Short-range denitrifying bacterium capable of selectively accumulating nitrite and application thereof

Abstract

The invention discloses short-cut denitrifying bacteria capable of selectively accumulating nitrite and application of the short-cut denitrifying bacteria, the denitrifying bacteria are Enterobacter cloacae USX-CL, the denitrifying bacteria are preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241769. The short-cut denitrifying bacteria can reduce NO3 <-> into NO2 <-> at a low C / N ratio, and efficient accumulation of NO2 <-> is realized; the discovery of the new strain can provide a substrate for an anaerobic ammonia oxidation process, and promote the coupling application of short-cut denitrification and anaerobic ammonia oxidation processes in actual sewage treatment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms for degrading organic pollutants, and more specifically to a short-range denitrifying bacterium capable of selectively accumulating nitrite and an application thereof. Background Art

[0002] Anaerobic ammonium oxidation does not require an external organic carbon source and can achieve the removal of ammonia nitrogen under autotrophic conditions. It is a green emission reduction biological denitrification process with a very broad application prospect. As an electron acceptor in the anaerobic ammonium oxidation process, NO 2 - The stable supply of NO is the key factor affecting the long-term stability of the anaerobic ammonium oxidation process. 2 - Yes NO 3 - The intermediate products in the biological denitrification process are highly toxic to microorganisms, and their selective accumulation requires strict control of the denitrifying bacteria and process conditions. For example, a high C / N ratio will result in complete denitrification, leading to nitrogen loss; while a low C / N ratio will inhibit nitrate reduction activity and reduce NO 2 - Therefore, screening can selectively accumulate NO 2 - Short-range denitrifying bacteria can provide a stable reaction substrate for anaerobic ammonium oxidation. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a short-range denitrifying bacterium that can selectively accumulate nitrite and its application. The denitrifying bacterium is a new strain of Enterobacter cloacae USX-CL that has natural growth advantages and uses nitrate as an electron acceptor. It can convert NO into nitrite at a low C / N ratio. 3 - Restore to NO 2 - , and realize NO 2 - The discovery of this new strain can provide a substrate for the anaerobic ammonium oxidation process and promote the coupling application of short-range denitrification and anaerobic ammonium oxidation processes in actual sewage treatment.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] A short-range denitrifying bacterium capable of selectively accumulating nitrite. The denitrifying bacterium is Enterobacter cloacae USX-CL, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241769.

[0006] Furthermore, the 16S rDNA sequence of the denitrifying bacteria is shown as SEQ ID NO.1.

[0007] Furthermore, the morphological characteristics of the denitrifying bacteria are: the colonies are white and have no spores.

[0008] Furthermore, the expression of the nitrate reductase of the denitrifying bacteria is restricted by dissolved oxygen conditions, and the denitrifying bacteria can use nitrate as the only nitrogen source and sodium acetate, sodium succinate or sodium citrate as the carbon source to perform short-range denitrification under anaerobic conditions to accumulate nitrite nitrogen.

[0009] Furthermore, the denitrifying bacteria are obtained by screening through the following method:

[0010] The activated sludge from the sewage treatment plant was inoculated into the enrichment medium for cultivation until the nitrate nitrogen content in the system was 0 and nitrite nitrogen had obvious accumulation. Then, the obtained culture solution was taken and added to the new enrichment medium at a volume ratio of 10% for domestication. The above steps were repeated for continuous domestication for several times. 2 mL of the final domestication solution was taken and heated at 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The culture medium was diluted with a gradient of 1 mL, and then 1 mL of the diluted acclimation solution was spread on the solid screening medium, and inverted culture was performed in a constant temperature incubator to obtain single colonies; the obtained single colonies were streaked and purified three times to obtain pure strains, which were inoculated into LB medium for enrichment, stored in glycerol tubes, and frozen.

[0011] Furthermore, the enrichment medium is composed of: CH 3 COONa 2.7g / L, NaNO 3 1.0g / L, KH 2 PO 4 1.0g / L, Na 2 HPO 4 12H 2 O 1.0g / L, MgSO 4 0.1g / L, trace elements 2mL / L, solvent is deionized water, pH=7.0;

[0012] The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 5g / L, CuSO 4 ·5H 2 O0.5g / L, CoCl 2 6H2 O 0.2g / L, the solvent is deionized water.

[0013] Furthermore, a new enrichment medium was taken and 2 wt % agar was added to obtain a solid screening medium.

[0014] Furthermore, the LB culture medium consists of 10.0 g / L NaCl, 10.0 g / L peptone, and 5.0 g / L yeast powder, the solvent is deionized water, and the pH value is 7.0.

[0015] Application of a short-range denitrifying bacterium capable of selectively accumulating nitrite in the treatment of nitrate-nitrogen wastewater.

[0016] Furthermore, the short-range denitrification operating conditions of the denitrifying bacteria in sewage treatment are: temperature 30°C, pH 7.0-7.5, and carbon-nitrogen ratio 2:1-8:1.

[0017] The beneficial effects of the present invention are:

[0018] 1. The bacteria has short-range denitrification function and can convert NO 3 - Reduction to NO 2 - , the conversion rate reached over 90%;

[0019] 2. The bacteria have good short-range denitrification function under the condition of C / N of 2-8;

[0020] 3. The bacteria grows relatively slowly and produces less sludge, which can reduce the amount of sludge produced and reduce treatment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a colony morphology diagram of Enterobacter cloacae USX-CL in this example;

[0022] Figure 2 This is a colony morphology of Enterobacter cloacae USX-CL under Gram staining microscopy in this example;

[0023] Figure 3 This is the phylogenetic tree of Enterobacter cloacae USX-CL in this example;

[0024] Figure 4 This is the anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL using sodium nitrate as the only nitrogen source;

[0025] Figure 5 This is the anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL using sodium nitrite as the only nitrogen source;

[0026] Figure 6This is the anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL using sodium acetate as the carbon source;

[0027] Figure 7 This is the anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL using sodium succinate as the carbon source;

[0028] Figure 8 This is the anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL using sodium citrate as the carbon source;

[0029] Fig. 9 Anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL under C / N ratio of 0;

[0030] Fig.10 Anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL under C / N ratio of 2;

[0031] Fig.11 Anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL under C / N ratio of 4;

[0032] Fig.12 Anaerobic denitrification performance diagram of Enterobacter cloacae USX-CL under C / N ratio of 6;

[0033] Fig.13 Anaerobic denitrification performance of Enterobacter cloacae USX-CL at a C / N ratio of 8. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1 Screening of anaerobic short-range denitrifying bacteria

[0036] Take 20mL of activated sludge (activated sludge is taken from the secondary sedimentation tank of the biochemical treatment process of the stainless steel pickling wastewater treatment plant) and add it to a triangular flask containing 150mL of enrichment culture medium. Place it on a shaker at 30°C and 150rpm for dark culture. Take samples every 48 hours until the nitrate nitrogen content in the system is 0 and nitrite nitrogen has obvious accumulation. Take the culture solution and add it to the new enrichment culture medium at a volume ratio of 10% for domestication; repeat the above steps for five consecutive domestications, take 2mL of the final domestication solution and add it to the new enrichment culture medium at a volume ratio of 10. -2 , 10 -3 , 10 -4, 10 -5 , 10 -6 Then take 1 mL of the diluted acclimation solution and evenly spread it on the solid screening medium (take the new enrichment medium, add 2wt% agar to obtain the solid screening medium), and invert it in a constant temperature incubator at 30℃ to obtain a single colony; the obtained single colony was streaked and purified 3 times to obtain a pure strain, which was inoculated into LB medium for enrichment culture and stored in a glycerol tube. Store it in a frozen state at -80℃.

[0037] The composition of the enrichment medium is: CH 3 COONa 2.7g / L, NaNO 3 1.0g / L, KH 2 PO 4 1.0g / L, Na 2 HPO 4 12H 2 O 1.0g / L, MgSO 4 0.1g / L, trace elements 2mL / L, the solvent is deionized water, pH=7.0; the trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

[0038] The composition of LB medium is: NaCl 10.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, the solvent is deionized water, pH=7.0.

[0039] All the above culture media were sterilized at 121°C for 20 min before use.

[0040] Example 2 Identification of Enterobacter cloacae USX-CL

[0041] a. Bacteriological morphological characteristics of Enterobacter cloacae USX-CL:

[0042] like Figure 1 , 2 As shown, the strain is a Gram-negative bacterium, the colonies are off-white, opaque, with relatively neat edges and a relatively smooth surface.

[0043] b. 16S rDNA sequence analysis and phylogenetic tree of Enterobacter cloacae USX-CL

[0044] The 16S rDNA sequence of denitrifying bacteria is:

[0045] (SEQ ID NO.1)

[0046] The sequence results were submitted to the NCBI database for homology comparison. Figure 3 The phylogenetic tree of the strain confirmed that the bacterium had 99.75% similarity with Enterobacter cloacae published in the database.

[0047] Based on the above results, the denitrifying bacteria strain of the present invention was identified as Enterobacter cloacae, and was named Enterobacter cloacae USX-CL. It was deposited in China Center for Type Culture Collection on August 9, 2024, with a deposit number of CCTCC NO: M 20241769, and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0048] Example 3 Denitrification performance of Enterobacter cloacae USX-CL

[0049] The strain Enterobacter cloacae USX-CL was inoculated at a 5% inoculum into a denitrification medium with nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, and cultured under anaerobic conditions at an initial nitrogen concentration of 100 mg / L, 30 ℃ and 150 rpm. Samples were taken at 0h, 4h, 8h, 12h, 24h, and 48h, and the supernatant was obtained by centrifugation to determine the concentrations of nitrate nitrogen, nitrite nitrogen, ammonia nitrogen and total nitrogen.

[0050] The composition of the culture medium (DM1) with nitrate as the sole nitrogen source is: NaNO 3 0.61g / L, C 4 H 4 Na 2 O 4 3.375g / L, KH 2 PO 4 0.1g / L, MgSO 4 0.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O0.2g / L, the solvent is deionized water.

[0051] The composition of the medium (DM2) with nitrite as the only nitrogen source is: NaNO 2 0.49g / L, C 4 H 4 Na 2 O 4 3.375g / L, KH 2 PO 4 0.1g / L, MgSO 40.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O0.2g / L, the solvent is deionized water.

[0052] like Figure 4 As shown, strain USX-CL has a good denitrification effect on the simulated 100 mg / L nitrate nitrogen wastewater. When nitrate is used as the only nitrogen source, the nitrate removal rate can reach 80% within 24 hours. The degradation rate is relatively slow between 24 hours and 144 hours, and the final nitrate removal rate is about 87%. In addition, it was found that nitrite accumulated rapidly within 0-24 hours. During the 24h-144h reaction stage, nitrite still did not degrade, indicating that strain USX-CL has good short-range denitrification performance and can selectively accumulate nitrite. When nitrite is used as the only nitrogen source, it was found that nitrite could not be further reduced. The results are shown in Figure 5 , further confirming that the strain has a good ability to accumulate nitrite.

[0053] Example 4 Denitrification performance of Enterobacter cloacae USX-CL under different carbon source conditions

[0054] Enterobacter cloacae USX-CL was inoculated at a 5% inoculum size into a denitrification medium with sodium nitrate as the nitrogen source and sodium acetate, sodium succinate or sodium citrate as the carbon source. The culture was carried out at an initial nitrogen concentration of 100 mg / L, anaerobic conditions, 30°C, and 150 rpm. Part of the culture solution was taken at 4h, 8h, 12h, 24h, 48h, 72h, and 96h, and the concentrations of nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen were measured.

[0055] The composition of the culture medium with sodium nitrate as the nitrogen source and sodium acetate as the carbon source is: NaNO 3 0.610g / L, CH 3 COONa3.417g / L, KH 2 PO 4 0.1g / L, MgSO 4 0.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2O 0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

[0056] The composition of the culture medium with sodium nitrate as the nitrogen source and sodium succinate as the carbon source is: NaNO 3 0.610g / L, C 4 H 4 Na 2 O 4 3.417g / L, KH 2 PO 4 0.1g / L, MgSO 4 0.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

[0057] The composition of the culture medium with sodium nitrate as the nitrogen source and sodium citrate as the carbon source is: NaNO 3 0.610g / L, C 6 H 5 Na 3 O 7 3.583g / L, KH 2 PO 4 0.1g / L, MgSO 4 0.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

[0058] Carbon source is also an important factor affecting the denitrification effect. Sodium acetate, sodium succinate and sodium citrate were used as carbon sources. Samples were taken at regular intervals to measure the concentrations of nitrate, nitrite and ammonium salt. Figure 6-Figure 8 As shown. When sodium acetate was used as the carbon source, it was found that the nitrate nitrogen content dropped sharply between 4h and 8h. When sodium succinate and sodium citrate were used as carbon sources, the removal rate of nitrate nitrogen in the denitrification medium was relatively slow. In these three culture media, after 96h of reaction, the final nitrate nitrogen removal rate was about 80%, and nitrite nitrogen was always in an accumulation state. In these three denitrification culture solutions, no ammonium ions were detected. This shows that in these three culture media, the denitrification pathway is to reduce nitrate to nitrite.

[0059] Example 5 Denitrification performance of Enterobacter cloacae USX-CL under different carbon-nitrogen ratios

[0060] C / N is an important factor affecting denitrification efficiency. The strain USX-CL was inoculated with 5% inoculum into denitrification medium with sodium nitrate as nitrogen source and C / N ratio of 0, 2, 4, 6, and 8 (sodium succinate was used as carbon source when C / N=2, 4, 6, and 8), and cultured under the conditions of initial nitrogen concentration of 100 mg / L, anaerobic, 30°C and 150 r / min. The reaction solution was taken at regular intervals and centrifuged to measure the concentrations of nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen in the reaction solution.

[0061] The denitrification culture medium in this example is composed of: NaNO 3 0.606g / L, C 4 H 4 Na 2 O 4 0-2.70g / L (when C / N=0, sodium succinate is 0; when C / N=2, sodium succinate is 0.674g / L; when C / N=4, sodium succinate is 1.35g / L; when C / N=6, sodium succinate is 2.02g / L; when C / N=8, sodium succinate is 2.70g / L), KH 2 PO 4 0.1g / L, MgSO 4 0.15g / L, trace elements 2mL / L, the solvent is deionized water; pH=7.0. The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O0.5g / L, CuSO 4 ·5H 2 O 0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

[0062] Using sodium nitrate as nitrogen source and sodium succinate as carbon source, the denitrification performance of Enterobacter cloacae USX-CL at different C / N ratios was shown in Figure 2. Figure 9-13 As shown, it was found that when C / N=8 (sodium nitrate 0.606g / L, sodium succinate 2.70g / L), the removal rate of nitrate nitrogen was 84% ​​after 5 days of reaction; when C / N=6 (sodium nitrate 0.606g / L, sodium succinate 2.02g / L), the removal rate of nitrate nitrogen was 80% after 5 days of reaction; when C / N=4 (sodium nitrate 0.606g / L, sodium succinate 1.35g / L), the removal rate of nitrate nitrogen was 87% after 5 days of reaction; when C / N=2 (sodium nitrate 0.606g / L, sodium succinate 0.674g / L), the removal rate of nitrate nitrogen was 89% after 5 days of reaction; when C / N=0 (sodium nitrate 0.606g / L, sodium succinate 0), after 5 days of reaction, the removal rate of nitrate nitrogen was found to be 47%. It can be seen that under the condition of low carbon-nitrogen ratio, the bacteria can still reduce about 89% of nitrate nitrogen. Under the condition of no carbon source, due to the lack of carbon source in the system, the growth of microorganisms is restricted, resulting in a decrease in the denitrification rate, and only 47% of nitrate nitrogen can be reduced. In summary, the bacteria still has a high nitrate nitrogen reduction ability under the condition of low carbon-nitrogen ratio, which can save carbon source, avoid secondary pollution of the environment by excessive carbon-containing compounds, and has high economic benefits.

[0063] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A short-range denitrifying bacterium that can selectively accumulate nitrite, It is characterized in that The denitrifying bacteria is Enterobacter cloacae USX-CL, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCCNO: M 20241769.

2. A short-range denitrifying bacterium capable of selectively accumulating nitrite according to claim 1, It is characterized in that The 16S rDNA sequence of the denitrifying bacteria is shown as SEQ ID NO.

1.

3. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 1, It is characterized in that The morphological characteristics of the denitrifying bacteria are: the colonies are white and have no spores.

4. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 1, It is characterized in that The expression of the nitrate reductase of the denitrifying bacteria is restricted by dissolved oxygen conditions. The denitrifying bacteria can use nitrate as the only nitrogen source and sodium acetate, sodium succinate or sodium citrate as the carbon source to perform short-range denitrification under anaerobic conditions to accumulate nitrite nitrogen.

5. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 1, It is characterized in that The denitrifying bacteria are obtained by screening through the following method: The activated sludge from the sewage treatment plant was inoculated into the enrichment medium for cultivation until the nitrate nitrogen content in the system was 0 and nitrite nitrogen had obvious accumulation. Then, the obtained culture solution was taken and added to the new enrichment medium at a volume ratio of 10% for domestication. The above steps were repeated for continuous domestication for several times. 2 mL of the final domestication solution was taken and heated at 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The culture medium was diluted with a gradient of 1 mL, and then 1 mL of the diluted acclimation solution was spread on the solid screening medium, and inverted culture was performed in a constant temperature incubator to obtain single colonies; the obtained single colonies were streaked and purified three times to obtain pure strains, which were inoculated into LB medium for enrichment, stored in glycerol tubes, and frozen.

6. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 5, It is characterized in that The enrichment medium consists of: CH 3 COONa 2.7g / L, NaNO 3 1.0g / L, KH 2 PO 4 1.0g / L, Na 2 HPO 4 12H 2 O1.0g / L, MgSO 4 0.1g / L, trace elements 2mL / L, solvent is deionized water, pH=7.0; The trace elements are: EDTA 1g / L, ZnSO 4 0.2g / L, MnCl 2 ·4H 2 O 5g / L, CuSO 4 ·5H 2 O0.5g / L, CoCl 2 6H 2 O 0.2g / L, the solvent is deionized water.

7. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 6, It is characterized in that Take new enrichment medium and add 2 wt % agar to obtain solid screening medium.

8. A short-range denitrifying bacteria capable of selectively accumulating nitrite according to claim 5, It is characterized in that The LB culture medium consists of 10.0 g / L NaCl, 10.0 g / L peptone, and 5.0 g / L yeast powder; the solvent is deionized water; and the pH value is 7.

0.

9. Use of the short-range denitrifying bacteria capable of selectively accumulating nitrite according to any one of claims 1 to 8 in the treatment of nitrate-nitrogen wastewater.

10. Use of a short-range denitrifying bacteria capable of selectively accumulating nitrite in the treatment of nitrate nitrogen wastewater according to claim 9, It is characterized in that The short-range denitrification operating conditions of the denitrifying bacteria in sewage treatment are: temperature 30°C, pH 7.0-7.5, and carbon-nitrogen ratio 2:1-8:1.

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