain R-1 occurred mainly through the assimilation pathway and not through nitrification. This result was similar to the results reported for Paracoccus denitrificans AC-3 and HY-1, which convert approximately 80.00% of NH 4 + into intracellular nitrogen [ 7 , 36 ] , but was not consistent with the results reported for Pseudomonas balearica UFV3 and Gordonia amicalis UFV4, in which approximately 55% of the NH 4 + is lost as N 2 (g) and 45% is assimilated [ 37 ] . These results show that ammonium assimilation is the main NH 4 + -N transformation pathway in ADB. However, the nitrogen balance detection method is based on nitrogen digestion, which unavoidably conceals the potential problems of nitrogen depletion during sample treatment. Moreover, the ammonium removed as a gas (N 2 or N 2 O) through aerobic denitrification was mostly determined through deduction calculations from the detectable nitrogen, and the amount of gas transformed from ammonium has not been accurately detected in the reaction system thus far [ 6 , 16 , 22 ] . Thus, there is a “bottleneck” in the techniques used to detect various substrates during the nitrogen transformation process, and integrated techniques need to be conducted to determine the exact nitrogen metabolism pathways of ADB. The 15 NH 4 + isotope tracer method was used to verify the NH 4 + transformation pathway of strain R-1, as shown in Fig. 2B. Verification of the 15 NH 4 + -N assimilation pathway was performed as follows: 15 N-NH 4 + in AM→ 15 N-organic nitrogen in R-1 cells→ 15 N-NO 3 − obtained from the digestion of R-1 cells→ 29 N 2 or 30 N 2 by reducing 15 N-NO 3 − by strain R-1 in DM (known as biomass group 15 N-ON). The culture supernatant was also used as a substrate for denitrification to detect the presence of NO 3 − or NO 2 − (known as supernatant group 15 N-IN or 15 NO 3 − + 15 NO 2 − ). After 24 h of cultivation, the concentration of 30 N 2 in the biomass group 15 N-ON increased from 0.06 to 1.56 µ·mol·L − 1 , while the concentration of 30 N 2 in the supernatant group was unchanged. The concentration of 29 N 2 was unchanged in both groups. These results further demonstrate that 15 NH 4 + was transformed into 15 N-ON (biomass nitrogen) in strain R-1 through ammonium assimilation, but not through nitrification. 3.3 The removal mechanism of nitrate by strain R-1 3.3.1 The reduction of nitrate coupled to the oxidation of electron donors To quantify the coupling relationship between the consumption of electron donors and the decrease in NO 3 − concentration, the oxidation of acetate or lactate and the reduction of NO 3 − were detected synchronously in DM with acetate or lactate as electron donors. The nearly complete disappearance of NO 3 − was accompanied by the consumption of acetate or lactate over time (Fig. 3A1 and B1). Moreover, a linear relationship was observed between the consumption of acetate or lactate and the decrease in NO 3 − concentration, with excess acetate or lactate and limited NO 3 − (Fig. 3A2 and B2). These results clearly show that the reduction of NO 3 − by strain R-1 depends entirely on the consumption of electron donors. The dissimilatory reduction of nitrate is an important nitrogen removal process that is usually required to provide electron donors for energy production. The redox potentials of various electron donors are related to the different efficiencies of nitrate reduction and energy conservation. The stoichiometric equation of acetate oxidation related to NO 3 − reduction was expressed by the metrological Eq. 1 [ 38 ] using the cell molecular formula C 5 H 7 NO 2 proposed by Hoover and Porgess [ 39 ] . The molar ratio of oxidized acetate and NO 3 − reduced in the reaction process was calculated to be 0.902, which is slightly higher than the theoretical value of 0.819 (Eq. 1). This result indicated that acetate was sufficiently oxidized for energy generation, and nitrate was eventually completely reduced to N 2 , which was similar to Fe (II) oxidation coupled with nitrate reduction [ 40 ] . According to the chemical equation between glucose and NO 3 −[ 38 ] , we speculated that the stoichiometric equation between lactate and nitrate is the metrological Eq. 2. The experimental results showed that the lac/N (ratio of lactate to nitrate, mol/mol) molar ratio was 0.691 between lactate and NO 3 − consumed in the reaction process, which was lower than the theoretical value of 0.811. \(\text{0.819C}{\text{H}}_{\text{3}}\text{COOH}\text{ }\text{+}{\text{ }\text{NO}}_{\text{3}}^{\text{-}}\text{ }\text{+}\text{ }\text{NH}\text{4}\text{+}\text{→}\text{0.068}{\text{C}}_{\text{5}}{\text{H}}_{\text{7}}\text{N}{\text{O}}_{\text{2}}\text{ }\text{+}\text{ }\text{HC}{\text{O}}_{\text{3}}^{\text{-}}\text{ }\text{+}\text{ }\text{0.466}{\text{N}}_{\text{2}\text{ }}\text{+}\text{ }\text{0.301C}{\text{O}}_{\text{2}}\text{ }\text{+}\text{ }\text{0.902}{\text{H}}_{\text{2}}\text{O}\) (Eq. 1) $$\text{0.811}{\text{C}}_{\text{3}}{\text{H}}_{\text{6}}{\text{O}}_{\text{3}}\text{+N}{\text{O}}_{\text{3}}^{\text{-}}\text{+0.932}{\text{H}}^{\text{+}}\text{→}\text{0.203}{\text{C}}_{\text{5}}{\text{H}}_{\text{7}}{\text{O}}_{\text{2}}\text{N}\text{ }\text{+}\text{ }\text{0.399}{\text{N}}_{\text{2}}\text{ }\text{+}\text{ }\text{1.419C}{\text{O}}_{\text{2}}\text{ }\text{+}\text{ }\text{2.189}{\text{H}}_{\text{2}}\text{O}$$ (Eq. 2) Under aerobic conditions, the values of OD 600 all reached approximately 2.0, both in AM and ADM with acetate as the electron donor (Fig. 1A-1 and C-1), suggesting that bacterial growth was not significantly different between the two media. Owing to the absolute advantage of O 2 in terms of redox potential, the growth of strain R-1 depends mainly on aerobic respiration with O 2 as an electron acceptor under aerobic conditions. Although nitrate reduction also occurred, it is possible that it was not the main pathway for energy conservation to support growth. However, the OD 600 value was significantly higher in ADM (1.1) than in AM (0.5) under anaerobic conditions, with acetate as the electron donor (Fig. 1A-2 and C-2), suggesting that the nitrate reduction process was able to generate energy to support bacterial growth. Thus, strain R-1 displayed remarkable characteristics of anaerobic nitrate respiration, which can conserve energy using nitrate as a terminal electron acceptor under anaerobic conditions. NO 3 − is reduced to N 2 through denitrification by accepting reducing power derived from the oxidization of acetate or lactate with the electron transport chain. Thermodynamic calculations also indicated that the oxidation of acetate or lactate, coupled with nitrate reduction and electron transmission, exhibited great potential to generate enough energy to maintain the growth and metabolism of strain R-1 (Supplemental Material 2). 3.3.2 The pathway of reduction of nitrate confirmed by 15 N isotope tracing Isotope tracing experiments were designed to verify the NO 3 − removal mechanism under anaerobic and aerobic conditions (Fig. 4). 30 N 2 was generated with a net increase of 5.78 µmol·L − 1 in ADM with 15 NH 4 + and 15 NO 3 − added and 4.36 µmol·L − 1 in DM with 15 NO 3 − added under anaerobic conditions (Fig. 4A), which was higher than 4.98 and 3.24 µ·mol·L − 1 , respectively, observed under aerobic conditions (Fig. 4B). 29 N 2 remained at a low level in all of the abovementioned experiments, and 30 N 2 was not detected in the control experiments without isotope-labeled nitrogen sources. These results suggested that strain R-1 removed NO 3 − through denitrification under both anaerobic and aerobic conditions. Moreover, the production of 30 N 2 was higher in ADM than in DM under both anaerobic and aerobic conditions (Fig. 4), indicating that the presence of NH 4 + promoted the transformation of NO 3 − . However, 30 N 2 was not directly produced in AM with 15 NH 4 + added as the only nitrogen source, which further demonstrated that NH 4 + was removed by ammonium assimilation and not nitrification by strain R-1. These results are consistent with the physiological and biochemical results of the strain R-1 nitrogen transformation process. 3.4 The genetic basis of nitrogen transformation by strain R-1 The nitrogen transformation processes and related functional genes (or enzymes) of strain R-1 were revealed using genomic analysis. KEGG annotation indicated that multiple nitrogen metabolic pathways were encoded in the genome of strain R-1, including denitrification, ammonia assimilation, and nitroalkane oxidation (Fig. S5). The enzyme names and the corresponding serial numbers shown in Figure S5 were listed in Table S1 . In nitrogen dissimilation pathways, nitrate is reduced to nitrite by dissimilatory nitrate reductase, followed by the transformation of nitrite into ammonia by dissimilatory nitrite reductase. Hydroxylamine and formadine are converted to ammonia by hydroxylamine reductase and formamidase, respectively. Ammonia is assimilated into glutamate by glutamine synthetase (GlnA), glutamate synthase (GltB,), and glutamate dehydrogenase (GdhA) via the nitrogen assimilation pathway. In the nitrification pathway, only nitrite oxidoreductase (NxrA and NxrB) was detected, which oxidizes nitrite into nitrate, whereas ammonia monooxygenase (Amo) and hydroxylamine oxidase (Hao), which are crucial enzymes for the oxidation of ammonia to hydroxylamine and hydroxylamine to nitrite, respectively, were not detected in the genome of strain R-1. Additionally, nitroalkane monooxygenase, which catalyzes nitrite formation, was encoded in the genome of strain R-1. The intermediate NO 2 − can then be transformed into gaseous nitrogen or assimilated into R-1 cells. Genome functional annotation revealed the presence of key enzymes involved in the transformation of nitrate and nitrite into gaseous nitrogen, including nitrate reductase (Nar GHI), nitrite reductase (Nir S), nitric oxide reductase (Nor B), and nitrous oxide reductase (Nos Z). Therefore, the gene annotation results indicated that strain R-1 could assimilate ammonia and complete denitrification (Fig. 5), but not nitrification, which was consistent with the physiological and biochemical results. The existence of N-related genes may provide direct evidence of the number of N-metabolism enzymes in bacteria. The presence of amo A in the genome of Halomonas sp. strain B01 indicates that it can conduct heterotrophic nitrification at high salt concentrations [ 41 ] . Exiguobacterium mexicanum SND-01 has been proposed to possess a heterotrophic nitrification pathway via the hao gene [ 42 ] . In contrast, Amo and Hao enzyme activities were not detected in Rhodococcus erythropolis Y10, suggesting that strain Y10 transforms ammonium through ammonium assimilation, rather than nitrification [ 6 ] . Based on metagenomics analysis, the presence of GlnA, GltB, and GdhA confirmed that strain R-1 could utilize NH 4 + as a nitrogen source via ammonium assimilation for cell propagation (Fig. S5). Ammonium can be transformed into glutamate via the glutamate dehydrogenase (GDH) pathway to promote cell propagation, which may take place preferentially in ADB at high ammonium concentrations [ 12 ] . It has been speculated that the expression of the GDH-related genes gln A (glutamate-ammonia ligase) and glt B (glutamate synthase large subunit) may be pivotal for the excellent nitrogen removal performance of strain R-1, which coincides with the novel ADB Acinetobacter sp. TAC-1 [ 43 ] . Ammonium assimilation is beneficial for the growth and propagation of bacteria and is convenient for the absorption and utilization of inorganic nitrogen [ 31 ] . Periplasmic nitrate reductase (Nap), which is mainly expressed under aerobic conditions, is involved in aerobic denitrification [ 44 , 45 ] . Nevertheless, a new electron transport chain, as a hypothesis for aerobic denitrification in the presence of Nar and Nir, may transmit electrons to NO 3 − - under aerobic conditions without the restriction of the presence or absence of oxygen [ 44 , 46 ] . Therefore, the presence of NarG, NirS, NorB, and NosZ in strain R-1, based on metagenomics analysis (Fig. S5), further confirmed that strain R-1 could execute the complete denitrification pathway (NO 3 − →NO 2 − →NO→N 2 O→N 2 ) to degrade NO 3 − under both aerobic and anaerobic conditions. 3.5 The biotechnological significance of understanding the mechanism of nitrogen removal by strain R-1 In summary, the overall mechanism of the simultaneous removal of NH 4 + and NO 3 − by strain R-1 is summarized in the model shown in Fig. 6. First, ammonium assimilation provides a nitrogen source for strain R-1, which supplies a nitrogen nutrient substance for cell biosynthesis. Second, the reduction in nitrate dissimilation in strain R-1 provides energy for cell growth and metabolism. In general, ammonium removal in bioreactors is frequently attributed to heterotrophic nitrification, but the significant role of ammonium assimilation has often been neglected because of the lack of understanding of the mechanism of microbial ammonia removal [ 16 , 35 ] . Our findings indicate that ammonia assimilation is an important pathway for nitrogen removal from bioreactors. Our research showed that strain R-1 can effectively remove ammonia and nitrate under both aerobic and anaerobic conditions, which inspired us to use strain R-1 to enhance nitrogen removal from biological reactors. In the aerobic stage, strain R-1 can effectively remove ammonia mineralized from organic matter, while some nitrate and nitrite may be lost due to aerobic denitrification. In the anaerobic or hypoxic stage, strain R-1 mainly removes nitrate. By strengthening both the aerobic and anaerobic stages, nitrogen removal efficiency can be greatly improved. The results of this study will facilitate the development of culturable ADB as promising candidates for nitrogen pollution control and bioremediation in various environments. 4. Conclusions In this study, the highly efficient denitrifying bacterium P. denitrificans R-1 was investigated. Strain R-1 exhibited prominent capacity for simultaneous ammonium and nitrate removal under aerobic and anaerobic conditions. Evidence provided through combined analysis of nitrogen balance, 15 N isotope tracing and metagenomic techniques demonstrated that strain R-1 utilizes ammonium through assimilation, rather than heterotrophic nitrification, and it simultaneously removes nitrate through denitrification under aerobic and anaerobic conditions. Moreover, ammonium assimilation and nitrate denitrification effectively promoted each other. In addition, nitrate reduction by strain R-1 was shown to be a biochemical or electron transport process coupled with the oxidation of electron donors through stoichiometric and thermodynamic analyses. In conclusion, our results provide novel insights into the nitrogen removal mechanism of ADB, which may be helpful in establishing a theoretical basis for the practical application of simultaneous ammonium and nitrate removal processes in the treatment of wastewater with a high inorganic nitrogen concentration in a single system. References Chen, J., Gu, S., Hao, H. & Chen, J. Characteristics and metabolic pathway of Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic denitrification. Appl. Microbiol. Biotechnol. 100 , 9787-9794 (2016). Sun, J. et al. High-concentration nitrogen removal coupling with bioelectric power generation by a self-sustaining algal-bacterial biocathode photo-bioelectrochemical system under daily light/dark cycle. 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Simultaneous removal of organic matter and nitrogen by heterotrophic nitrification-aerobic denitrification bacteria in an air-lift multi-stage circulating integrated bioreactor. Bioresource Technol. 363 , 127888 (2022). Kong, Q. X., Wang, X. W., Jin, M., Shen, Z. Q. & Li, J. W. Development and application of a novel and effective screening method for aerobic denitrifying bacteria. FEMS Microbiol. Lett. 260 , 150-155 (2006). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementmaterial1.docx Supplement material 1 Supplementmaterial2CB.docx Supplement material 2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3890763","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268796987,"identity":"83da1e00-6896-4c15-b947-4b39a0004d22","order_by":0,"name":"Yiguo 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1\u003c/p\u003e","description":"","filename":"Supplementmaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3890763/v1/721734e6953921da4c8d58bb.docx"},{"id":50168393,"identity":"57200be5-3f68-4f87-b993-f81cbbe3aa46","added_by":"auto","created_at":"2024-01-25 15:24:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19975,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement material 2\u003c/p\u003e","description":"","filename":"Supplementmaterial2CB.docx","url":"https://assets-eu.researchsquare.com/files/rs-3890763/v1/d9c0a7b71957034d695efd8c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1 under aerobic and anaerobic conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNitrogen pollution is becoming increasingly severe in aquatic environments due to improper treatment of industrial wastewater, excessive discharge of domestic sewage, agricultural contamination, animal husbandry, and the metabolism of aquatic organisms\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Nitrogen overload leads to water eutrophication\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, destroys ecosystem function, and threatens human health\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Microbiological nitrogen removal (MNR) is an economical and sustainable approach that causes no secondary pollution\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, traditional nitrogen removal approaches involve two sequential reactions of aerobic nitrification coupled with anaerobic denitrification, which must be conducted in two separate pieces of equipment with independent conditions. This results in deficient performance when dealing with pollution with high concentrations of inorganic and organic nitrogen\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Thus, the high treatment costs have limited the development of MNR technology.\u003c/p\u003e \u003cp\u003eFortunately, with the continuous development of MNR technology, diverse microorganisms have been isolated and identified for nitrogen removal from both engineered and natural ecosystems. Aerobic denitrifying bacteria (ADB) show higher growth rates and nitrogen removal efficiencies than autotrophic, traditional denitrifying bacteria\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and can simultaneously utilize various organic substrates\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e; therefore, aerobic denitrification technology overcomes the disadvantages of traditional nitrogen removal methods and exhibits prominent application value in the engineering process of wastewater treatment\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo improve nitrogen removal efficiency, microbial strains that can simultaneously remove ammonium and nitrate have been isolated and identified from various environments. Most of these strains are ADB. The nitrogen removal mechanisms of these strains are often defined as heterotrophic nitrification and aerobic denitrification\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e; however, the evidence for this definition is insufficient. The nitrogen metabolic processes and mechanisms of ADB have not yet been clarified\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, especially in regard to ammonium removal. Although ammonium is an important and preferable nitrogen source for the growth of most prokaryotes\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, its removal is generally attributed to heterotrophic nitrification, and little attention has been paid to the role of microbial ammonium assimilation\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Cell growth and biomass accumulation are directly correlated with ammonium assimilation, which significantly affects nitrogen removal efficiency during aerobic denitrification\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Ammonium assimilation is an important driving force of aerobic denitrification\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Based on the current research in this field, the mechanism responsible for the simultaneous removal of ammonium and nitrate by ADB remains unclear. Thus, the nitrogen metabolic mechanism of ADB warrants exploration as it will be helpful for improving the nitrogen removal efficiency and application of ADB in wastewater treatment.\u003c/p\u003e \u003cp\u003eIn a previous study, we reported the genome sequence of \u003cem\u003eParacoccus denitrificans\u003c/em\u003e R-1 isolated from the activated sludge of a sewage treatment plant in Taiwan\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. This strain was show to be an efficient ADB. In the present study, we investigated the performance and metabolic mechanisms of nitrogen removal by \u003cem\u003eParacoccus denitrificans\u003c/em\u003e R-1. The specific objectives were to: (1) examine the nitrogen removal performance of strain R-1 using growth characteristics and various forms of inorganic nitrogen under aerobic and anaerobic conditions and (2) clarify the metabolic pathways involved in the simultaneous removal of ammonium and nitrate by strain R-1 based on the production of metabolic intermediates, nitrogen balance analysis, stoichiometry between the carbon and nitrogen sources, thermodynamic analysis, \u003csup\u003e15\u003c/sup\u003eN-metabolic flux analysis, and metagenome analysis. A series of combined methodologies was used to elucidate the bacteria-mediated mechanism of simultaneous ammonium and nitrate removal, which will provide a deeper understanding of the nitrogen removal mechanism in ADB and promote its application in wastewater treatment.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Strain, medium, and cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eParacoccus denitrificans\u003c/em\u003e strain R-1 was isolated from the activated sludge originating from the Xinfeng Sewage Treatment Plant in Taiwan\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The denitrification medium (DM) used in this study comprised1.2 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KNO\u003csub\u003e3\u003c/sub\u003e, 7.9 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 4.7 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CH\u003csub\u003e3\u003c/sub\u003eCOONa\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 2 mL\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e trace element solution, as reported previously\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The above medium supplemented with only 0.3 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eCl (AM) or 0.3 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eCl\u0026thinsp;+\u0026thinsp;1.2 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KNO\u003csub\u003e3\u003c/sub\u003e (ADM) as replacement for KNO\u003csub\u003e3\u003c/sub\u003e in DM was used to test the capability of nitrogen removal by strain R-1.\u003c/p\u003e \u003cp\u003eThe R-1 bacterial solution was added to the DM and cultured for 12 h until the logarithmic phase of bacterial growth was attained. The culture was centrifuged, the supernatant was removed, and the bacterial precipitate was washed three times with sterile water. Then, 1 mL of the DM solution was added to form a bacterial suspension, which was used as the seed liquid. The equation OD600 (seed liquid) \u0026times; V1\u0026thinsp;=\u0026thinsp;100 mL * 0.05 was used to calculate the inoculation volume, V\u003csub\u003e1\u003c/sub\u003e, which ensured that the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of strain R-1 was consistently 0.05 in the culture media for the experimental groups at the initial cultivation stage.\u003c/p\u003e \u003cp\u003eThe prepared seed culture was added to a 250 mL conical flask with 100 mL sterile medium and incubated for 24 h at 30\u0026deg;C with a rotational speed of 150 rpm for aerobic cultivation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-A). The conical flasks were replaced with anaerobic bottles to ensure an anaerobic culture system (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-B). Anaerobic culture technologies were based on those reported in a previous study\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. High-purity helium (He) was subjected to sterile filtration before use. The prepared medium was split into 100 mL anaerobic bottles sealed with septa and flushed with sterilized high-purity He for 10 min. After inoculation with seed liquid, the bottles were cultured at 30\u0026deg;C. The cultures were sampled at different time points (2 and 4 h or 0, 6, 12, 18, and 24 h). The biomass was determined by measuring the OD\u003csub\u003e600\u003c/sub\u003e, and the concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were determined using a rapid spectrophotometry method\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Effect of different electron donors on strain R-1 growth and nitrogen removal under aerobic and anaerobic conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the coupling of the growth and nitrogen transformation of strain R-1 with the oxidation of electron donors, R-1 cells were cultured in AM, DM, and ADM containing different electron donors (formate, acetate, succinate, pyruvate, lactate, and glucose) with an initial C/N ratio of 9.65. The concentrations of electron donors were determined by ion chromatography, as described in a previous report \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The stoichiometric equation for denitrification with the selected carbon sources was deduced by measuring the content of electron donors and nitrates as acceptors in the determination system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Nitrogen removal processes of R-1 under aerobic and anaerobic conditions\u003c/h2\u003e \u003cp\u003eThe nitrogen removal kinetics of R-1 were explored in AM, DM, and ADM using sodium acetate as an electron donor under aerobic and anaerobic conditions. The nitrogen removal efficiency (RE) and rate (RR) were calculated according to the following formulae: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{R}\\text{E} \\left(\\text{%}\\right)=\\frac{\\left(\\text{A}-\\text{B}\\right)}{\\text{A}}\\times 100\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{R}\\text{R} \\left(\\text{m}\\text{g}\u0026middot;\\text{L}\\text{-}\\text{1}\u0026middot;\\text{h}\\text{-}\\text{1}\\right)=\\frac{\\left(\\text{C}-\\text{D}\\right)}{\\text{T}},\\)\u003c/span\u003e\u003c/span\u003ewhere A is the initial ammonium or nitrate concentration, B is the observed ammonium or nitrate concentration, C and D are the observed ammonium or nitrate concentrations between adjacent sampling times, and T is the time phase between adjacent samplings. All analyses were performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Nitrogen balance analysis of ammonium removal\u003c/h2\u003e \u003cp\u003eNitrogen balance analysis was used to identify the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e removal pathway. The seed liquid of strain R-1 was inoculated into 250 mL triangular flasks containing 100 mL of AM and grown for 28 h at 30\u0026deg;C with a shaking speed of 150 rpm. Bacterial samples were collected every 4 h to determine the biomass concentration, OD\u003csub\u003e600\u003c/sub\u003e, and total nitrogen (TN) concentration of the culture. The concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, and TN\u0026acute; in the liquid supernatant were determined after centrifugation (8,000 \u0026times; g, 10 min), and the dissolved organic nitrogen (DON) concentration was calculated as the TN concentration minus the sum of the inorganic nitrogen concentrations. Biomass N was calculated as TN minus TN\u0026acute; concentrations \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Nitrogen loss (N-loss) was calculated as the initial TN concentration minus the final TN concentration after cultivation in non-centrifuged medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Ammonium removal based on isotope tracing analysis\u003c/h2\u003e \u003cp\u003eThe seed liquid of strain R-1 was inoculated into 250 mL triangular flasks containing 100 mL of AM, with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e replaced with the stable isotope \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. After 24 h of culture, R-1 cells and supernatants were collected by centrifugation. The R-1 cells were washed three times with ultrapure water and transferred to a clean colorimetric tube. After the addition of an alkaline potassium persulfate solution, the cells were digested at 121℃ for 30 min. If \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N assimilation occurred during AM cultivation, the organic nitrogen in the R-1 cells was labeled with \u003csup\u003e15\u003c/sup\u003eN (\u003csup\u003e15\u003c/sup\u003eN-ON) and released as \u003csup\u003e15\u003c/sup\u003eN-labeled inorganic nitrogen after digestion. The digested solutions were adjusted to pH 7 and then transferred to a 60 mL serum bottle, which was then used as the nitrogen source in DM and inoculated with R-1 seed liquid for 24 h of cultivation (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Meanwhile, the AM supernatants obtained after centrifugation were also used as a nitrogen source in DM. They were directly added to the serum bottle, and then inoculated with the seed liquid of strain R-1. After 24 h of cultivation, they were referred to as \u003csup\u003e15\u003c/sup\u003eN-IN (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Subsequently, a membrane injection mass spectrometer (MIMS) was used to determine the generation of \u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e in the culture medium \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The variation in \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e concentration after cultivation in these two groups of experiments was used to determine whether NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N was assimilated into the organic nitrogen of R-1 cells or nitrified to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the supernatants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Analysis of nitrate and ammonium removal mechanism based on isotope tracing\u003c/h2\u003e \u003cp\u003e \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (200 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (200 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), or both were added into the AM, DM, and ADM, respectively, as substitute nitrogen sources under aerobic and anaerobic conditions (Fig. S3). Sterilized media were injected into 60 mL serum bottles with no headspace and aerated with high-purity He for 10 min. The seed liquid of R-1 was inoculated into serum bottles sealed with butyl rubber stoppers and cultured at 30\u0026deg;C. The culture systems used for the aerobic experiments comprised 30 mL of culture medium and 30 mL of headspace to ensure dissolved oxygen in the media. The experimental settings were the same as those used for the anaerobic experiments described above. Blank control experiments were included to test the effects of the medium in the absence of \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. At 0 and 8 h, 2 mL of 50% ZnCl\u003csub\u003e2\u003c/sub\u003e solution was injected using a syringe to inactivate the bacterial cells in the serum bottles. The serum bottles were then transferred to a dark environment for 24 h of precipitation. A MIMS was used to determine the contents of \u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e produced in the culture systems, as previously described \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Genome sequencing and gene annotations to nitrogen transformation pathways\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from strain R-1 using a HiPure soil DNA kit (Magen, Guangzhou China). DNA was fragmented into 300\u0026ndash;350 bp segments to construct a paired-end library. The end repair of sequences (including phosphorylation of the 5\u0026prime; end and the addition of A to the 3\u0026prime; end) was carried out using End Prep Enzyme Mix(NGS Fast DNA Library Prep Set for Illumina, Baiolaibo, Beijing China), and sequencing adapters were added to both ends. Magnetic beads were used to purify the fragments, and 341F/805R primers were used to amplify the V3-V4 variable region of the \u003cem\u003e16S rRNA\u003c/em\u003e gene \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. The NovaSeq PE150 platform (Illumina, San Diego, CA, USA) was used to conduct paired-ended sequencing (Azenta Biotech, Suzhou, China) after mixing the DNA libraries labeled with different indices. Raw data processing was performed as described in detail in a previous report \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenomic DNA (5\u0026thinsp;~\u0026thinsp;10 \u0026micro;g) was used to construct a PacBio sequencing library. An Agilent 2100 Biological Analyzer (Agilent Technologies, Palo Alto, CA, USA) was used to determine the library quality and a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA) was used to determine the concentration of the library. Sequencing was performed using a PacBio Sequel instrument (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) combined the SMRTbell library with the sequencing primers and enzymes. Sequencing data were analyzed by Azenta Biotech. The raw genome reads were deposited in GenBank under accession numbers CP087986, CP087987, and CP087988 \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The key genes encoding important enzymes closely related to bacterial nitrogen metabolism were identified by annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The simultaneous removal of ammonium and nitrate by strain R-1 under aerobic and anaerobic conditions\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Effective electron donors for the removal of ammonium and nitrate\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. denitrificans\u003c/em\u003e R-1 strain was cultured in AM, DM, and ADM supplemented with six different carbohydrates (formate, acetate, pyruvate, succinate, lactate, and glucose) under anaerobic conditions (Fig. S4). The results showed that strain R-1 could use the selected carbohydrates to remove NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N from AM with an RE of 17.73\u0026ndash;52.42%. RE values greater than 50% were achieved with formate, acetate, and glucose as the carbon sources, and the growth curves of R-1 in AM were all linear with OD\u003csub\u003e600\u003c/sub\u003e values of 0.28\u0026ndash;0.62 (Fig. S4A-1\u0026ndash;A-6). Comparatively, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was almost completely reduced (with an RE of almost 100%) within 12\u0026thinsp;~\u0026thinsp;18 h in DM with acetate, pyruvate, succinate, lactate, and glucose, but not sodium formate, and the OD\u003csub\u003e600\u003c/sub\u003e values of 0.62\u0026ndash;1.67 were higher in DM than that in AM under the same conditions (Fig. S4B-1\u0026ndash;B-6). The RE values of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were 80\u0026ndash;99% and almost 100%, respectively, within 12\u0026thinsp;~\u0026thinsp;18 h in ADM with acetate, pyruvate, succinate, lactate, and glucose, but not sodium formate, and the OD\u003csub\u003e600\u003c/sub\u003e values of 1.02\u0026ndash;1.68 were higher than those for AM and DM under the same conditions (Fig.S4 C-1\u0026ndash;C-6). Moreover, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal curves were consistent with the changing trend with culture time, suggesting that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were simultaneously removed from ADM. Therefore, acetate, pyruvate, succinate, lactate, and glucose could be used as carbon sources to support the growth and nitrogen metabolism of strain R-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e removal performance and growth of strain R-1 under aerobic and anaerobic conditions\u003c/h2\u003e \u003cp\u003eThe removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and the growth of strain R-1 were analyzed in AM for 24 h under aerobic conditions, and the results are shown in Fig.\u0026nbsp;1 (A-1). The concentration of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in AM decreased from 70.56 to 64.25 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during 0\u0026ndash;4 h, with an RR of 1.79 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e- N. The concentration of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in AM decreased from 64.25 to 0 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the average RR of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N by R-1 was 5.35 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during 4\u0026ndash;16 h. The highest RR was 9.94 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 16 h, which was close to the RR of \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e strain Y10 (9.69 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e and higher than the RR of \u003cem\u003eVibrio\u003c/em\u003e sp. Y1-5 (2.65 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eRhodococcus\u003c/em\u003e sp. CPZ24 (3.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003ePseudomonas mendocina\u003c/em\u003e TJPU04 (4.69 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. These results indicated that strain R-1 has a strong ammonium conversion ability. The growth curve with the OD\u003csub\u003e600\u003c/sub\u003e values was consistent with a decrease in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, the removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e by strain R-1 and its growth in AM were obviously different under anaerobic conditions (Fig.\u0026nbsp;1A-2). The RE of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reached 75.09% at 24 h, with the highest RR of 2.91 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 20 h. This was lower than the RR under aerobic conditions. This may be due to the lack of available electron acceptors in AM under anaerobic conditions\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Energy derived only from the oxidation of organic carbon may be inefficient for ammonium assimilation during heterotrophic growth or respiration. A similar result was reported in a previous study, in which the removed NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was mainly transformed into biological nitrogen through ammonium assimilation, with cell synthesis rates greatly inhibited under anaerobic conditions\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. These results are not consistent with those of \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e T13, which cannot utilize NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e as the sole nitrogen source to grow under anaerobic conditions\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, the accumulation of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was not observed in the AM of strain R-1 under aerobic or anaerobic conditions (Fig.\u0026nbsp;1A-1 and A-2), suggesting that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was used as a nitrogen source for cell synthesis in the AM of strain R-1. This result is in accordance with those of previous studies of \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e T13\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eOchrobactrum anthropic\u003c/em\u003e LJ81\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e Y10\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. These results suggest that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was possibly removed through the assimilation pathway, but not through heterotrophic nitrification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e removal performance and growth of strain R-1 under aerobic and anaerobic conditions\u003c/h2\u003e \u003cp\u003eThe kinetics of nitrate removal and growth of strain R-1 were analyzed in DM for 24 h under aerobic conditions, as shown in Fig.\u0026nbsp;1 (B-1). The OD\u003csub\u003e600\u003c/sub\u003e value of strain R-1 increased slowly during 0\u0026ndash;4 h, and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR of strain R-1 reached 6.17 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 4 h. The OD\u003csub\u003e600\u003c/sub\u003e value in DM then increased exponentially during 4\u0026ndash;16 h, and the highest NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR was 18.05 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 12 h. During 16\u0026ndash;20 h, the OD\u003csub\u003e600\u003c/sub\u003e value remained stable, and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR decreased from 11.30 to 0.74 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Finally, the OD\u003csub\u003e600\u003c/sub\u003e value decreased in the period 20\u0026ndash;24 h, and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR of strain R-1 decreased to the lowest value of 0.74 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RE reached 95.04% at 24 h.\u003c/p\u003e \u003cp\u003eThe nitrate removal performance and bacterial growth in DM under anaerobic conditions are shown in Fig.\u0026nbsp;1 (B-2). The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration decreased from 171.23 to 132.45 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR reached 5.59 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 12 h. The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration decreased from 132.45 to 12.45 mg \u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during 12\u0026ndash;20 h and the highest NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR was 19.76 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 20 h. The RE of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reached 94.90% at 24 h under anaerobic conditions. The OD\u003csub\u003e600\u003c/sub\u003e was 0.97 in the stable period (20\u0026ndash;24 h), which was lower than the OD\u003csub\u003e600\u003c/sub\u003e value observed under aerobic conditions (16 h, OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.82). Similarly, \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e T13 and \u003cem\u003eParacoccus denitrificans\u003c/em\u003e HY-1 have been reported to assimilate less nitrate into their biomass under anaerobic conditions than under aerobic conditions\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. These results showed that strain R-1 could adopt NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as both a nitrogen source and an electron acceptor when it was supplied as the sole source under aerobic and anaerobic conditions. The maximal NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RRs of strain R-1 were 18.05 and 19.76 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under aerobic and aerobic conditions, respectively, in DM, which were higher than those of \u003cem\u003eBacillus cereus\u003c/em\u003e GS-5 (2.70 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eParacoccus denitrificans\u003c/em\u003e HY1 (14.56 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e strain Y10 (5.43 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, which suggested that strain R-1 has a strong ability for NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e conversion. During the whole NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e removal process, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were almost undetected, which was in accordance with the previously reported results for \u003cem\u003ePseudomonas mendocina\u003c/em\u003e X49 without NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e T13\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e strain Y10\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e accumulate NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e during NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e removal. These differences may be due to the different RRs among different ADB, different initial NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations, or different detection times for the immediate products.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.1.4 The simultaneous removal of NH\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e \u003c/sub\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand the growth of strain R-1 under aerobic and anaerobic conditions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe growth curve and simultaneous ammonium and nitrate removal by strain R-1 were determined in ADM cultured for 24 h under aerobic conditions, as shown in Fig.\u0026nbsp;1 (C-1). R-1 was in the lag phase in the first 4 h, with a slow increase in the OD\u003csub\u003e600\u003c/sub\u003e from 0.05 to 0.07, which was accompanied by an NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e RE of 15.20% and no decrease in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration at 4 h. The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration then decreased rapidly from 65.33 to 2.15 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the following 12 h, with the maximum RR of 9.72 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between 12 and 16 h, and the OD\u003csub\u003e600\u003c/sub\u003e of strain R-1 rapidly increasing from 0.07 to 1.66, which suggested that R-1 was in the logarithmic phase. At the same time, a rapid decline in the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration was observed from 149.49 to 9.23 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between 12 and 16 h, with a maximum RR of 34.89 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content rapidly increased from 0.23 to 85.65 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In the final 8 h, the growth of strain R-1 reached the stationary phase, with a slow increase in OD\u003csub\u003e600\u003c/sub\u003e from 1.66 to 1.97. Moreover, the RE of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were 98.37% and 100%, respectively, at 24 h and the NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration rapidly decreased from 85.65 to 0.24 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during 16\u0026ndash;20 h. The detection of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e at the logarithmic phase of R-1 growth may be due to the accumulation of intermediate products produced during denitrification, while the small amount of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e regenerated after 20 h was mainly due to microbial autolysis when infertile conditions occurred after nutrient depletion \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration decreased slowly from 72.25 to 63.70 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as it was utilized by the limited biomass increase from 0.02 to 0.04 in the initial 4 h under anaerobic conditions, as shown in Fig.\u0026nbsp;1 (C-2). The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration then rapidly decreased from 63.70 to 26.89 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between 4 and 16 h, with an RR of 3.98 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from 8 to 12 h, and the OD\u003csub\u003e600\u003c/sub\u003e increased from 0.04 to 0.61. Meanwhile, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration sharply decreased from 179.29 to 3.95 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a maximum RR of 31.16 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from 12 to 16 h, while NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e began to be detected from 8 h and reached a peak value of 74.52 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 16 h, with an average rate of increase of 9.32 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In the final 8 h, strain R-1 reached the stationary phase, with a slow increase in the OD\u003csub\u003e600\u003c/sub\u003e value from 0.61 to 0.88, and a sharp reduction in the NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration from 74.52 to 0 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At the same time, the concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e decreased, with an RE of 100% at 24 h.\u003c/p\u003e \u003cp\u003eThe maximal NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RRs of strain R-1 were 34.89 and 31.16 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under aerobic and aerobic conditions, respectively, in ADM (Fig.\u0026nbsp;1C-1, C-2), which were obviously higher than that RRs in DM with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as the sole nitrogen source, suggesting that the addition of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e increased the denitrification rate and N\u003csub\u003e2\u003c/sub\u003e production for strain R-1 under both aerobic and anaerobic conditions. Similar results were found in studies of \u003cem\u003eParacoccus denitrificans\u003c/em\u003e HY-1 and \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e strain Y10, which reported that the presence of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e increased the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e denitrification rates\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, a superior nitrogen source for most prokaryotes, is easily absorbed by cells to participate in the synthesis of biological macromolecules \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Thus, bacterial growth and reproduction are directly correlated with ammonium assimilation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Increasing the initial NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration supplied more nitrogen for cell synthesis during ammonium assimilation and promoted the production of biomass nitrogen\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, which provided sufficient material for NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e denitrification. Although oxygen is absent under anaerobic conditions, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e could also be effectively adopted as a nitrogen source through assimilation with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as an electron acceptor, along with nitrate respiration to provide energy for assimilation\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Moreover, the aerobic and anaerobic denitrification efficiency enhanced by ammonium assimilation may be related to genes associated with the nitrogen metabolism pathway and transporters and other metabolism-related genes\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe maximal NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e RR of strain R-1 was 5.92 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under anaerobic conditions in ADM, which was two times higher than the RR with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e as the sole nitrogen source under anaerobic conditions (Fig.\u0026nbsp;1A-2 and C-2), suggesting that the existence of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e promotes the removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e to biomass nitrogen. Similarly, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e RR increased from 3.75 to 5.70 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e when adding NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to the novel denitrification bacteria \u003cem\u003eOchrobactrum Anthropic\u003c/em\u003e LJ81\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Because cell synthesis is a heterotrophic growth process, organic carbon can serve as an electron donor to provide electrons for NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduction and produce energy support for ammonium assimilation. Moreover, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e must be converted into available nitrogen sources via assimilative NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduction for cell synthesis\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, such a process may occur when NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is exhausted, because strain R-1 prefers NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e over NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as a nitrogen source, similar to \u003cem\u003eP. stutzeri\u003c/em\u003e T13, \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e Y10, \u003cem\u003eAcinetobacter haemolyticus\u003c/em\u003e ZYL, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e P-1\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 The mechanism of ammonium removal by strain R-1 based on analysis of nitrogen balance and isotope tracing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAlthough heterotrophic nitrification coupled with aerobic denitrification is widely accepted for ADB, some ADB may not follow this pathway. The nitrogen metabolic pathways for ADB are often deduced by conducting nitrogen intermediate product measurements corresponding to the key functional genes, as well as detection of the expressed enzyme activity and nitrogen balance calculations\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Due to the complex biological process of nitrogen transformation and the lack of reference genomes, the metabolic mechanisms whereby ADB utilize only one of the conventional methods remain elusive\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Therefore, it was necessary to integrate various methods to investigate the exact pathway of ammonium transformation by strain R-1.\u003c/p\u003e \u003cp\u003eThe NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e metabolic pathway of strain R-1 was analyzed using a nitrogen balance experiment. Figure\u0026nbsp;2A presents the changes in different nitrogen species with time during the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e removal process. Within 0\u0026ndash;8 h, the inorganic nitrogen concentration decreased slowly, and the biomass-N increased from 3.33 to 8.16%. During 8\u0026ndash;24 h, the inorganic nitrogen concentration decreased rapidly from 91.84 to 18.17%, and the biomass-N increased from 8.17 to 65.25%. During the final stage of the incubation, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration decreased to 0.32 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with no accumulation of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e or NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, but a slight accumulation of DON (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The variations in TN concentration in the entire incubation system showed that 77.06% NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was transformed into biomass-N, 8.10% NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was transformed into DON, and nitrogen loss was only 14.42% through gaseous or other forms (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These results suggest that the removal of ammonium by strain R-1 occurred mainly through the assimilation pathway and not through nitrification. This result was similar to the results reported for \u003cem\u003eParacoccus denitrificans\u003c/em\u003e AC-3 and HY-1, which convert approximately 80.00% of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e into intracellular nitrogen\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, but was not consistent with the results reported for \u003cem\u003ePseudomonas balearica\u003c/em\u003e UFV3 and \u003cem\u003eGordonia amicalis\u003c/em\u003e UFV4, in which approximately 55% of the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is lost as N\u003csub\u003e2\u003c/sub\u003e (g) and 45% is assimilated\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. These results show that ammonium assimilation is the main NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N transformation pathway in ADB.\u003c/p\u003e \u003cp\u003eHowever, the nitrogen balance detection method is based on nitrogen digestion, which unavoidably conceals the potential problems of nitrogen depletion during sample treatment. Moreover, the ammonium removed as a gas (N\u003csub\u003e2\u003c/sub\u003e or N\u003csub\u003e2\u003c/sub\u003eO) through aerobic denitrification was mostly determined through deduction calculations from the detectable nitrogen, and the amount of gas transformed from ammonium has not been accurately detected in the reaction system thus far \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Thus, there is a \u0026ldquo;bottleneck\u0026rdquo; in the techniques used to detect various substrates during the nitrogen transformation process, and integrated techniques need to be conducted to determine the exact nitrogen metabolism pathways of ADB.\u003c/p\u003e \u003cp\u003eThe \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e isotope tracer method was used to verify the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e transformation pathway of strain R-1, as shown in Fig.\u0026nbsp;2B. Verification of the \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N assimilation pathway was performed as follows: \u003csup\u003e15\u003c/sup\u003eN-NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in AM\u0026rarr;\u003csup\u003e15\u003c/sup\u003eN-organic nitrogen in R-1 cells\u0026rarr;\u003csup\u003e15\u003c/sup\u003eN-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e obtained from the digestion of R-1 cells\u0026rarr;\u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e or \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e by reducing \u003csup\u003e15\u003c/sup\u003eN-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e by strain R-1 in DM (known as biomass group \u003csup\u003e15\u003c/sup\u003eN-ON). The culture supernatant was also used as a substrate for denitrification to detect the presence of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e or NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (known as supernatant group \u003csup\u003e15\u003c/sup\u003eN-IN or \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e + \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). After 24 h of cultivation, the concentration of \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e in the biomass group \u003csup\u003e15\u003c/sup\u003eN-ON increased from 0.06 to 1.56 \u0026micro;\u0026middot;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the concentration of \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e in the supernatant group was unchanged. The concentration of \u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e was unchanged in both groups. These results further demonstrate that \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was transformed into \u003csup\u003e15\u003c/sup\u003eN-ON (biomass nitrogen) in strain R-1 through ammonium assimilation, but not through nitrification.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The removal mechanism of nitrate by strain R-1\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 The reduction of nitrate coupled to the oxidation of electron donors\u003c/h2\u003e \u003cp\u003eTo quantify the coupling relationship between the consumption of electron donors and the decrease in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration, the oxidation of acetate or lactate and the reduction of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were detected synchronously in DM with acetate or lactate as electron donors. The nearly complete disappearance of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was accompanied by the consumption of acetate or lactate over time (Fig.\u0026nbsp;3A1 and B1). Moreover, a linear relationship was observed between the consumption of acetate or lactate and the decrease in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration, with excess acetate or lactate and limited NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;3A2 and B2). These results clearly show that the reduction of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e by strain R-1 depends entirely on the consumption of electron donors.\u003c/p\u003e \u003cp\u003eThe dissimilatory reduction of nitrate is an important nitrogen removal process that is usually required to provide electron donors for energy production. The redox potentials of various electron donors are related to the different efficiencies of nitrate reduction and energy conservation. The stoichiometric equation of acetate oxidation related to NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduction was expressed by the metrological Eq.\u0026nbsp;1\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e using the cell molecular formula C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e proposed by Hoover and Porgess\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. The molar ratio of oxidized acetate and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduced in the reaction process was calculated to be 0.902, which is slightly higher than the theoretical value of 0.819 (Eq.\u0026nbsp;1). This result indicated that acetate was sufficiently oxidized for energy generation, and nitrate was eventually completely reduced to N\u003csub\u003e2\u003c/sub\u003e, which was similar to Fe (II) oxidation coupled with nitrate reduction\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. According to the chemical equation between glucose and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, we speculated that the stoichiometric equation between lactate and nitrate is the metrological Eq.\u0026nbsp;2. The experimental results showed that the lac/N (ratio of lactate to nitrate, mol/mol) molar ratio was 0.691 between lactate and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e consumed in the reaction process, which was lower than the theoretical value of 0.811.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{0.819C}{\\text{H}}_{\\text{3}}\\text{COOH}\\text{ }\\text{+}{\\text{ }\\text{NO}}_{\\text{3}}^{\\text{-}}\\text{ }\\text{+}\\text{ }\\text{NH}\\text{4}\\text{+}\\text{\u0026rarr;}\\text{0.068}{\\text{C}}_{\\text{5}}{\\text{H}}_{\\text{7}}\\text{N}{\\text{O}}_{\\text{2}}\\text{ }\\text{+}\\text{ }\\text{HC}{\\text{O}}_{\\text{3}}^{\\text{-}}\\text{ }\\text{+}\\text{ }\\text{0.466}{\\text{N}}_{\\text{2}\\text{ }}\\text{+}\\text{ }\\text{0.301C}{\\text{O}}_{\\text{2}}\\text{ }\\text{+}\\text{ }\\text{0.902}{\\text{H}}_{\\text{2}}\\text{O}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;1)\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{0.811}{\\text{C}}_{\\text{3}}{\\text{H}}_{\\text{6}}{\\text{O}}_{\\text{3}}\\text{+N}{\\text{O}}_{\\text{3}}^{\\text{-}}\\text{+0.932}{\\text{H}}^{\\text{+}}\\text{\u0026rarr;}\\text{0.203}{\\text{C}}_{\\text{5}}{\\text{H}}_{\\text{7}}{\\text{O}}_{\\text{2}}\\text{N}\\text{ }\\text{+}\\text{ }\\text{0.399}{\\text{N}}_{\\text{2}}\\text{ }\\text{+}\\text{ }\\text{1.419C}{\\text{O}}_{\\text{2}}\\text{ }\\text{+}\\text{ }\\text{2.189}{\\text{H}}_{\\text{2}}\\text{O}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e(Eq.\u0026nbsp;2)\u003c/p\u003e \u003cp\u003eUnder aerobic conditions, the values of OD\u003csub\u003e600\u003c/sub\u003e all reached approximately 2.0, both in AM and ADM with acetate as the electron donor (Fig.\u0026nbsp;1A-1 and C-1), suggesting that bacterial growth was not significantly different between the two media. Owing to the absolute advantage of O\u003csub\u003e2\u003c/sub\u003e in terms of redox potential, the growth of strain R-1 depends mainly on aerobic respiration with O\u003csub\u003e2\u003c/sub\u003e as an electron acceptor under aerobic conditions. Although nitrate reduction also occurred, it is possible that it was not the main pathway for energy conservation to support growth. However, the OD\u003csub\u003e600\u003c/sub\u003e value was significantly higher in ADM (1.1) than in AM (0.5) under anaerobic conditions, with acetate as the electron donor (Fig.\u0026nbsp;1A-2 and C-2), suggesting that the nitrate reduction process was able to generate energy to support bacterial growth.\u003c/p\u003e \u003cp\u003eThus, strain R-1 displayed remarkable characteristics of anaerobic nitrate respiration, which can conserve energy using nitrate as a terminal electron acceptor under anaerobic conditions. NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e is reduced to N\u003csub\u003e2\u003c/sub\u003e through denitrification by accepting reducing power derived from the oxidization of acetate or lactate with the electron transport chain. Thermodynamic calculations also indicated that the oxidation of acetate or lactate, coupled with nitrate reduction and electron transmission, exhibited great potential to generate enough energy to maintain the growth and metabolism of strain R-1 (Supplemental Material 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 The pathway of reduction of nitrate confirmed by \u003csup\u003e15\u003c/sup\u003eN isotope tracing\u003c/h2\u003e \u003cp\u003eIsotope tracing experiments were designed to verify the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e removal mechanism under anaerobic and aerobic conditions (Fig.\u0026nbsp;4). \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e was generated with a net increase of 5.78 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in ADM with \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e added and 4.36 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DM with \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e added under anaerobic conditions (Fig.\u0026nbsp;4A), which was higher than 4.98 and 3.24 \u0026micro;\u0026middot;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, observed under aerobic conditions (Fig.\u0026nbsp;4B). \u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e remained at a low level in all of the abovementioned experiments, and \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e was not detected in the control experiments without isotope-labeled nitrogen sources. These results suggested that strain R-1 removed NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e through denitrification under both anaerobic and aerobic conditions. Moreover, the production of \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e was higher in ADM than in DM under both anaerobic and aerobic conditions (Fig.\u0026nbsp;4), indicating that the presence of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e promoted the transformation of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. However, \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e was not directly produced in AM with \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e added as the only nitrogen source, which further demonstrated that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was removed by ammonium assimilation and not nitrification by strain R-1. These results are consistent with the physiological and biochemical results of the strain R-1 nitrogen transformation process.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The genetic basis of nitrogen transformation by strain R-1\u003c/h2\u003e \u003cp\u003eThe nitrogen transformation processes and related functional genes (or enzymes) of strain R-1 were revealed using genomic analysis. KEGG annotation indicated that multiple nitrogen metabolic pathways were encoded in the genome of strain R-1, including denitrification, ammonia assimilation, and nitroalkane oxidation (Fig. S5). The enzyme names and the corresponding serial numbers shown in Figure S5 were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. In nitrogen dissimilation pathways, nitrate is reduced to nitrite by dissimilatory nitrate reductase, followed by the transformation of nitrite into ammonia by dissimilatory nitrite reductase. Hydroxylamine and formadine are converted to ammonia by hydroxylamine reductase and formamidase, respectively. Ammonia is assimilated into glutamate by glutamine synthetase (GlnA), glutamate synthase (GltB,), and glutamate dehydrogenase (GdhA) via the nitrogen assimilation pathway. In the nitrification pathway, only nitrite oxidoreductase (NxrA and NxrB) was detected, which oxidizes nitrite into nitrate, whereas ammonia monooxygenase (Amo) and hydroxylamine oxidase (Hao), which are crucial enzymes for the oxidation of ammonia to hydroxylamine and hydroxylamine to nitrite, respectively, were not detected in the genome of strain R-1. Additionally, nitroalkane monooxygenase, which catalyzes nitrite formation, was encoded in the genome of strain R-1. The intermediate NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e can then be transformed into gaseous nitrogen or assimilated into R-1 cells. Genome functional annotation revealed the presence of key enzymes involved in the transformation of nitrate and nitrite into gaseous nitrogen, including nitrate reductase (Nar GHI), nitrite reductase (Nir S), nitric oxide reductase (Nor B), and nitrous oxide reductase (Nos Z). Therefore, the gene annotation results indicated that strain R-1 could assimilate ammonia and complete denitrification (Fig.\u0026nbsp;5), but not nitrification, which was consistent with the physiological and biochemical results.\u003c/p\u003e \u003cp\u003eThe existence of N-related genes may provide direct evidence of the number of N-metabolism enzymes in bacteria. The presence of \u003cem\u003eamo\u003c/em\u003eA in the genome of \u003cem\u003eHalomonas sp. strain\u003c/em\u003e B01 indicates that it can conduct heterotrophic nitrification at high salt concentrations\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eExiguobacterium mexicanum\u003c/em\u003e SND-01 has been proposed to possess a heterotrophic nitrification pathway via the \u003cem\u003ehao\u003c/em\u003e gene\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. In contrast, Amo and Hao enzyme activities were not detected in \u003cem\u003eRhodococcus erythropolis\u003c/em\u003e Y10, suggesting that strain Y10 transforms ammonium through ammonium assimilation, rather than nitrification\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on metagenomics analysis, the presence of GlnA, GltB, and GdhA confirmed that strain R-1 could utilize NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e as a nitrogen source via ammonium assimilation for cell propagation (Fig. S5). Ammonium can be transformed into glutamate via the glutamate dehydrogenase (GDH) pathway to promote cell propagation, which may take place preferentially in ADB at high ammonium concentrations\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. It has been speculated that the expression of the GDH-related genes \u003cem\u003egln\u003c/em\u003eA (glutamate-ammonia ligase) and \u003cem\u003eglt\u003c/em\u003eB (glutamate synthase large subunit) may be pivotal for the excellent nitrogen removal performance of strain R-1, which coincides with the novel ADB \u003cem\u003eAcinetobacter\u003c/em\u003e sp. TAC-1\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Ammonium assimilation is beneficial for the growth and propagation of bacteria and is convenient for the absorption and utilization of inorganic nitrogen\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeriplasmic nitrate reductase (Nap), which is mainly expressed under aerobic conditions, is involved in aerobic denitrification\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, a new electron transport chain, as a hypothesis for aerobic denitrification in the presence of Nar and Nir, may transmit electrons to NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e- under aerobic conditions without the restriction of the presence or absence of oxygen\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Therefore, the presence of NarG, NirS, NorB, and NosZ in strain R-1, based on metagenomics analysis (Fig. S5), further confirmed that strain R-1 could execute the complete denitrification pathway (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026rarr;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026rarr;NO\u0026rarr;N\u003csub\u003e2\u003c/sub\u003eO\u0026rarr;N\u003csub\u003e2\u003c/sub\u003e) to degrade NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e under both aerobic and anaerobic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The biotechnological significance of understanding the mechanism of nitrogen removal by strain R-1\u003c/h2\u003e \u003cp\u003eIn summary, the overall mechanism of the simultaneous removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e by strain R-1 is summarized in the model shown in Fig.\u0026nbsp;6. First, ammonium assimilation provides a nitrogen source for strain R-1, which supplies a nitrogen nutrient substance for cell biosynthesis. Second, the reduction in nitrate dissimilation in strain R-1 provides energy for cell growth and metabolism. In general, ammonium removal in bioreactors is frequently attributed to heterotrophic nitrification, but the significant role of ammonium assimilation has often been neglected because of the lack of understanding of the mechanism of microbial ammonia removal\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Our findings indicate that ammonia assimilation is an important pathway for nitrogen removal from bioreactors.\u003c/p\u003e \u003cp\u003eOur research showed that strain R-1 can effectively remove ammonia and nitrate under both aerobic and anaerobic conditions, which inspired us to use strain R-1 to enhance nitrogen removal from biological reactors. In the aerobic stage, strain R-1 can effectively remove ammonia mineralized from organic matter, while some nitrate and nitrite may be lost due to aerobic denitrification. In the anaerobic or hypoxic stage, strain R-1 mainly removes nitrate. By strengthening both the aerobic and anaerobic stages, nitrogen removal efficiency can be greatly improved. The results of this study will facilitate the development of culturable ADB as promising candidates for nitrogen pollution control and bioremediation in various environments.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, the highly efficient denitrifying bacterium \u003cem\u003eP. denitrificans\u003c/em\u003e R-1 was investigated. Strain R-1 exhibited prominent capacity for simultaneous ammonium and nitrate removal under aerobic and anaerobic conditions. Evidence provided through combined analysis of nitrogen balance, \u003csup\u003e15\u003c/sup\u003eN isotope tracing and metagenomic techniques demonstrated that strain R-1 utilizes ammonium through assimilation, rather than heterotrophic nitrification, and it simultaneously removes nitrate through denitrification under aerobic and anaerobic conditions. Moreover, ammonium assimilation and nitrate denitrification effectively promoted each other. In addition, nitrate reduction by strain R-1 was shown to be a biochemical or electron transport process coupled with the oxidation of electron donors through stoichiometric and thermodynamic analyses. In conclusion, our results provide novel insights into the nitrogen removal mechanism of ADB, which may be helpful in establishing a theoretical basis for the practical application of simultaneous ammonium and nitrate removal processes in the treatment of wastewater with a high inorganic nitrogen concentration in a single system.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen, J., Gu, S., Hao, H. \u0026amp; Chen, J. Characteristics and metabolic pathway of Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic denitrification. \u003cem\u003eAppl. Microbiol. Biotechnol.\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 9787-9794 (2016).\u003c/li\u003e\n\u003cli\u003eSun, J. et al. 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Strain R-1, isolated from the activated sludge of a sewage treatment plant, was identified as \u003cem\u003eParacoccus denitrificans\u003c/em\u003e, and was found to efficiently remove ammonium and nitrate under anaerobic and aerobic conditions. The maximal NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e removal rate (RR\u0026thinsp;=\u0026thinsp;9.94 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was significantly higher under aerobic conditions than under anaerobic conditions (RR\u0026thinsp;=\u0026thinsp;2.91 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Analysis of the nitrogen balance and isotope tracers indicated that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was consumed through assimilation, but not nitrification. The maximal NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e RR of strain R-1 was 18.05 and 19.76 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under aerobic and anaerobic conditions, respectively, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduction was able to support the growth of R-1 under anaerobic conditions. The stoichiometric consumption ratios of acetate and lactate to nitrate were 0.902 and 0.691, respectively. The \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e isotopic tracer experiment demonstrated that NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was reduced to N\u003csub\u003e2\u003c/sub\u003e by aerobic and anaerobic denitrification. These results indicated that the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e reduction by strain R-1 was a respiratory process coupled with the oxidation of electron donors. Genomic analysis showed that strain R-1 contained complete genes for the nitrogen metabolism pathways of ammonium assimilation and denitrification, but not for nitrification, which is consistent with the physiological process of inorganic nitrogen metabolism in strain R-1. Moreover, we found that ammonium assimilation and nitrate denitrification effectively promoted each other. Our findings demonstrate that the mechanism of the simultaneous removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e by strain R-1 involves ammonium assimilation and denitrification under aerobic and anaerobic conditions. These findings provide new insights into microbial nitrogen transformation and facilitate the simultaneous removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in a single reaction system.\u003c/p\u003e","manuscriptTitle":"Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1 under aerobic and anaerobic conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 15:24:54","doi":"10.21203/rs.3.rs-3890763/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0cf34362-0c29-4926-83ce-22cc579a8bd2","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28317361,"name":"Biological sciences/Biological techniques/Microbiology techniques"},{"id":28317362,"name":"Biological sciences/Biochemistry/Metabolomics"}],"tags":[],"updatedAt":"2024-02-02T12:15:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-25 15:24:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3890763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3890763","identity":"rs-3890763","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}