A method for aerobic biological nitrogen removal and its application

Abstract

This invention discloses a method for aerobic denitrification and its application. Glutamine in N Under the catalysis of oxidases, using oxidoreductase / NADPH or methyl phenolate / ascorbic acid as electron carriers, nitrogen gas can be formed as the final product under the action of amidotransferases. The method of this invention can be used to treat wastewater, playing a particularly important role in the treatment of wastewater containing ammonia nitrogen.

Description

Technical Field

[0001] This invention relates to the fields of microbial biotechnology, wastewater treatment technology, and genetic engineering technology, specifically to a method for aerobic biological denitrification and its application, and more specifically to an aerobic nitrogen production bioconversion and its application in wastewater treatment processes. Background Technology

[0002] Nitrogen is a crucial element for promoting sustainable agricultural development. Microbial nitrogen transformation pathways are a vital link in the global nitrogen cycle and a primary mechanism for nitrogen removal from wastewater. Traditional ammonia-to-nitrogen conversion mainly involves two pathways: 1) aerobic nitrification-anaerobic denitrification, the most well-known classic denitrification pathway; and 2) anaerobic ammonia oxidation, which uses nitrite as an electron acceptor to directly convert ammonia nitrogen into nitrogen gas. Both of these denitrification pathways require the synergistic action of multiple bacterial strains or aerobic-anaerobic conditions to complete, and are dependent on the substrate NH4. + Both nitrogen (N) and COD are relatively sensitive. Traditional denitrification processes are no longer sufficient to meet the demand for efficient removal of high-ammonia nitrogen wastewater.

[0003] Therefore, researching and developing new denitrification technologies and constructing new denitrification processes are urgent problems to be solved. Summary of the Invention

[0004] The novel aerobic nitrogen bioconversion method provided by this invention can directly oxidize -3 valent N to nitrogen under aerobic conditions using a single strain. It has high conversion efficiency, low energy consumption, and almost no accumulation of the greenhouse gas nitrous oxide. It is a green, clean, efficient, and low-energy bioconversion pathway from ammonia to nitrogen.

[0005] The purpose of this invention is to provide a novel aerobic nitrogen production biotransformation. The novel aerobic nitrogen production metabolic pathway provided by this invention can be derived from Alcaligenes species such as Alcaligenes sammonioxydans HO-1, Pseudomonas species, Delftella species, Microvirgula species, etc., or from novel aerobic nitrogen production metabolic engineered strains or in vitro enzyme reaction systems constructed by combining N-oxidase and amidotransferase.

[0006] This invention provides a method for aerobic biological denitrification, in which glutamine, under the catalysis of N-oxidase, uses oxidoreductase / NADPH or methyl phenolate / ascorbic acid as electron carriers, and amidotransferase as the final product nitrogen gas can be formed.

[0007] The reaction system includes glutamine, Tris HCl, oxidase protein, oxidase reductase protein / NADPH or methyl phenolate / ascorbic acid, and amidotransferase; the reaction is carried out at 25-35°C; preferably, the Tris HCl is 20 mM Tris HCl.

[0008] Preferably, the reaction system includes glutamine, O2 as substrate, Tris HCl, N-oxidase, oxidase reductase protein / NADPH or methyl phenazine / ascorbic acid; the reaction is carried out at 25-35°C. Preferably, the Tris HCl is 20 mM Tris HCl.

[0009] Optionally, it also includes the step of harvesting the intermediate product glutamate γ-hydroxyoxime.

[0010] Preferably, the reaction system also includes amidotransferase to convert glutamine into nitrogen gas and harvest hydroxylamine.

[0011] Specifically, the NCBI accession numbers for N-oxidases are WP_012205541.1, WP_042561895.1, WP_028499013.1 or WP_054594875.1; the NCBI accession numbers for oxidases and oxidoreductases are WP_028499014.1, WP_054594876.1, WP_047304347.1, WP_203399393.1 or WP_061288733.1; and the NCBI accession numbers for amidotransferases are WP_028499015.1, WP_054594877.1, WP_047304349.1, WP_203399392.1 or WP_016446024.1.

[0012] In the embodiments, the dosage or concentration range of each component in the reaction system is as follows: N-oxidase 1-5 mg / ml, oxidase reductase 0.4-0.8 mg / ml, amidotransferase dosage of 1 / 16-1 / 32 N-oxidase, glutamine 1-10 mM, NADPH 4-8 mM; or N-oxidase 1-5 mg / ml, amidotransferase 1 / 16-1 / 32 N-oxidase, glutamine 1-10 mM, methyl methacrylate 0.1-0.3 mM, ascorbic acid 4-8 mM.

[0013] Preferably, the oxidase protein, oxidase reductase protein, or amidotransferase is obtained by expression in genetically engineered bacteria, preferably by introducing the encoding gene into the same strain for expression; or by aerobic biological denitrification catalyzed by the whole cell of the genetically engineered bacteria.

[0014] In the field of genetic engineering, plasmid expression vectors, viral expression vectors, and yeast expression vectors, such as the pGEX series, pET series, and pBAD vectors, can be used to construct recombinant expression vectors containing the encoding genes of the aforementioned functional enzymes and oxidases / reductases. A suitable expression host is selected, and appropriate inducers are chosen for induction culture. When the host is bacteria, the aforementioned functional enzyme and oxidase / reductase genes can be ligated into vectors such as pET21a(+), transformed into competent *E. coli* cells, cultured in LB medium or other media, and screened for resistance with ampicillin or other antibiotics. Colonies containing the ligated gene fragment are considered positive transformants. Transformants with correct sequencing are induced to express using inducers such as IPTG. Positive transformants can be used for the production of novel aerobic nitrogen-producing pathway functional enzymes, or for the detoxification and transformation of -3-valent N or hydroxylamine.

[0015] The present invention also provides the application of the method in denitrification. Specifically, the application is for treating wastewater requiring denitrification, preferably for treating high-ammonia-nitrogen wastewater such as domestic sewage.

[0016] The expression levels of the functional enzymes of this invention in *E. coli* can reach 100-500 mg / L. Enzymatic experiments show that glutamine and other substances, under the catalysis of these functional enzymes, can form nitrogen gas as the final product using ferroredoxin / NADPH or cofactors such as methyl phenolate / ascorbic acid as electron carriers. The novel aerobic nitrogen-producing bioconversion enzyme system of this invention can not only use glutamine as the initial substrate, but also ammonia, arginine, isoleucine, aspartic acid, etc., as substrates. This invention provides a novel aerobic nitrogen-producing bioconversion for the treatment of ammonia-containing nitrogen wastewater. This bioconversion can directly catalyze the oxidation of ammonia, glutamine, etc., to nitrogen gas, with a conversion rate >80%. This novel aerobic nitrogen-producing bioconversion plays an important role in hydroxylamine detoxification and the treatment of wastewater containing ammonia nitrogen and organic amines. Attached Figure Description

[0017] Figure 1 Identification and reaction flowchart of intermediates such as glutamate γ-hydroxyoxime acid (GluHXM).

[0018] Figure 2 Diagram showing the formation of hydroxylamine from hydrolyzed hydroxamic acid by amidotransferase.

[0019] Figure 3 In vitro enzymatic reactions producing hydroxylamine and nitrogen from novel aerobic nitrogen-producing biotransformation pathways from different sources.

[0020] Figure 4Nitrogen production diagrams of novel aerobic nitrogen-producing biotransformation pathways in *E. coli* positive clones from different sources. Negative control: *E. coli* containing the pBAD and pET-21a(+) empty plasmids. From *D. acidovorans* CGMCC 1.3363 T D. tsuruhatensis CGMCC 1.3659 T ,D. lacustris CGMCC1.15624, and P. parafulva CGMCC 1.15634 dnfABC operons cloned to pBAD / HisA(Amp r Invitrogen (USA) obtained plasmids pBAD-DA, pBAD-DT, pBAD-DL, and pBAD-PP and transformed them into E. coli BL21. dnfABCoperons from P. fluorescens AU11114, P. fulva 82B1, and M. aerodenitrificans DSM 15089 were cloned into pET-21a(+)(Amp r (TIANGEN, China) obtained plasmids pet21a-PF, pet21a-Ps, and pet21a-MA and transformed them into Escherichia coli BL21.

[0021] Figure 5 A diagram showing nitrogen production in wild-type strains using glutamine as a substrate.

[0022] Figure 6 Diagram showing the formation of hydroxamic acid intermediates in Alcaligenes HO1 strain using ammonium sulfate as a substrate. Blue represents the wild-type Alcaligenes HO-1 strain, yellow represents the engineered strain HO-1-2-29 with the inactivated dnfR gene, the regulatory gene for dnfABC in HO-1. This inactivation of dnfR results in the loss of both the hydroxamic acid intermediate and the nitrogen-producing phenotype. Yellow also represents the complemented strain HO-1-2-29 with restored normal dnfR function. STD represents the standard for glutamate hydroxamic acid.

[0023] Figure 7 A graph showing the denitrification rate of engineered bacteria with a novel aerobic nitrogen-producing biotransformation pathway in nitrogen-containing wastewater. Detailed Implementation

[0024] Example 1: Obtaining the functional gene for aerobic nitrogen production biotransformation of the present invention

[0025] Using the coding sequences of glutamine hydroxylase DnfA (Accession No. QXX79842.1), oxidase reductase DnfB (Accession No. QXX79843.1), and amidotransferase DnfC (Accession No. QXX79844.1) from the genus *Alcaligenes* as templates, BLAST was performed on the NCBI database to screen for homologous N-hydroxylases and amidotransferases. Most of these were derived from *Pseudomonas* and *Delphae* species. These functional enzyme genes (clusters) were amplified by PCR or synthesized and ligated into vectors such as pET28a+, pET21a+, and pBAD. After transduction into *Escherichia coli* DH5α host cells, the cells were cultured at 37°C for 30 minutes and plated onto solid LB agar plates containing 50 μg·mL⁻¹ kanamycin or streptomycin resistance. Positive single clones were selected, sequenced, and stored at -80°C.

[0026] Hydroxylamine has a maximum absorption peak at 705 nm. Referring to the citation (Shurong Liu et al. A highly sensitive method for the determination of hydroxylamine in soils, Geoderma 232-234 (2014) 117-122), a colorimetric method was used to initially screen positive clones with hydroxylamine as the reaction product. A green reaction indicates a positive clone producing hydroxylamine. Plasmids from the positive clones were extracted and sequenced. The sequencing results were analyzed, and Blastx alignment was performed on NCBI to analyze the gene sequences encoding functional enzymes. These enzymes were derived from *D. acidovorans* CGMCC1.3363. T D. tsuruhatensis CGMCC 1.3659 T ,D.lacustris CGMCC 1.15624,P.parafulvaCGMCC 1.15634,P.fluorescens AU11114,P.fulva 82B1,M.aerodenitrificans DSM15089.

[0027] Positive clones were cultured in anaerobic flasks at 20% oxygen partial pressure (O2 / He2). Using 15-N-labeled ammonium sulfate as a substrate and nitrogen, hydroxylamine, and nitrite as indicators, the denitrification activity of novel aerobic nitrogen production pathways mediated by functional enzymes from different sources in E. coli positive clones was investigated (see Example 4 and...). Figure 4 ).

[0028] Example 2: Construction of the oxidase expression vector of the present invention and its expression in Escherichia coli

[0029] Alcaligenes HO-1 (CGMCC No. 16549, see Chinese Patent 201811423813.6), Pseudomonas (P. parafulva CGMCC 1.15634, P. fluorescens AU11114, P. fulva 82B1, purchased from the China General Microbiological Culture Collection Center), and Delftella (D. acidovorans CGMCC 1.3363) were used. T D. tsuruhatensis CGMCC 1.3659 T Genomic DNA of *D. lacustris* CGMCC 1.15624 (purchased from the China General Microbiological Culture Collection Center), and *M. aerodenitrificans* DSM 15089 (purchased from the China General Microbiological Culture Collection Center), or plasmids containing positive clones of *E. coli* containing functional enzymes from different sources, was used as templates for PCR amplification of homologous functional enzyme genes, including the N-oxidase (Accession No. WP_012205541.1, WP_042561895.1, WP_028499013.1 or WP_054594875.1) enzyme genes screened in this invention, and the oxidoreductase (Accession No. WP_012205541.1, WP_042561895.1, WP_028499013.1 or WP_054594875.1) enzyme genes of oxidases. The enzyme genes for aminotransferases (Accession No. WP_028499014.1, WP_054594876.1, WP_047304347.1, WP_203399393.1 or WP_061288733.1) and aminotransferases (Accession No. WP_028499015.1, WP_054594877.1, WP_047304349.1, WP_203399392.1 or WP_016446024.1).

[0030] The reaction system consisted of: template 6-10 ng / ul; dNTP mix (25 mM) 8ul; forward primer (10 mM) 1ul; reverse primer (10 mM) 1ul; 10× buffer 5ul; FastPfu 0.4ul; and ddH2O to a final volume of 50ul. The high-fidelity enzyme and buffer were purchased from TransGen Biotech. PCR conditions included: 95℃ for 2 min pre-denaturation, 95℃ for 20 sec, 55℃ for 30 sec, and 72℃ for 1 min, for a total of 30 cycles; followed by a complete extension at 72℃ for 10 min. The PCR products were amplified by 1% agarose gel electrophoresis. The three PCR systems yielded bands of approximately 1000 bp, 1100 bp, and 700 bp, respectively, consistent with the expected sizes. The homologous recombination method was used to clone pET21a(+), constructing the pET21a(+)-N oxidase gene, the pET21a(+)-oxidase oxidoreductase gene, and the pBAD-amidole transferase gene. These genes were then transformed into Escherichia coli DH5α competent cells, cultured in ampicillin-resistant LB medium, and the cloned colonies were selected for PCR verification.

[0031] The pET21a(+)-N oxidase gene, the pET21a(+)-oxidase oxidoreductase gene, and the pBAD-amidole transferase gene were transformed into *E. coli* BL21(DE3) using recombinant plasmids. The bacteria were cultured in ampicillin-resistant LB medium, and positive clones were verified by colony PCR. Positive transformants containing pET21a(+)-N oxidase, pET21a(+)-oxidase oxidoreductase, and pBAD-amidole transferase were cultured in LB liquid medium at 37°C with shaking at 150 rpm until the bacterial concentration reached OD500. 600 At 0.8°C, IPTG or arabinose was added and the mixture was induced overnight at low temperature to express the various functional enzymes. The enzymes were then purified using a His Tag column (His Bind Resin Chromatography, Novagen, USA), following the instructions in the product manual. The expression levels of each enzyme were approximately 100-200 mg / L for N-oxidase, approximately 50-100 mg / L for oxidase and reductase, and approximately 500 mg / L for amidolytic enzyme.

[0032] Example 3: Determination of the aerobic nitrogen production bioconversion activity of the present invention

[0033] Taking glutamine as an example, this paper elucidates a novel method for determining the biotransformation activity of aerobic nitrogen production.

[0034] Glutamine, catalyzed by N-oxidase, uses oxidoreductases such as NADPH and methyl phenolate / ascorbic acid as electron carriers, and amidotransferases as the final product nitrogen gas. In enzyme activity assays, glutamine or... 15Using N-labeled glutamine as a substrate, the determination of glutamate γ-hydroxyoxime, hydroxylamine, and N2 can be achieved. 15 The activity of the novel aerobic nitrogen-producing biotransformation was determined by measuring N2.

[0035] The reaction system includes 5mM glutamine or 15 N-glutamine, 20 mM Tris-HCl pH 8.5, 1-3 mg of the above oxidase protein, oxidase reductase protein / 6 mM NADPH (or 0.05 mM methyl phenazine / 6 mM ascorbic acid), amidotransferase at a concentration less than 1 / 16 of N-oxidase, total reaction volume 300 μL, reaction at 30°C for 100 min. One unit of enzyme activity is defined as the production of 1 μmol N2 or... 15 The amount of enzyme required for N2. The electron transport system includes oxidoreductases and / or reducing forces, indicated by slashes.

[0036] With 5mM glutamine or 15N glutamine, O2 or 18 Using O2 as the substrate, 20 mM Tris HCl pH 8.5, and adding 3 mg of N-oxidase, with an electron transport chain of oxidase-reductase protein / 6 mM NADPH, or 0.5 mM methyl methacrylate / 6 mM ascorbic acid, after reacting at 30°C for 30 minutes, the amount of nitrogen generated in a 300 μL enzymatic reaction system was detected. This novel aerobic nitrogen-producing biotransformation in vitro enzyme system can convert glutamine and accumulate the intermediate product γ-hydroxyoxime glutamate. Figure 1 (China AG).

[0037] Using 5 mM γ-hydroxyxamic acid as a substrate, 0.007 mg of the above-mentioned amidotransferase was added to 20 mM Tris HCl at pH 8.5. After reacting at 30°C for 100 minutes, the amount of hydroxylamine generated in a 300 μL enzymatic reaction system was measured. The method described in Example 1 and HPLC-MS analysis showed that the amidotransferase system can rapidly hydrolyze γ-hydroxyxamic acid and accumulate hydroxylamine. Figure 2 ).

[0038] Using 5 mM glutamine as a substrate, 20 mM Tris HCl at pH 8.5, and 3 mg of N-oxidase system (oxidase / reductase protein / 6 mM NADPH electron transport chain), with amidotransferase at <1 / 16 the amount of N-oxidase, nitrogen gas was generated in a 300 μL enzymatic reaction system after reacting at 30℃ for 100 minutes. Detection showed that this novel aerobic nitrogen-producing biotransformation enzyme system can convert more than 80% of glutamine into nitrogen gas. Figure 3 ), and accumulate the intermediate product hydroxylamine ( Figure 3 ).

[0039] The nitrogen detection method is as follows: N2 or nitrogen gas is detected by GC / MS (model 7890A / 5975C, Agilent). 15 Gaseous products such as N2 and N2O are produced. A 50% O2 / He mixed gas is introduced into the anaerobic tube. The reaction products are injected into the anaerobic tube through a syringe. After the reaction, the gas in the tube is extracted by the syringe. 15 N2 was detected using a CP-Molsieve 5A (25m × 0.32mm × 30μm, Agilent, USA) column, and N2O was detected using a GS–Carbon Plot (30m 0.32mm 3.0μm, Agilent, USA) column.

[0040] The glutamine hydroxylated products were identified using HPLC-MS (6500QTRAP triple quadrupole MS coupled to an ExionLC™ system, Applied Biosystems / Sciex) and a Pntulips QS-C18 Plus column (5-μm, 4.6×250mm).

[0041] Example 4

[0042] A recombinant *E. coli* strain containing tandem expression of pET21a(+)-N oxidase, pET21a(+)-oxidase oxidoreductase, and pET21a(+)-amidotransferase was constructed. The strain was cultured in an anaerobic flask with 20% oxygen at 37°C and 150 rpm in HNM liquid medium until the bacterial concentration reached OD500. 600 At 0.8, 0.5 mM IPTG was added and the cells were induced to express various functional enzymes at 30°C for 5 days. *E. coli* containing pBAD and pET-21a(+) empty plasmids served as a blank control; *D. acidovorans* CGMCC 1.3363 was used as a control. T D. tsuruhatensis CGMCC 1.3659 T The dnfABC operons (a gene cluster consisting of N-oxidase, oxidase, oxidoreductase, and amidotransferase genes) of D. lacustris CGMCC 1.15624 and P. parafulva CGMCC 1.15634 were cloned into pBAD / HisA (Amp rInvitrogen (USA) obtained plasmids pBAD-DA, pBAD-DT, pBAD-DL, and pBAD-PP and transformed them into E. coli BL21. dnfABC operons from P. fluorescens AU11114, P. fulva 82B1, and M. aerodenitrificans DSM 15089 were cloned into pET-21a(+)(Amp r The plasmids pet21a-PF, pet21a-Ps, and pet21a-MA were obtained in Tianjin, China and transformed into *E. coli* BL21. The protein products of the gene cluster consisting of N-oxidase, oxidase, oxidoreductase, and amidotransferase include N-oxidase (Accession No. WP_012205541.1, WP_042561895.1, WP_028499013.1 or WP_054594875.1), oxidase oxidoreductase (Accession No. WP_028499014.1, WP_054594876.1, WP_047304347.1, WP_203399393.1 or WP_061288733.1), and amidotransferase (Accession No. WP_012205541.1, WP_042561895.1, WP_028499013.1 or WP_054594875.1), and amidotransferase (Accession No. WP_028499014.1, WP_054594876.1, WP_047304347.1, WP_203399393.1 or WP_06128873 ...). No.WP_028499015.1, WP_054594877.1, WP_047304349.1, WP_203399392.1 or WP_016446024.1).

[0043] GS / MS detection of headspace gas showed that the induced culture could 15 N-labeled ammonium sulfate / glutamine is converted to nitrogen gas with a conversion rate of 1-4%. Figure 4 ).

[0044] The HNM culture medium formula (g / L) is as follows: (NH4)2SO4 0.66, sodium succinate hexahydrate 7.88, KH2PO4 0.5, Na2HPO4·12H2O 1.25, MgSO4·7H2O 0.2, trace element solution 2mL, pH 8.0.

[0045] Trace element solution formula (g / L): EDTA·2Na 57.1, ZnSO4·7H2O 3.9, CaCl2·2H2O 7.0, MnCl2·4H2O 5.1, FeSO4·7H2O 5.0, (NH4)6Mo7O 24 ·4H2O 1.1, CuSO4·5H2O 1.6, CoCl2·6H2O1.6, pH6.0.

[0046] Identification of nitrogen-containing gases was performed by GC / MS (model 7890A / 5975C, Agilent) to detect N2 or... 15 Gaseous products such as N2 and N2O were introduced into an anaerobic flask containing HNM medium and filled with a 20% O2 / He mixed gas. Positive clones stored at -80℃ were activated by LB+ampicillin plates and LB+ampicillin liquid, and then inoculated into liquid medium containing HNM+ampicillin with a syringe. After about 5 hours, they were induced with IPTG or arabinose. After culturing for 96 hours, the gas in the tube was withdrawn with a syringe. 15 N2 was detected using a CP-Molsieve 5A (25m × 0.32mm × 30μm, Agilent, USA) column, and N2O was detected using a GS–Carbon Plot (30m 0.32mm 3.0μm, Agilent, USA) column.

[0047] This embodiment demonstrates that the recombinant Escherichia coli strain containing the novel aerobic nitrogen-producing biotransformation functional enzymes of the present invention can convert ammonia / glutamine molecules into nitrogen gas. Figure 4 ).

[0048] Example 5

[0049] The wild-type strain containing novel aerobic nitrogen-producing biotransformation is derived from *D. acidovorans* (CGMCC 1.3363), belonging to the genera *Alcaligenes*, *Pseudomonas*, *Delphinium*, and *Microbranchium*. T *D. lacustris* CGMCC 1.18821, *P. parafulva* CGMCC 1.15634, *M. aerodenitrificans* DSM 15089 were cultured in an anaerobic flask at 20% oxygen concentration, 30°C, and 150 rpm shaking for 2 days using HNM liquid medium. 15 N-labeled ammonium sulfate or glutamine is converted into nitrogen gas, with a conversion rate of up to approximately 50%. Figure 5 ).

[0050] Identification of nitrogen-containing gases was performed by GC / MS (model 7890A / 5975C, Agilent) to detect N2 or... 15 Gaseous products such as N2 and N2O were introduced into an anaerobic bottle containing HNM medium and filled with a 20% O2 / He mixed gas. The wild-type strains were stored at -80℃. After activation by LB plates and LB liquid, the strains were inoculated into liquid medium containing HNM using a syringe. After culturing for 48 hours, the gas in the syringe was withdrawn. 15N2 was detected using a CP-Molsieve 5A (25m × 0.32mm × 30μm, Agilent, USA) column, and N2O was detected using a GS–Carbon Plot (30m 0.32mm 3.0μm, Agilent, USA) column.

[0051] Example 6: In vivo glutamine hydroxylation to generate glutamate γ-hydroxyoxime

[0052] The wild-type Alcaligenes sp. strain HO-1 (CGMCC No. 16549) containing a novel aerobic nitrogen-producing metabolic pathway of the present invention can convert glutamine into glutamine hydroxamic acid. Alcaligenes sp. HO-1 was obtained from wild-type Alcaligenes sp. strain HO-1, stored at -80℃, activated by LB agar and LB liquid, and then cultured in a wide-mouth bottle in HNM liquid medium (20% volume) at 20% oxygen concentration, 30℃, and 150 rpm shaking for 10 h. This not only allows the Alcaligenes sp. strain to convert glutamine into glutamine hydroxamic acid. 15 N-labeled glutamine is converted into nitrogen gas, and glutamate γ-hydroxyoxime acid is also accumulated. Figure 6 ).

[0053] The glutamine hydroxylated products were identified using HPLC-MS (6500QTRAP triple quadrupole MS coupled to an ExionLC™ system, Applied Biosystems / Sciex) and a Pntulips QS-C18 Plus column (5-μm, 4.6×250mm).

[0054] Example 7: Application of novel aerobic nitrogen-producing biotransformation strains in wastewater treatment

[0055] An engineered strain was constructed by expressing the N-oxidase, reductase, amidotransferase, or regulatory gene of the oxidase described in Examples 1 and 2 above in (1) a wild-type strain. A normally functioning dnfR was reintroduced into HO-1-2-29 to construct HO-1-2-29-dnfR, which restored the phenotype of producing hydroxylamine and nitrogen (HO-1-2-29 is a uracil auxotrophic strain of the wild-type strain HO-1; due to the loss of function of the regulatory gene dnfR, this strain lacks the ability to release hydroxylamine and nitrogen). The nitrogen production per unit cell was 30-10 times that of HO-1. The denitrification capacity of the engineered strain containing the novel aerobic nitrogen-producing biotransformation strain of this invention is applied to the treatment of high-ammonia nitrogen industrial wastewater. Engineered bacteria, such as HO-1-2-29-dnfR, were inoculated into rural domestic sewage (COD 480 mg / L, concentration approximately 120 mg / L). Using a simulated reactor, with the pH adjusted to around 7, an inoculum size of 5%, and DO controlled at 3 mg / L, after culturing at 30℃ for 24 hours, the ammonia nitrogen removal rate reached over 90%, and the total nitrogen removal rate also reached over 85%. Figure 7 ).

[0056] The detection methods for total nitrogen and ammonia nitrogen were as follows: The ammonia nitrogen determination method in the reactor using the salicylic acid method was based on the national standard "Determination of Ammonia Nitrogen in Water Quality - Continuous Flow-Salicylic Acid Spectrophotometric Method HJ 665-2013". For the preparation of the ammonia nitrogen standard stock solution (ρ = 1000.0 mg / L), 3.8190 g of ammonium chloride (NH4Cl, analytical grade), dried at 100–105℃ for 2 h and cooled to constant weight, was weighed, dissolved in an appropriate amount of water, transferred to a 1000 ml volumetric flask, diluted to the mark, and stored at 4℃. Low-concentration ammonia nitrogen standard solutions were obtained through serial dilution.

[0057] Total nitrogen was determined in accordance with the national standard "Determination of Total Nitrogen in Water Quality - Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometry HJ 636—2012".

[0058] The results showed that the novel aerobic nitrogen-producing biotransformation engineered bacteria could grow in actual rural domestic wastewater and had a certain ability to remove ammonia nitrogen and COD.

Claims

1. A method for aerobic biological nitrogen removal, characterized in that, Glutamine in N - Under the catalysis of oxidases, with oxidoreductase / NADPH or methyl phenolate / ascorbic acid as electron carriers, nitrogen gas can be formed as the final product under the action of amidotransferases. The reaction system includes glutamine, Tris HCl, oxidase protein, oxidase reductase protein / NADPH, and amidotransferase; or the reaction system includes glutamine, Tris HCl, oxidase protein, methyl phenolate / ascorbic acid, and amidotransferase. Furthermore, it uses O2 as a substrate and reacts at 25-35℃.

2. The method as described in claim 1, characterized in that, The Tris HCl was 20 mM Tris HCl.

3. The method as described in claim 1 or 2, characterized in that, The N The NCBI accession numbers for oxidases are WP_012205541.1, WP_042561895.1, WP_028499013.1, or WP_054594875.1; the NCBI accession numbers for oxidoreductases are WP_028499014.1, WP_054594876.1, WP_047304347.1, WP_203399393.1, or WP_061288733.1; and the NCBI accession numbers for amidotransferases are WP_028499015.1, WP_054594877.1, or WP_047304349.

1. WP_203399392.1 or WP_016446024.

1.

4. The method as described in claim 1 or 2, characterized in that, The dosage or concentration range of each component in the reaction system is as follows: N-oxidase 1-5 mg / ml, oxidase reductase 0.4-0.8 mg / ml, aminotransferase dosage is 1 / 16-1 / 32 of N-oxidase, glutamine 1-10 mM, NADPH 4-8 mM; Alternatively, N-oxidase 1-5 mg / ml, oxidase reductase 0.4-0.8 mg / ml, amidotransferase 1 / 16-1 / 32 N-oxidase, glutamine 1-10 mM, methyl phenazine 0.01-0.03 mM, and ascorbic acid 4-8 mM.

5. The method as described in claim 1 or 2, characterized in that, The oxidase protein, reductase protein of the oxidase, or amidotransferase is obtained by expression in genetically engineered bacteria.

6. The method as described in claim 5, characterized in that, The genetically engineered bacteria are obtained by introducing the encoding genes of oxidase protein, oxidase reductase protein and amidotransferase into the same strain and expressing them; or by performing aerobic biological denitrification through whole-cell catalysis of the genetically engineered bacteria.

7. The application of the method according to any one of claims 1 to 6 in denitrification, characterized in that, Genes encoding oxidase proteins, reductase proteins of oxidases, and amidotransferases were introduced into genetically engineered bacteria and expressed.

8. The application as described in claim 7, characterized in that, The application is for treating wastewater that requires denitrification.

9. The application as described in claim 8, characterized in that, Used for the treatment of domestic sewage with high ammonia nitrogen content.

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