CN114369544A

CN114369544A - Denitrifying bacteria and application thereof in treatment of nitrogen-containing sewage

Info

Publication number: CN114369544A
Application number: CN202111561362.4A
Authority: CN
Inventors: 何腾霞; 张漫漫
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2021-12-18
Filing date: 2021-12-18
Publication date: 2022-04-19
Anticipated expiration: 2041-12-18
Also published as: CN114369544B

Abstract

The invention belongs to the technical field of wastewater treatment, and particularly relates to a denitrifying bacterium and application thereof in treatment of nitrogen-containing wastewater. The strain isPseudomonas taiwanensisEN-F2, preservation number CCTCC NO: M20211515. The strain of the invention shows excellent denitrification capability in both heterotrophic nitrification and aerobic denitrification processes, and has good application prospect in nitrogenous wastewater treatment.

Description

Denitrifying bacteria and application thereof in treatment of nitrogen-containing sewage

Technical Field

The invention belongs to the technical field of wastewater treatment, and particularly relates to a denitrifying bacterium and application thereof in treatment of nitrogen-containing sewage.

Background

Nitrogen compounds play an important role in life metabolism. However, inorganic nitrogen discharged from agricultural production and rural life enters rivers and lakes, threatens the survival of aquatic organisms, causes the propagation of a large amount of bacteria, further deteriorates water resources, emits unpleasant odor, and finally threatens human health (Zheng, z., Li, w., Zhang, d., Qin, w., Zhao, y., Lv, l.2019.effect of ion and mangene on ammonium removal from micro-polar water source by immobilized HITLi7(T) at 2degrees c. bioresource technique. 285, 121367). It was found that the treatment efficiency of drinking water is lowered when the concentration of ammonium exceeds 0.5 mg/L. However, some contaminated surface waters in China have been found to have ammonium contents in excess of 2mg/L (Chu, H.Q., Cao, D.W., Dong, B.Z., Qiang, Z.M.2010. Bio-catalyst membrane bioreactor for micro-poluted surface water treatment. Water Res.44(5), 1573. 1579; Stef. a. n.D., Erd. lyi, N., Izs. k, B.A.Z. Z. ray, G.and Vargha, M.2019.Formation of precipitation by-products in drying water treatment plants using breaking point chlorine treatment. micro journal.104149, 008). Meanwhile, ammonium is used as a substrate for nitrification, and is converted into hydroxylamine and nitrite by nitrifying bacteria under aerobic or anaerobic conditions, and further converted into nitrate. Previous reports have shown that hydroxylamine is toxic to microorganisms or has a strong mutagenic effect on bacteria, plants and viruses as an intermediate in the nitration process (Gross, p.1985. biological activity of hydroxyamine: a review. critical reviews in the toxicology 14(1),87-99), and that the function of denitrifying bacteria can also be inhibited by disrupting the ribosome translation process (Xing, c.y., Fang, y.c., Chen, x, Guo, j.s., Shen, y., Yan, p., Fang, Chen, y.p.2020.effect of hydrolysis of ANAMMOX slab.41 (7), 3365-3372). To date, only a few nitrifying bacteria have the ability to survive in the presence of hydroxylamine. Nitrites are intermediate products of nitrification or denitrification. The accumulation of Nitrite can prevent the normal delivery of blood oxygen, cause hypoxia and even asphyxia, and may harm aquatic organisms when water contains a large amount of Nitrite (Jia, w.l., Wang, q., Zhang, j., Yang, w.h., Zhou, x.w.2016.nutrients removal and nitrate oxide emission modification, differentiation, and phosphor removal process: effect of iron. environmental Science and Pollution research.23(15), 15657-. The accumulation of nitrite also inhibits the nitrification process. In addition, nitrite can be oxidized to nitrate under the action of certain microorganisms (Zeng, j., Liao, s., Qiu, m., Chen, m., Ye, j., Zeng, j. and Wang, a.2020.effects on the removal of ammonium, nitrite and nitrite reagent by the red treated spore bacteria technology us y1. bioresoure technology.312, 123593). The nitrates have the following characteristics: the water solubility is high, and the relative stability in water is strong; nitrate, when converted to nitrosamines, can lead not only to eutrophication of water bodies, but also to carcinogenesis in humans (devin, j.f., edidy, r.i., Butler, b.j.2000.the effects of electron dono and nuclear iron on transformation rates in section from a microbial water supply. journal of environmental hydrology.46(1), 81-97; Zhao, y.x., Feng, c.p., Wang, q.h., Yang, y.n., Zhang, z.y., sugiro, n.2011.nitrate transformation with aqueous acidic nitrile, biological ion, 1033.192), material j.3. h. Therefore, how to remove ammonium and hydroxylamine efficiently and simultaneously and control the accumulation of nitrite and nitrate has become an urgent problem to be solved.

The bacteria with heterotrophic nitrification-aerobic denitrification capability have good application prospect in wastewater treatment and are widely concerned by people. The discovery of well-maintained denitrifying bacteria breaks through the traditional denitrification theory, and has the advantages of high efficiency, high growth speed, low consumption, environmental protection and the like (Yao, S, Ni, J, Chen, Q.and Borthwick, A.G.2013. Environment and characterization of a bacterial consortium able of a bacterial nitrification and aerobic nitrification at low temperature Bioresource technology.127, 151-157). More importantly, the autotrophic denitrifying bacteria are capable of effectively reducing the accumulation of hydroxylamine and nitrite. For example, Pseudomonas taiwanensis J488 can remove ammonium or hydroxylamine at low temperature and no accumulation of nitrite is observed with ammonium as the sole nitrogen source (He, t, Xie, d, Ni, J, Li, z 2020. nitrate oxide produced direct from ammonium, nitrate and nitrate dual ion nitride and nitride ion journal of Hazardous materials388, 122114).

However, in the case where a plurality of nitrogen sources coexist, few bacteria are capable of efficiently and simultaneously removing hydroxylamine or ammonium of high intensity. For example, under optimized conditions, Pseudomonas sp.DM02 can only remove 10mg/L of ammonium within 12 hours (Deng, M., ZHao, X., Senbai, Y., Song, K., He, X.2021.Nitrogen Removal by basic photosynthetic bleaching and aerobic differentiating bacterium Pseudomonas sp.DM02: Removal Performance, mechanism and immobilized application for real aqueous waste treatment. BioResourcer technique.322, 124555). The strains Zobella taiwanensis DN-7 and Pseudomonas mendocina X49, although highly effective in removing ammonium at high concentrations, do not have hydroxylamine-removing ability (Joo, H.S., Hirai, M., Shoda, M.2005.characteristics of ammonium removal by biochemical inactivation by anaerobic inactivation by Alcaligenes faecies No.4.J Biosci Bioeng 100(2), 184. 191; Xie, F., Thiri, M.Wang, H.2021.Simultaneous hydrolytic inactivation and aerobic inactivation by a non-isolated Pseudomonas mendocina X49, 319). Pseudomonas stutzeri C3 was shown to have the ability to remove nitrate efficiently, but it lacks the amoA gene and is unable to undergo heterotrophic nitrification (Ji, B., Yang, K., Wang, H.Y., Zhou, J., Zhang, H.N.2014.Aerobic nitrification by Pseudomonas stutzeri C3 in cable of heterologous nitrification, bioprocess and Biosystems engineering.38(2), 407-. Pseudomonas stutzeri GEP-01 has heterotrophic nitrification capacity, but the removal rate of ammonium is relatively low (3.73mg/L/h) (Gao, J., Zhu, T., Liu, C., Zhang, J., Gao, J., Zhang, J., Cai, M.and Li, Y.2020.ammonium removalcharacteristics of heterogeneous catalysis bacterium Pseudomonas stutzeri GEP-01with biological biosystem Eng 43(6), 959-. Furthermore, even though both strains Photobacterium sp.NNA4 and Alcaligenes faecalis No.4 were able to remove hydroxylamine, cells were unable to grow under these conditions (Joo, H.S., Hirai, M.S., Shoda, M.2005.characteristics of ammonium removal by cationic mutagenesis by alkaline reagents 1. evaluation No.4.JBiosci Bioeng 100(2), 184. 191; Liu, Y.A., Ai, G.M., Wu, M.R., Li, S.S., Miao, L.L., Liu, Z.P.2019.Photobacterium sp.NNA4, amino-degrading reagent J.71, Biocement J.71).

In conclusion, the existing heterotrophic nitrification-aerobic denitrification bacteria have limitations in the denitrification process, can not utilize hydroxylamine for propagation, can not grow in the presence of hydroxylamine and nitrite at the same time, and the nitrification capacity is to be further improved so as to improve the treatment effect of the nitrogen-containing sewage.

Disclosure of Invention

In view of the above, the present invention aims to provide a denitrifier and an application thereof in the treatment of nitrogen-containing wastewater.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention aims to provide a strain, which is Pseudomonas taiwanensis EN-F2, and the strain is deposited in China center for type culture Collection at 12 months and 07 days 2021, and has the following addresses: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, with preservation number CCTCC NO: m20211515.

The inventor finds that the Pseudomonas taiwanensis EN-F2 strain can simultaneously and efficiently remove various inorganic nitrogen in water body in the research process.

The invention also aims to provide a screening method of the strain, which comprises the following steps:

collecting a soil sample, and culturing in a sterilized primary screening culture medium; and (3) coating 5 mu L of bacterial suspension on a re-screening culture medium, streaking, continuing to culture, and selecting and purifying bacteria capable of changing bromothymol blue solid culture medium into blue as candidate bacteria.

Further, the prescreening medium comprises: HONH₃Cl、NaNO₂、CaCI₂、K₂HPO₄、MgSO₄、KH₂PO₄、C₆H₅Na₃O₇·2H₂O and Fe₂(SO₄)₃And the pH of the primary screening medium was 7.20.

Further, the prescreening medium comprises: 0.0496g/L HONH₃Cl、0.0986g/L NaNO₂、0.014g/L CaCI₂、3.5g/L K₂HPO₄、0.04g/L MgSO₄、1.5g/L KH₂PO₄、3.676g/LC₆H₅Na₃O₇·2H₂O and 0.009g/L Fe₂(SO₄)₃。

Further, the rescreening medium comprises: (NH)₄)₂SO₄、MgSO₄、KH₂PO₄、Na₃C₆H₅O₇.2H₂O、FeSO₄·7H₂O,、CaCl₂Agar and bromothymol blue reagent.

Further, the rescreening medium comprises: 0.236g (NH)₄)₂SO₄,0.488g MgSO₄、1gKH₂PO₄、8.5g Na₃C₆H₅O₇.2H₂O、0.592g FeSO₄.7H₂O、0.094g CaCl₂18g agar and 1mL bromothymol blue reagent.

Further, the concentration of bromothymol blue reagent was 1.5g/100ml ethanol.

The invention also aims to provide the application of the strain in the treatment of nitrogen-containing sewage.

The invention has the beneficial effects that:

the strain of the invention shows excellent denitrification capability in both heterotrophic nitrification and aerobic denitrification processes.

The strain has good application prospect in the treatment of the nitrogen-containing wastewater.

Drawings

FIG. 1 shows the morphology of strain EN-F2; wherein a is colony morphology on LB culture medium; b is the colony morphology on the double-screening culture medium plate; c is the gram stain colony morphology under an optical microscope; d is cell morphology under a scanning electron microscope;

FIG. 2 is a phylogenetic tree;

FIG. 3 is a graph showing the results of measurement of heterotrophic nitrification activity, in which A is a graph showing the results of measurement using ammonium sulfate as a single nitrogen source; b is a detection result graph with hydroxylamine as a single nitrogen source; c is a result graph of the effect of hydroxylamine on ammonium; the results of the test are the mean. + -. SD (standard error) of the triplicates.

FIG. 4 is a graph showing the results of measurement of denitrification capacity, wherein A is a graph showing the results of measurement using nitrate as a single nitrogen source; b is a detection result graph with nitrite as a single nitrogen source; c is a result graph of the influence of nitrite on nitrate; the results of the test are the mean. + -. SD (standard error) of the triplicates.

FIG. 5 is a graph showing the results of measurement of simultaneous nitrification and denitrification, in which A is a graph showing the results of measurement using ammonium salts and nitrates as nitrogen sources; b is a detection result graph with ammonium and nitrite as nitrogen sources;

FIG. 6 is a graph showing the results of measurement of simultaneous nitrification and denitrification of hydroxylamine and nitrate/nitrite, in which A is a graph showing the results of measurement using hydroxylamine and nitrate as nitrogen sources, and B is a graph showing the results of measurement using hydroxylamine and nitrite as nitrogen sources.

Detailed Description

The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention. In the present invention, the term "wt%" means a mass content.

Example 1

Isolation and characterization

Collecting soil samples from vegetable field (silk screen, Guizhou, China), transferring 1g of wet soil into a 250mL conical flask filled with 100mL of sterilized primary screening medium, and then culturing for 3d at 25 ℃ and 150r/min by a shaking table; the culture was continued three times under the culture conditions. Wherein, the formula of the primary screening culture medium is as follows: 0.0496g/L HONH3Cl, 0.0986g/L sodium nitrite (the nitrite used in this example is sodium nitrite, as long as the nitrogen content reaches 50mg/L, other nitrite can be used), 0.014g/L CaCI₂、3.5g/L K₂HPO₄、0.04g/L MgSO₄、1.5g/L KH₂PO₄、3.676g/L C₆H₅Na₃O₇·2H₂O and 0.009g/L Fe₂(SO₄)₃And adjusting the pH of the medium to 7.2;

taking 5 mu L of bacterial suspension obtained by culture, coating and marking on a double-screen culture medium flat plate, and culturing for 3d at 25 ℃; selecting and purifying bacteria capable of changing the rescreened culture medium into blue as candidate bacteria; the strain with the highest removal efficiency of hydroxylamine and nitrite (hereinafter referred to as the strain EN-F2) is preserved in a glycerol solution with the concentration of 30 percent (volume ratio) for a long time at the temperature of-20 ℃; wherein, the formula of the re-screening culture medium is as follows: 0.236g (NH)₄)₂SO₄,0.488gMgSO₄、1gKH₂PO₄、8.5g Na₃C₆H₅O₇.2H₂O、0.592g FeSO₄.7H₂O、0.094g CaCl₂18g of agar and 1mL of bromothymol blue reagent;

the morphology of strain EN-F2 was observed using a scanning electron microscope (SU8100, Hitachi, Tokyo, Japan) and an optical microscope (Olympus BX53-DIC, Japan) and, as shown in FIG. 1, the formulation of LB medium used in the course of observing the morphology of strain EN-F2 was: 10.0g/L tryptone, 10g/L NaCl and 5.0g/L yeast extract, and adjusting the pH of the culture medium to 7.20, wherein the solid culture medium is prepared by adding 18% agar to a liquid culture medium (liquid LB culture medium is used for activating and expanding bacteria, and the solid LB culture medium is used for coating and streaking strains for purification).

DNA was extracted using bacterial DNA extraction kit (magenta) and used as template for polymerase chain reaction amplification (PCR); the F27 and R1492 universal primers (wherein the nucleotide sequence of the F27 universal primer is 5'-AGAGTTTGATCCTGGCTCAG-3' and the nucleotide sequence of the R1492 universal primer is 5'-GGTTACCTTGTTACGACTT-3') were selected for PCR amplification (Wei, R., Hui, C., Zhang, Y., Jiang, H., ZHao, Y.and Du, L.2020.Nitrogen removal characteristics and compressed conversion schemes of a heterologous mutation-inhibitory bacterium, Pseudomonas aeruginosa P-1.Environmental Science and polarization Research 28 (6)), 7503-. PCR amplification was performed using a 25. mu.l reaction system containing 12.5. mu.l 2 × Tap Plus Master mix ll, 1. mu.l Primer1, 1. mu.l Primer 2, 0.5. mu.l DNA, 10. mu.l sterile water; PCR conditions of 94 ℃ denaturation for 5min, 30 cycles per minute, 55.5 ℃ annealing for 30s, and 72 ℃ extension for 10 min;

carrying out gel electrophoresis on the amplified PCR product, and then entrusting Shanghai Biotechnology company to carry out sequencing; wherein, the gel electrophoresis comprises the following specific steps: weighing 0.4g of agar into 40ml of AE (acrylic acid) buffer solution, melting the agar by using a microwave oven, adding 5ul of coloring agent, shaking the mixture evenly, pouring the mixture into an electrophoresis plate, inserting an 11-hole comb, pulling out the agar after solidification, adding 5ul of DNA into each hole, using a maker as a control, putting the mixture into an electrophoresis tank, carrying out electrophoresis under the conditions of 150v and 30/40min, and observing the DNA to the middle of the gel plate.

Trusting Shanghai Biotechnology company to sequence the full-length 16S rRNA gene sequence of the amplification product, and submitting NCBI to obtain a login number;

phylogenetic trees were constructed using the MEGA 7.0 program by using the neighbor joining method and 1000 repetitions of bootstrap analysis, and the results are shown in FIG. 2.

As can be seen from FIG. 1, strain EN-F2 can rapidly remove hydroxylamine and nitrite nitrogen, and the colony morphology of the strain on LB and BTB plates is yellow and round, regular in edge, convex in middle, smooth and moist in surface, and opaque (FIGS. 1a and b); strain EN-F2 was gram negative, short rod-shaped, flagellated (fig. 1c, d), and its 16s rrna gene sequence was submitted to the GenBank database under accession number OK 638151.

As can be seen from FIG. 2, homology search with BLAST revealed that the strain EN-F2 has a 99% similarity with Pseudomonas taiwanensis. The phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain EN-F2 and other related strains, further demonstrating that strain EN-F2 is very close to P5, Pseudomonas taiwanensis (FIG. 2). Although He et al reported the species of pseudomonas taiwan, the denitrification properties of strain EN-F2 were different from those of previously reported strain J488 (He et al, 2021 a). In addition, the denitrification capability of EN-F2 is obviously stronger than that of the strain J488, and a plurality of mixed nitrogen sources can be removed simultaneously, so that the method has greater application potential in wastewater treatment.

Detection of denitrification capability of EN-F2 strain

Strain EN-F2 was inoculated into LB liquid medium (formula as described above) at 25 deg.C150r/min of shaking bed activation for 24 hours; centrifuging at 6500rpm for 5min, washing the strain with 20ml sterilized pure water for 3 times, and respectively inoculating into 100ml nitrification, denitrification, and synchronous nitrification-denitrification culture medium; wherein, the formula of the nitrification culture medium (per liter) is as follows: 0.236g (NH)₄)₂SO₄/0.0992g HONH₃Cl、0.04gMgSO₄、3.5g K₂HPO₄、0.009g Fe₂(SO₄)₃、1.5g KH₂PO₄,0.014g CaCI₂、3.064g/1.225gC₆H₅Na₃O₇·2H₂O (nitration process with ammonium salt or hydroxylamine, respectively, as nitrogen source, if 0.236g (NH) is used₄)₂SO₄As nitrogen source, 3.064g C is required₆H₅Na₃O₇·2H₂O, if 0.0992g HONH₃Cl as nitrogen source, 1.225gC was required₆H₅Na₃O₇·2H₂O), and adjusting the pH of the medium to 7.20; the formulation of the denitrification medium (per liter) is as follows: 0.361g of Nitrate (Nitrate, potassium Nitrate used in this example)/0.246 g of Nitrate (Nitrite, sodium Nitrite used in this example), 0.361g of KNO₃、0.246g NaNO₂、3.5g K₂HPO₄、1.5g KH₂PO₄、0.014g CaCl₂、0.009g Fe₂(SO₄)₃、0.04g MgSO₄And 3.064g/6.127gC₆H₅Na₃O₇.2H₂O (nitrate or nitrite can be used as nitrogen source in the denitrification process, respectively, and 3.064gC is required when 0.361g nitrate is used as nitrogen source₆H₅Na₃O₇.2H₂O, 6.127gC if 0.246g of nitrite is used as nitrogen source₆H₅Na₃O₇.2H₂O), and adjusting the pH of the culture medium to 7.20; the formula of the simultaneous nitrification-denitrification culture medium (per liter) is as follows: 0.236g (NH)₄)₂SO₄and 0.361g KNO₃/0.246g NaNO₂and 0.0496g HONH₃Cl/0.0496g HONH₃Cl and 0.361g KNO₃/0.0496g HONH₃Cl and 0.246g NaNO₂(Simultaneous Nitrification and Denitrification can be respectively ammonium salt and nitrate, ammonium salt and nitrite, hydroxylamine and nitrate, hydroxylamine and nitrite as mixed nitrogen source), 0.361g KNO₃、3.5g K₂HPO₄、1.5g KH₂PO₄、0.04g MgSO₄、6.127g/3.676g C₆H₅Na₃O₇·2H₂O (6.127 g C is needed if ammonium and nitrate, ammonium and nitrite are respectively used as mixed nitrogen sources for simultaneous nitrification and denitrification₆H₅Na₃O₇·2H₂O, if hydroxylamine and nitrate, hydroxylamine and nitrite are used as nitrogen source, 3.676g C is required₆H₅Na₃O₇·2H₂O, other cultures unchanged), 0.009g Fe₂(SO₄)₃And 0.014g of CaCl₂And adjusting the pH of the culture medium to 7.20;

initial cell Optical Density (OD) of Strain EN-F2₆₀₀) Controlled at 0.2 to remove ammonium, nitrate and nitrite, and at 0.3 for hydroxylamine; samples of different nitrogen sources were collected every 6h to determine cell density and concentrations of ammonium, hydroxylamine, nitrate, nitrite and total nitrogen;

a spectrophotometer (Shanghai Metash Instruments, UV-6000, Shanghai, China) for the detection of cellular optical density and inorganic nitrogen;

the total nitrogen is calculated by subtracting 2 times of 275nm background absorbance from the absorbance value of the potassium sulfate after digestion at 220 nm; the total nitrogen, ammonium, hydroxylamine, nitrate and nitrite are measured by taking the supernatant of a sample in a culture medium, and the total nitrogen, ammonium, hydroxylamine, nitrate and nitrite are respectively measured by adopting an alkaline potassium persulfate digestion-ultraviolet spectrophotometry (measuring the total nitrogen), an indophenol blue ultraviolet spectrophotometry (measuring the ammonium salt), an 8-hydroxyquinoline ultraviolet spectrophotometry (measuring the hydroxylamine), an ultraviolet spectrophotometry and an N- (1-naphthyl) -ethylenediamine ultraviolet spectrophotometry (measuring the nitrite); the alkaline potassium persulfate digestion ultraviolet spectrophotometry comprises the following specific steps: taking 1ml of culture medium in a 25ml colorimetric tube → diluting with deionized water (18.2) to 10ml → adding 5ml of alkaline potassium persulfate (needing recrystallization) → 121 ℃ for digestion for 30min → adding 1ml of diluted hydrochloric acid (hydrochloric acid: water: 1: 9) → diluting with water to 25ml → measuring at 220nm and 275nm respectively after cooling; the indoxyl blue ultraviolet spectrophotometry method comprises the following specific steps: taking 0.5ml of centrifuged (6500rpm, 5min) supernatant for incubation in a 50ml cuvette → diluting to 30ml with deionized water (18.2) → adding 5ml of a phenol solution, sodium hypochlorite solution → standing for 1h → adding 1ml of a masking agent → metering to 50ml → measuring absorbance at a wavelength of 625nm in visible light; the 8-hydroxyquinoline ultraviolet spectrophotometry method comprises the following specific steps: taking 1ml of supernatant to culture in a 10ml colorimetric tube → adding 1ml of phosphoric acid buffer solution and fixing the volume to 5ml → adding 0.2ml of trichloroacetic acid → adding 1ml of 8-hydroxyquinoline → shaking → adding 1ml of sodium carbonate to shake → boiling water bath for 1min (generating green) → standing for 15min and then measuring the absorbance value at the wavelength of 750 nm; the ultraviolet spectrophotometry and the N- (1-naphthyl) -ethylenediamine ultraviolet spectrophotometry comprise the following specific steps: taking 1ml of supernatant to cultivate the supernatant on the basis of the culture in a 50ml colorimetric tube → adding 1ml of color developing agent → standing for 20min → detecting the light absorption value at the wavelength of 540 nm; the denitrification rate is calculated by the formula Ef ═ R1-R2)/h, where Ef, R1, R2 and h represent the inorganic nitrogen removal rate, the initial nitrogen concentration over time, the final nitrogen concentration and the time interval, respectively.

Detection of heterotrophic nitrification capability of EN-F2 strain

Ammonium sulfate is used as a single nitrogen source to detect the ammonium oxidation rate of the strain EN-F2, and the method comprises the following specific steps: 8ml of ammonium sulfate culture medium is taken every 6h, the medium is centrifuged at 6500rpm for 5min, 0.5ml of supernatant is taken and put into a 50ml colorimetric tube, the colorimetric tube is diluted to 30ml by deionized water, 5ml of phenol and 5ml of sodium hypochlorite solution are added, the mixture is kept stand for 1h, 1ml of masking agent is added, finally the wavelength of the mixture is measured at 625nm, the OD value is calculated by taking the OD value into the standard curve y which is 1.2157x +0.0062, the obtained concentration is multiplied by 50, and the obtained concentration is divided by the sampling amount, and the result is shown in figure 3A.

As can be seen from FIG. 3A, the strain EN-F2 entered the logarithmic phase directly at the first 6h without lag phase, 52.90mg/L of initial ammonium nitrogen was almost completely removed (98.89%), the maximum removal rate 6 hours after inoculation was 8.72mg/L/h, and the maximum removal rate of ammonium could be higher than 8.72mg/L/h, since ammonium nitrogen could be removed 98.89% before 6 h. Compared with the reported strains, the maximum removal rate of ammonium nitrogen of the strain EN-F2 is higher than that of Exiguobacterium mexicanum SND-01(2.24mg/L/h) (Cui, Y., Cui, Y.W.and Huang, J.L.2021.A novel halogenated Exiguobacterium mexicanum strand removal reagent from saline water viral diagnostic and biological diagnostic. Bioresource technical 333,125189), and Vibrio diobolicus SF16(2.29mg/L/h) (Duan, J.journal, Fang, H.journal, B.Chen, J.and Lin, J.journal of saline water diagnostic 2015, C.421, 421. 12. and 179), pseudomonas torasii Y-11(2.04mg/L/h) (He, T., Li, Z., Sun, Q., Xu, Y.and Ye, Q.2016. heterologous differentiation and aerobic differentiation by Pseudomonas torasii Y-11with out cleavage degradation control. BioResourcer Technol 200,493 499).

Ideonella sp.TH17(1.4mg/L/h) (Zhang, L.J., Xie, Y., Ding, L.Y., Qiao, X.J.and Tao, H.C.2020 b.Highley impact ammonium removal of a root cause-oxidizing bacteria, Ideonella sp.TH17.environ Res 191,110059) and Ochrobactrum anti-inflammatory LJ81(3.85mg/L/h) (Lei, X., Jia, Y., Y.and Hu, Y.2019. Silulans ni and Denitrin iron oxide, 442, Biotech 442, Biotech, 442, and Biotech, U.S. Pat. No. 3,85 mg/L/h) (Lei, X., J., Y., Y.and Hu., Y.2019. Siltanium hardness and Denitrin, J.S. III, N.S. S. and D.S. IV, J.S. IV, N.S. S. N. S. N.S. N. M.₆₀₀When the value increased from 0.25 to 0.98, 100% of the ammonium was removed over 12 h. Ammonium nitrogen concentration increases after 12h inoculation due to the release of ammonium by dead bacteria (Li, C., Yang, J., Wang, X., Wang, E., Li, B., He, R.and Yuan, H.2015.Removal of nitrogen by basic biochemical determination of a phosphate accumulating bacteria Pseudomonas stutzeri YG-24. BioResourcer Technol 182,18-25), while the total nitrogen concentration decreases from 51.55 to 3.68mg/L, with a removal efficiency and maximum removal rate of 92.86% and 7.98mg/L/h, respectively. Furthermore, it was observed that the pH of the system increased from 7.19 to 9.00 over time (not shown in FIG. 3A) throughout the ammonium removal process, similar to that of Zobella taiwanensis DN-7 (Lei, Y., Wang, Y., Liu, H., Xi, C.and Song, L.2016.A novel heterocyclic nifrifying and aerobic Denitrifying bacterium, Zobella taiwanensis DN-7, can remove high-strength ammoniacrobiol Biotechnology 100(9), 4219-. Only 0.11mg/L nitrite was detected 6h after inoculation and disappeared after 12h, indicating that the EN-F2 strain can undergo denitrification during nitrification. Hydroxylamine was not detected in this procedure, but 0.61mg/L nitrate was observed, which is consistent with the detection of 12.95mg/L nitrate by strain Ochrobactrum anthropic LJ81 (Lei, X., Jia, Y., Chen, Y.and Hu, Y.2019.Simultaneous nitrile and mutation by non-nitrile incubation by a novel isolated Ochrobactum anthropic LJ81. BioResourcer Technol 272, 442-450). The above results show that the strain EN-F2 can efficiently convert most of ammonium salt into nitrogen-containing gas (N)₂O and N₂)。

In order to confirm the hydroxylamine removal capacity of the strain EN-F2 and further explore the nitration pathway of the strain EN-F2, hydroxylamine is used as a single nitrogen source for detection, and the specific steps are as follows: 8ml of hydroxylamine culture medium is taken every 6h and centrifuged at 6500rpm for 5min, 0.5ml of supernatant is taken and put into a 50ml colorimetric tube, diluted to 30ml by deionized water, then 5ml of phenol and 5ml of sodium hypochlorite solution are added, the mixture is kept stand for 1h, 1ml of masking agent is added, finally the wavelength is measured at 625nm, the OD value is substituted into the standard curve y which is 1.2157x +0.0062 for calculation, and the obtained value is multiplied by 10 to be divided by the sampling amount, and the result is shown in figure 3B.

As can be seen from FIG. 3B, OD of the strain EN-F2 after 18 hours of cultivation₆₀₀The value increased from 0.35 to 0.58. Meanwhile, the hydroxylamine concentration is remarkably reduced from 23.32 to 1.48mg/L, and the removal efficiency and the maximum removal rate are 93.69 percent and 2.12mg/L/h respectively. The maximum removal rate of hydroxylamine is much higher than that of Pseudomonas taiwanensis J488(0.28mg/L/H) (He, T., Xie, D., Ni, J., Li, Z.and Li, Z.2020.Nitro oxide produced direct from ammonium, nitrate and nitrate reducing and reducing reagents. journal of Hazardous Materials388), Glutaminobacter aritification EM-H8(0.21mg/L/H) (Liu, Y., Hu, T., Song, Y., Chen, H.and Lv, Y.2015. hetrophtic reagent removed from Acetobacter by Ysp 1 ist)From make plant water.journal of Bioscience and Bioengineering 120(5),549- & 554) and Photobacterium sp NNA4(0.7mmol/L/h) (Liu, Y., Ai, G.M., Wu, M.R., Li, S.S., Miao, L.L.and Liu, Z.P.2019.Photobacterium sp.NNA4, an efficient hydroamidane-transforming biochemical triode/aerobiotic densifier.J Biosci Bioeng 128(1), 64-71). When the incubation time was extended to 30h, 100% of hydroxylamine was removed. In addition, 62.95% of the total nitrogen is converted into other inorganic nitrogen or gaseous nitrogen, and the maximum removal rate is 1.82mg/L/h, which is also obviously higher than that of the strain. The accumulated amount of nitrite reaches 14.81mg/L after 18 hours of culture, and decreases to 0.53mg/L after 30 hours of culture. The pH increased from 7.15 to 8.05. It is noteworthy that nitrite and nitrate can be detected and reduced during hydroxylamine oxidation, which means that denitrification occurs simultaneously. Furthermore, the growth of cells and accumulation of nitrite of strain EN-F2 was not observed with strain Photobacterium sp.nna4(Liu, y, Ai, g.m., Wu, m.r., Li, s.s., Miao, l.l.and Liu, z.p.2019.Photobacterium sp.nna4, an effective hydroxylamine-transforming heterologous nitrite/aerobic Denitrifier.j bioscience 128(1),64-71) and Alcaligenes faecis No.4 (Joo, h.s., Hirai, m.and Shoda, m.2005. rheological science of ammonium regenerative nitrite, biochemical nitrite, 191, and hydroxylamine, wherein accumulation of nitrite was not observed with strain Photobacterium sp.nnna 4 (4.J, y., al, 191), even though accumulation of cell growth of ammonium nitrite, biochemical nitrite was observed with strain. In conclusion, EN-F2 can be grown in the presence of hydroxylamine and completely removed.

To further study the effect of hydroxylamine on the ammoxidation process, 10mg/L hydroxylamine was added to the above-mentioned nitrification medium, and ammonium nitrogen and hydroxylamine and the nitrification intermediate were measured every 6 hours, and the results are shown in FIG. 3C; wherein the detection step of ammonium nitrogen is (the same as the nitrification step using ammonium sulfate as a nitrogen source); the hydroxylamine detection step was (as in the nitrification detection step using hydroxylamine as the nitrogen source); the detection steps of the nitrifying intermediate products are (the nitrifying intermediate products generate nitrate and nitrite, and the steps are the same as the denitrification steps which are respectively carried out by taking the nitrate and the nitrite as nitrogen sources).

As can be seen from FIG. 3C, hydroxylamine was completely converted with an average removal rate of 1.59 mg/L/h; the ammonium nitrogen removal efficiency was 85.46%. Addition of 10mg/L hydroxylamine to the ammonium salt medium resulted in OD higher than when ammonium was the sole nitrogen source₆₀₀The improvement is 0.40. After 12h of culture, the ammonium nitrogen and total nitrogen concentrations are respectively reduced to 0.48 and 11.01mg/L from 48.13 and 57.17mg/L, and the removal efficiency is 99.00% and 80.74%. The system pH increased from 7.19 to 9.05. Meanwhile, the peak of nitrite (6.65mg/L) appeared at 6h and then remained relatively unchanged (about 2.40 mg/L). Nitrate was detected at a low concentration (1.18mg/L) during this process, in contrast to the results obtained with Enterobacter cloacae CF-S27, strain EN-F2 did not accumulate nitrate and nitrite even in the presence of hydroxylamine (Padhi, S.K., Tripathy, S.A., Mohanty, S.and Maiti, N.K.2017.Aerobic and hydrotopic nitrile removal by Enterobacter cloacae CF-S27 with efficacy evaluation of hydrolxamine Bioresour Technol 232, 285-. In addition, due to the addition of hydroxylamine, the maximum removal rate of ammonium after 30h of incubation decreased from 8.72 to 6.8mg/L/h, indicating that hydroxylamine was able to retard the oxidation of ammonium nitrogen. Also, the removal efficiency of total nitrogen (80.74%) was lower than that of ammonium as a single nitrogen source (92.84%). In conclusion, the EN-F2 strain was able to completely remove hydroxylamine and ammonium nitrogen by prolonged culture.

Aerobic denitrification capability detection

Nitrate is used as a unique nitrogen source, and the denitrification capacity of the strain EN-F2 under aerobic conditions is detected, and the result is shown in FIG. 4A; wherein, the detection step is as follows: 8ml of potassium nitrate medium was centrifuged (6500rpm, 5min), 1ml of the supernatant was placed in a 25ml cuvette and the OD was measured directly at a wavelength of 220,275, and the OD value measured at 220 was subtracted by two times the OD value of 275, and the value obtained when the standard y was 0.2396x-0.0038 was multiplied by 25 and divided by the sample volume. As can be seen from FIG. 4A, the strain EN-F2 rapidly decreased the nitrate concentration and removed 88.95% after culturing for 12h, and the maximum removal rate was 5.80mg/L/h, and then remained around 6.01 mg/L. The maximum nitrate removal rate is much higher than 1.90mg/L/h of Streptomyces mediolani EM-B2 (He, T., Wu, Q., Ding, C., Chen, M.and Zhang, M.2021B. Hydroxylamine and nitrile earth removed effect by Streptomyces mediolani strain EM-B2. ecooxicol Environ Saf 224,112693), 1.04mg/L/h of Acinetobacter tandoii MZi-5 (Ouyang, L., Wang, K., Liu, X., Wong, M.H., Hu, Z., Chen, H., Yang, X.and Li, S.A. udson nitrogen recovery effect, M.H., Hu, Z., Young, H., Young, X.and Li, S.A. medium recovery coefficient recovery, M.H., III, Cu, M.H., III, S.A. D. M.A. medium recovery strain, M.H. and III, C.A. M.A. medium recovery, M.H., Cu, M.20, M.7. and M.7. bacterium, C.7. and M.A. A. medium, M.7, C. bacterium, C. 7. and III, C.7. bacterium, C.7. A. 1.7, C. A. bacterium, C. 1.7. bacterium, C. A. 1.A. 1, C. A. 1, C. A. bacterium, A. 1, A. 1, A. 1, C. A. 1, A. M. A. M. A. M. A. M. M.

To further clarify whether or not the strain EN-F2 can remove nitrate at a low concentration, 10mg/L of nitrate was used as the sole nitrogen source (to examine the denitrification ability of the strain EN-F2 under aerobic conditions, the examination step was carried out by culturing the strain EN-F2 in a medium containing 10mg/L of potassium nitrate, taking 2ml of the supernatant of the medium every 6 hours, and then carrying out the denitrification step using potassium nitrate as the nitrogen source as described above.

Only 10mg/L nitrate consumption was detected at the time of the experiment, and no nitrate was detected at 6h, indicating that all nitrate was consumed and no other inorganic nitrogen detection was performed.

As can be seen from the test results, the strain EN-F2 can completely remove 10mg/L of nitrate. Thus, it can be concluded that strain EN-F2 requires more nutrients such as Ca²⁺Or Fe2⁺To remove high concentrations of nitrates. The system pH increased from 7.21 to 9.11. Furthermore, total nitrogen was reduced from 58.29 to 13.10mg/L, the removal efficiency was 77.53%, the maximum removal rate was 4.42mg/L/h, significantly higher than Acinetobacter tandoii MZ-5(1.06mg/L/h) (Ouyang, L., Wang, K., Liu, X., Wong, M.H., Hu, Z., Chen, H., Yang, X.and Li, S.2020.A study on the nitrogen removal efficiency of bacterium Acinetobacter tandoir MZ-5from a regulated converter of Shenzhen, Guougdong provice, China. Bioresr technique 32) and Streptomyces media-2 (1.60mg/L, Zolmine, Zolmin, Wylone, and 2. Zone, M.12, and Zone, M.2. and M.12. and W.E.D. Nitrous acidSalt accumulated to 6.79mg/L peak after 6h of culture and then dropped to 2.40mg/L, similar to Paracoccus densificans Z195(Zhang, H., Li, S., Ma, B., Huang, T., Qiu, H., Zhao, Z., Huang, X.and Liu, K.2020a. Nickel removability and 13) C metallic pathways of biological densifier Technology 307,123230) and F.solani radar F-77(Cheng, H.Y., Xu, A.A. origin, M.D., chemical J.D., chemical J.S. 19, S.S., S.J., Wang.S.S. 83, S.D.D.D.S. 52, S.S.83, D.S.A. pacifier, K.D.A. origin, M.D., chemical J.S.S. 12, and S.S.S.83, D.A. 12, D.S.A. pacification, D.D.A. 12, S.S.A. 12, S.A. 12, D.A. 12, S.A. 12, D.A. pacific and D. 12, D.D. 12, D. 12, D.A. 12, D. and D. 12, D. and D. 12, D. and D. origin, D. 12, D. origin, D. and D. origin, D. 12, D. origin, D. 12, and D. origin, D. 12, and D. 12, D. origin, and D. 12, D. origin, D. 12, D. origin, and D. origin, and D. origin, and D. origin, and D. origin, and 2, and D. origin, and, the ammonium concentration increased from 0 to 5.34mg/L, which is likely to result from the lysis of dead cells. This phenomenon is consistent with that of the previously reported strain EN-B2 (He, T., Wu, Q., Ding, C., Chen, M.and Zhang, M.2021B. Hydroxylamine and nitrile area removed effect by Streptomyces media strain EM-B2. ecotoxol Environ Saf 224,112693).

In order to further research the denitrification characteristics, the strain EN-F2 is cultured by taking nitrite as a unique nitrogen source under aerobic conditions, and the denitrification capability is detected, wherein the detection steps are as follows: and (3) centrifuging 8ml of nitrite culture medium, putting 0.2ml of centrifuged supernatant into a 50ml colorimetric tube, diluting the supernatant to 50ml by using deionized water, adding 1ml of color developing agent, standing for 20min, measuring the OD value at the wavelength of 540nm, and obtaining the nitrite concentration when the OD value is equal to y which is 0.0613x + 0.0024. The results are shown in FIG. 4B.

As can be seen from FIG. 4B, the growth of strain EN-F2 was positively correlated with nitrite and total nitrogen removal. As the bacteria grew from 0.18 to 0.67, nitrite nitrogen and total nitrogen were removed 86.31% and 68.15%, respectively, after 12h of culture, with maximum removal rates of 4.55 and 3.31mg/L/h, respectively. After 18h of culture, the removal efficiency of nitrite and total nitrogen is 87.47% and 73.62%, respectively, which is similar to 89.80% when nitrate is used as a unique nitrogen source, but is lower than the removal efficiency (100%) of ammonia or hydroxylamine, and the phenomenon shows that the heterotrophic nitrification capacity of the strain EN-F2 is higher than that of aerobic denitrification. Although, as such, the nitrite removal rate of Strain EN-F2 is also significantly higher than that of most strains, such as P.putida Y-12(1.60mg/L/h) (Ye, Q., Li, K., Li, Z., Xu, Y., He, T., Tang, W.and Xiang, S.2017. heterologous Nitric nitrile-aqueous Denitrification Perform of Strain Y-12under Low Temperature and High Concentration of Inorgnic Nitrogen Conjugation, Water 9(11)), P.putida 5(1.33mg/L/h) (Yang, L., Wang, X.H., Cui, S., Ren, Y.X., Yu, J., Chen, N, Gui, Q., W.12, W.D., and H. (12, W.S., D. 1, J.P.P., Q., W.S., D. 12, N.S., D. 3, W., D., 12, N.S., P.S. 1, J., P.S. 12, J., P.S. 12, U.S. Pat. No. 3, N.S. 1, N.A., 12, N.S. 1, N. 1, N.S. 1, N. 1, N.S. 3, N.S. 1, N.S, kumar Awasthi, M., Kong, D.D., Chen, J.S., Wang, Y.F.and Xu, P.2020.Aerobic Density performance and nitro removal analysis of a novel important bacterial solani RADF-77.Bioresource Technology 295) and Ochrobactrum anthropic LJ81(4.12mg/L/h) (Lei, X.A., Jia, Y.Chen, Y.and Hu, Y.2019. Silinaneou nification and Denitrification with a thionitrile amino acid side a isolated O. In addition, 2.12mg/L nitrate was detected during nitrite removal and was then almost completely removed at 30h, which is inconsistent with the strains Arthrobacter ariliaexisting Y-10(He, T., Xie, D., Li, Z., Ni, J.and Sun, Q.2017.ammonium nitrate reduction and differentiation process by Arthrobacter ariliaexisting Y-10. BioResource Technol 239,66-73) and Sporidiobolus paradoxus Y1 (Zeng, J.Liao, S., Qiu, M., Chen, M., Ye, J.and J.J.and J.J.2020. Electron of nitrite reduction and regeneration, 312,123593). The pH of the system increased from 7.20 to 9.04. The above results further show that the strain EN-F2 can be subjected to denitrification under aerobic conditions.

With 50mg/L nitrite and 50mg/L nitric acidSalt (0.361g KNO)₃,0.246g NaNO₂,3.5gK₂HPO₄,1.5g KH₂PO₄,0.014g CaCl₂,0.009g Fe₂(SO₄)₃,0.04g MgSO₄,6.127gC₆H₅Na₃O₇.2H₂O, pH 7.20.) is a mixed nitrogen source, the influence of the addition of nitrite on nitrate is detected, and the detection steps are as follows: culturing the strain in LB liquid for 24h, centrifuging, washing, adding into mixed culture medium containing nitrate and nitrite, taking culture medium supernatant every 6h to determine the content of nitrate, nitrite, total nitrogen and ammonium salt, wherein the determination method is the same as that when nitrate or nitrite is used as single nitrogen source. The detection results are shown in fig. 4C.

As can be seen from FIG. 4C, EN-F2 rapidly propagated within a period of 0-18h, OD₆₀₀The value increased from 0.20 to 1.24 without lag phase and then reached 1.43 at 30 h. The system pH increased from 7.18 to 9.25. Notably, strain EN-F2 grew better in mixed nitrite and nitrate nitrogen media than when nitrate was the sole nitrogen source, indicating that supplementation with nitrite in the nitrate removal system did not affect the cell growth of EN-F2. In addition, the nitrate of 59.50 and the nitrite of 57.28mg/L sharply dropped to 25.06 and 8.34mg/L after 18h of cultivation, corresponding maximum removal rates of 3.94 and 4.34mg/L/h, respectively. The total nitrogen removal efficiency is 61.60%, the maximum removal rate is 7.07mg/L/h, which is significantly higher than the total nitrogen with nitrate (4.42mg/L/h) or nitrite (3.31mg/L/h) as the sole nitrogen source. After nitrite was added to the nitrate medium, the nitrate removal efficiency and maximum removal rate dropped from 89.82% and 5.80mg/L/h to 57.88% and 3.94 mg/L/h. Nevertheless, the maximum nitrite removal rate in the mixed nitrogen system was 4.34mg/L/h, similar to the 4.55mg/L/h value for a single nitrite source. The above results show that nitrate has no effect on the reduction of nitrite, and nitrite can be preferentially reduced during denitrification.

Simultaneous nitrification-denitrification performance detection

In order to further research the synchronous nitrification-denitrification capability of the strain EN-F2, 50mg/L of ammonium salt and nitrate are used as mixed nitrogen sources, and the detection steps are as follows: the bacterial strain activated for 24 hours in the LB liquid culture medium is inoculated into a mixed culture medium containing ammonium salt and nitrate after centrifugation, and a supernatant liquid culture medium is taken every 6 hours for measurement, wherein the measurement method is the same as the method of respectively taking the ammonium salt or the nitrate as a single nitrogen source. The results are shown in FIG. 5A;

as can be seen from FIG. 5A, OD was observed when ammonium and nitrate were used as the mixed nitrogen source₆₀₀The maximum value can reach 1.39, which is higher than 0.94 of single ammonium nitrogen and 0.65 of single nitrate; after the strain is cultured for 12h, 100% of ammonium is removed, and the average removal rate is 8.11mg/L/h, which shows that the addition of nitrate does not influence the removal rate of the ammonium by EN-F2. Meanwhile, the concentration of the nitrate gradually decreases from 53.18 to 21.62mg/L, the maximum removal rate is 4.64mg/L/h, and then the relative stability is kept. The addition of ammonium did not increase the nitrate removal rate, contrary to the Arthrobacter aritificinsis Y-10 conclusion (He, T., Xie, D., Li, Z., Ni, J.and Sun, Q.2017.ammonium salts reduction in the same area nitrile and condensation process by Arthrobacter aritificinsis Y-10. BioResourceTechnol 239, 66-73). After 30h of culture, the pH value of the system is remarkably increased from 7.18 to 9.36. Furthermore, when ammonium and Nitrate are used as the mixed Nitrogen source, Nitrification is preferentially carried out, which is consistent with Pseudomonas toraasii Y-11(He, T., Li, Z., Xie, D., Sun, Q., Xu, Y., Ye, Q. and Ni, J.2018.Simultaneous nitrile and differentiation with differential mixed nitrile loads by a hydrolytic Aerobic bacterial Strain of biological biodegradation 29(2), 159. 170) and Rhodococcus sp HY-1(Li, W.2013.study on rheological properties in the regenerative Process of ammonium Nitrate and Nitrate biochemical reaction Strain of biological hydrolysis of Escherichia coli Strain of biological hydrolysis of Strain of biological hydrolysis of lactic acid 79. 74. 79, but differs from strain CF-S9(Padhi, S.K., Tripathy, S., Sen, R., Mahapatra, A.S., Mohanty, S.and Maiti, N.K.2013.Characterisation of rheological nitrile and aerobic attenuation Klebsiella pneumoniae CF-S9 strain for biornemeration of water.&Biodegradation 78, 67-73). Furthermore, only 0.28mg/L nitrite was detected in this study, which is in conjunction with nitrateAlmost equal in the removal system. The maximum removal rate of total nitrogen was 6.72mg/L/h, lower than that of ammonium as the sole nitrogen source (7.98mg/L/h), but higher than that of nitrate as the sole nitrogen source (4.42 mg/L/h). The above results show that the strain EN-F2 can simultaneously carry out nitrification and denitrification in the presence of mixed ammonium and nitrate.

50mg/L of ammonium and 50mg/L of nitrite are used as mixed nitrogen sources, and the synchronous nitrification-denitrification performance of the strain EN-F2 is detected, wherein the detection steps are as follows: the strains respectively activated and expanded in LB liquid medium for 24 hours were inoculated into a mixed medium containing ammonium and nitrite, and the supernatant liquid medium was taken every 6 hours for detection in the same manner as the method using ammonium or nitrite as a single nitrogen source, respectively, with the results shown in FIG. 5B.

As can be seen from fig. 5B, as the cell growth increased from 0.17 to 1.20, the removal efficiency of ammonium nitrogen reached 94.85% at 12h, and then was completely removed after 18 hours of incubation. The maximum removal rate of ammonium achieved between 0 and 6 hours was calculated to be 6.24mg/L/h, significantly lower than 8.72mg/L/h for a single ammonium nitrogen removal system. This result indicates that the addition of nitrite negatively affects the removal of ammonium, which is reported in analogy to the carbon source of the strains Pseudomonas stutzeri D6(Yang, X., Wang, S.and Zhou, L.2012.Effect of carbon source, C/N ratio, nitrate and dissolved oxygen control on nitrate and ammonium production from condensation process by Pseudomonas stutzeri D6.Bioresour technique 104,65-72) and Paracoccus Veritus LYM (Shi, Z., Zhang deficiency, Y., Zhou, J., Chen, M.and Wang, X.2013.Biologiceremometer of nitrate and ammonium anion exchange 148. under Bioresourc condition 148. Bioresourc technique 148. the results are reported. It is noted that the nitrite concentration increased slightly during the first 6h, which may be due to ammonium oxidation. Accumulation of nitrite indicates preferential removal of ammonium salts, consistent with the case of mixed ammonium and nitrate nitrogen sources, but contradictory to simultaneous removal of ammonium and nitrite by pseudomonas torasii Y-11(He, t, Li, z, Xie, d, Sun, q, Xu, Y, Ye, q.and Ni, j.2018.simultaneous nitrogen and denitrification with differential nitrogen using a biochemical analysis by a hypotherma aerobic bacteria biological analysis 29(2), 159. 170. maximum removal rate with nitrite as the sole nitrogen source (4.55mg/L/h) is substantially equal to 4.80mg/L/h, inconsistent with the result of nitrite removal rate enhancement by strain aerobic supplemented ammonium salt LJ81 (jii, x, Y, 2019. and 12. environmental analysis by biochemical analysis).

In addition, nitrate accumulation was observed during 24h of strain culture, and then was completely removed as the culture time was extended to 30 hours. The pH of the system increased from 7.20 to 9.32 and these results indicate that denitrification occurred simultaneously with nitrate as the nitrogen source. The maximum removal rate of the total nitrogen is 5.09mg/L/h, which is obviously lower than the total nitrogen of 7.98mg/L/h when the ammonium salt is used as a single nitrogen source, but higher than the total nitrogen of 3.31mg/L/h of nitrite. These results further indicate that EN-F2 can simultaneously perform nitrification and denitrification, and that ammonium can be preferentially removed.

In conclusion, the EN-F2 strain was able to completely remove ammonium salts even in the presence of high concentrations of nitrate or nitrite in the wastewater. Incomplete nitrate and nitrite removal may be the result of the medium being depleted of some nutrients other than inorganic nitrogen.

Detection of simultaneous nitrification and denitrification performance of hydroxylamine and nitrate/nitrite

Detecting the capability of the strain EN-F2 for synchronously removing mixed nitrogen source of hydroxylamine (10mg/L) and nitrate (50mg/L), wherein the detection steps are as follows: the strain which is activated by LB liquid culture medium and expanded for 24 hours is inoculated into a mixed culture medium containing hydroxylamine and nitrate, and detection is carried out every 6 hours, wherein the detection method is the same as that of taking hydroxylamine and nitrate as nitrogen sources respectively; the results are shown in FIG. 6A.

As can be seen in FIG. 6A, the OD was determined by adding 10mg/L hydroxylamine to the nitrate nitrogen removal system compared to nitrate alone₆₀₀The value is increased by 0.44. 11.15mg/L of hydroxylamine was completely consumed, and the average removal rate was 1.86mg/L/h, indicating that low concentrations of hydroxylamine did not hinder the cell growth and denitrification capability of strain EN-F2. Nitrate as an intermediate product of hydroxylamine oxidation, with hydroxyl groups in the first 6h incubationThe amine consumption increased by 2.78 mg/L. After 18h of culture, the nitrate removal efficiency is 77.40%, the maximum removal rate is 5.07mg/L/h, which is obviously lower than 5.80mg/L/h taking nitrate as a unique nitrogen source, and the phenomenon is similar to low-concentration hydroxylamine (hydroxylamine) (C)<35mg/L) did not agree with the conclusion that nitrate removal was promoted (Zhang, X., Xia, Y., Wang, C., Li, J., Wu, P., Ma, L., Wang, Y., Da, F., Liu, W.and Xu, L.2020c.enhancement of nitride production vision addition of hydrolamine to partial condensation (PD) biological: Functional genes dynamics and enzymic activity. Bioresourc technology 318,124274). Nitrite accumulation (0.48 → 7.91mg/L) was observed between 0 and 6h, and then was completely removed within 18 h. The maximum removal rate of the total nitrogen is 5.09mg/L/h, which is obviously higher than that of a single nitrate removal system. The system pH increased from 7.15 to 9.11. In conclusion, the strain EN-F2 can simultaneously carry out nitrification and denitrification with hydroxylamine and nitrate as mixed nitrogen sources, and can preferentially and completely remove hydroxylamine.

Detecting the removal characteristics of hydroxylamine and nitrite of the strain EN-F2, wherein the detection steps are as follows: the strain activated by LB liquid culture medium and expanded for 24h is inoculated into mixed culture medium containing hydroxylamine and nitrate, and detection is carried out every 6h, the detection method is the same as that respectively using hydroxylamine and nitrite as nitrogen source. The results are shown in FIG. 6B.

As can be seen in FIG. 6B, the OD600 increased by 0.45 after addition of 10mg/L hydroxylamine in the nitrite removal system compared to nitrite alone. The system pH increased from 7.18 to 9.22. With the propagation of the strain EN-F2, the hydroxylamine concentration of 13.24mg/L was reduced to 2.85mg/L in the first 6h, and was completely removed after 12h of culture. Calculated, the maximum removal rate of hydroxylamine was 1.73mg/L/h, lower than 2.21mg/L/h with hydroxylamine as the sole nitrogen source. Nevertheless, hydroxylamine can be removed efficiently in the presence of nitrite. After hydroxylamine consumption, nitrite decreased significantly from 53.50 to 10.71mg/L within 18h and then remained constant. The maximum nitrite removal rate was 6.65mg/L/h, significantly higher than 4.55mg/L/h for nitrite as the sole nitrogen source, which indicates that the nitrite removal capacity of strain EN-F2 was enhanced by hydroxylamine supplementation, which contradicted the report that hydroxylamine is a chemical inhibitor of nitrite-oxidizing bacteria (notably, the maximum removal rate of total nitrogen (6.90mg/L/h) in the case of mixed nitrogen sources was significantly higher than 3.31mg/L/h for total nitrogen using a single nitrite as the nitrogen source), which further demonstrates that the addition of hydroxylamine can promote nitrite removal, since both hydroxylamine oxidation and nitrite reduction processes can produce nitrate continuously, the peak value of 32.56mg/L was detected at 12h, fortunately, after 18h of culture, nitrate was completely removed by aerobic denitrification, to summarize, when hydroxylamine and nitrite coexist in the wastewater, the strain EN-F2 can effectively carry out nitrification and denitrification simultaneously.

The above results show that hydroxylamine can be completely and preferentially removed in both nitrate and nitrite supplementation systems. In particular, the removal rates for nitrite and total nitrogen were both significantly increased due to the addition of hydroxylamine, which delayed the removal rate for nitrate.

Enzyme activity detection

To further understand the denitrification mechanism of strain EN-F2 in the nitrification-denitrification process, the specific activity of Ammonia Monooxygenase (AMO) of strain EN-F2, the specific activity of hydroxylamine oxidoreductase (HAO) of strain EN-F2, the specific activity of Nitrate Reductase (NR) of strain EN-F2 and the specific activity of nitrite reductase (NIR) of strain EN-F2 were tested, and the results are shown in Table 1; wherein the specific Ammonia Monooxygenase (AMO) activity of the strain was determined by the company Wela, Guiyang, China;

detecting the specific activity of nitrite reductase (NIR) of the strain by using a nitrite reductase activity determination kit (COMIN), and reflecting the enzyme activity of NIR by the reduction of nitrite; the specific enzyme activity analysis method of hydroxylamine oxidoreductase (HAO) of the strain comprises the following steps: the strain EN-F2 was disrupted under the conditions of nitrite reductase kit, and the reaction system (20ml) contained enzyme extract, K₃[Fe(CN)₆](0.01mol/L)、EDTA(0.04mmol/L)(Xu,N.,Liao,M.,Liang,Y.,Guo,J.,Zhang,Y.,Xie,X.,Fan,Q.and Zhu,Y.2021.Biological nitrogen removal capability and pathways analysis of a novel low C/N ratio heterotrophic nitrifying and aerobic denitrifying bacterium(Bacillus thuringiensis strain WXN-23).EnvironRes 195,110797), Tris-HCl (10mmol/L)), and HONH₃Cl-N (15mg/L), after reacting at 25 ℃ for 45min, HAO enzyme activity was reacted by hydroxylamine reduction; the reaction system (20ml) for Nitrate Reductase (NR) contained enzyme extract, Tris-HCl (10mmol/L), nitrate (20mg/L), NADH (0.2mmol/L), and the reduction of nitrate was used to evaluate the enzymatic activity of NR.

TABLE 1 specific Activity of the strain EN-F2

Enzyme	Specific activity(U/mg)
		AMO	0.95±0.00
HAO	0.31±0.01
		NR	0.42±0.13
NIR	1.54±0.02

As can be seen from Table 1, the specific activity of AMO of EN-F2 is as high as 0.95U/mg protein, is significantly higher than Pseudomonas taiwanensis J488(0.65U/mg protein) (He, T, Xie, D, Ni, J., Li, Z.and Li, Z.2020.Nitro oxide produced direct from ammonium, nitrate and nitrate discharge and concentration discharge. journal of Hazardous Materials388), Bacillus thuringiensis WXN-23(0.11U/mg protein) (Xu, N., Liao, M., Liang, Y., Guo, J., Zhang, Y., Xie, X., Fan, Q.and Zhu, Y.2021.biological regeneration and delivery and moisture discharge and concentration discharge, respectively) (Xu, N., D, S.85, D.85, III, IV, III, IV, III, IV, III. EN-F2 has an AMO specific activity of 0.31U/mg protein, much higher than Glutaminobacter argentens EM-H8(0.07U/mg protein) (Chen, M., Ding, C., He, T., Zhang, M.and Wu, Q.2021. effective hydrogel removal of sodium hydroxide replacement by non-ferrous cellulose sodium argentation EM-8.M. Cheshows 288(Pt 1),132475), Acinetobacter calcoaceticus HNR (0.05U/mg protein) (Zhao, B., He, Y.L., Gheses No. J.and Zhang, X.F.2010. Heterophyllic protein HN NO: 02, III. 12. and III. Biocalcium carbonate replacement by S.5202. M.02, M.5202. Biocalcium chloride, M.5201. and M.5202. Biocalcium chloride, M.5202. 12. calcium carbonate calcium chloride, M.02. 12. Biocalcium chloride, M.5202. 12. calcium chloride, M.02, M.5202. calcium chloride, M.12. 12. calcium chloride, III, these results further demonstrate that ammonium and hydroxylamine can be rapidly removed by strain EN-F2. The strain EN-F2 has an NR (0.42U/mg protein) specific activity slightly lower than that of Pseudomonas verticitus LYM (0.47U/mg protein) (Shi, Z., Zhang, Y., ZHOUU, J., Chen, M.and Wang, X.2013.biological removal of nitrate and ammonium together with kinetic bottom by Paracoccus verticus verticitus LYM.Bioresourc technique 148, 144. the activity of this strain is obviously higher than that of Pseudomonas vertica NP5 (Yang, L., Wang, X.H., Cui, S., Ren, Y.X., Yu, J., Chen, N.Xiao, Q.Guo, K.K., Wang, W.H.201H., Curie, U.S., III, U.S. Pat. No. 7, D.7. 12. D.12. and S. Pat. No. 3. 7. D.12. biological removal No. 12. and No. 12. biological removal of Bacillus specimen No. 7. 12. biological reagent No. 7. biological reagent No. 12, D.12. 12. environmental reagent No. 7. 12. D.12. and No. 7. biological reagent No. 7. 12. biological reagent No. 7. 12. reagent No. 7. 12. reagent No. 7, C, S. 7. reagent No. 3. reagent No. 7, S. 3. reagent No. 7, S. 7. reagent No. 7, S. 3. reagent No. 7, S. 7. reagent, EN-F2 has the highest NIR specific activity (1.54U/mg protein), which is much higher than 0.04U/mg protein in providencianrettgeriyl (ZHAO, B., He, Y.L., Huang, J., Taylor, S.and Hughes, J.2010a. heterologous nitrogene removal by Providencia rettgeri strain J. Ind Microbiol Biotechnol 37(6),609 616), 0.10U/mg protein of Pseudomonas tarWagnensis J488 (He, T., Xie, D.Ni., J., Li, Z.and Li, Z.2020. Nitrosuooxide produced directly from strain, III. and K. moisture of tissue strain K. J. repair, K.3. repair, K.52. moisture, S.32, S.52. moisture, S.52. and S.3. moisture, K. of tissue strain, moisture and K. of moisture. The results show that the four specific activities of the strain EN-F2 are all higher than those of most reported aerobic denitrifying bacteria. NIR exhibits higher specific activity and lower nitrite removal capacity compared to the specific activity of AMO, HAO and NR, which may be due to suboptimal culturing or detection conditions for AMO, HAO and NR. In conclusion, the successful expression of all enzymes further indicates that the strain EN-F2 is a heterotrophic nitrification-aerobic denitrification bacterium.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Guizhou university

<120> a denitrifier and its application in treatment of nitrogen-containing wastewater

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> artificial seqnence

<220>

Claims

1. The strain is characterized in that the strain is a strain Pseudomonas taiwanensis EN-F2 with a preservation number of CCTCC NO: m20211515.

2. The method for screening a strain according to claim 1, comprising the steps of:

3. The screening method of claim 2, wherein the prescreening medium comprises: HONH₃Cl、NaNO₂、CaCI₂、K₂HPO₄、MgSO₄、KH₂PO₄、C₆H₅Na₃O₇·2H₂O and Fe₂(SO₄)₃And the pH of the primary screening medium was 7.20.

4. The screening method of claim 2, wherein the prescreening medium comprises: 0.0496g/L HONH₃Cl、0.0986g/L NaNO₂、0.014g/L CaCI₂、3.5g/L K₂HPO₄、0.04g/L MgSO₄、1.5g/L KH₂PO₄、3.676g/L C₆H₅Na₃O₇·2H₂O and 0.009g/L Fe₂(SO₄)₃。

5. The screening method according to claim 2, characterized in that the rescreening medium (BTB) comprises: (NH)₄)₂SO₄、MgSO₄、KH₂PO₄、Na₃C₆H₅O₇.2H₂O、FeSO₄·7H₂O,、CaCl₂Agar and bromothymol blue reagent.

6. The screening method of claim 5, wherein the rescreening medium comprises: 0.236g (NH)₄)₂SO₄,0.488g MgSO₄、1gKH₂PO₄、8.5g Na₃C₆H₅O₇.2H₂O、0.592g FeSO₄.7H₂O、0.094g CaCl₂18g agar and 1mL bromothymol blue reagent.

7. The screening method according to claim 6, wherein the concentration of the bromothymol blue reagent is 1.5g/100ml of ethanol.

8. The use of the strain of claim 1 in the treatment of nitrogen-containing wastewater.