A Short-Term Denitrifying Bacteria and Its Application in Wastewater Treatment

Abstract

The present invention relates to the field of microbial wastewater treatment, and more particularly to a short-range denitrifying bacterium and its application in wastewater treatment. The bacterium is Pseudomonas stutzeri WH-01. The strain is deposited in the China Center for Type Culture Collection and is classified as Stutzerimonas stutzeri WH-01, with a deposit number of CCTCC NO: M 20222100. The Pseudomonas stutzeri can grow rapidly under aerobic conditions to produce a large amount of bacterial agent, which is suitable for short-range denitrification of municipal sewage, industrial sewage, etc.

Description

Technical Field

[0001] The present invention relates to the field of sewage treatment by microorganisms, and in particular to a short-range denitrifying bacterium and its application in sewage treatment. Background Art

[0002] The short-cut denitrification-anaerobic ammonium oxidation process significantly reduces carbon source costs because it only requires a short-cut denitrification of nitrate nitrogen to nitrite (while avoiding further reduction of nitrite). Nitrite then reacts with ammonia nitrogen to produce nitrogen gas through anaerobic ammonium oxidation. Currently, this process is a hot topic in wastewater biological denitrification research and is beginning to be applied in practice.

[0003] Previous research primarily aimed to achieve short-term denitrification by controlling parameters such as nitrate-nitrogen concentration, carbon-nitrogen ratio, and pH. However, in practice, fluctuations in wastewater quality make precise and real-time control of these parameters difficult (particularly challenging for upgrading existing plants). Furthermore, from an application perspective, microbial agents are widely used to enhance the denitrification effect of actual wastewater. Therefore, developing a short-term denitrifying agent is of great significance for its production and application. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, provide a novel short-range denitrifying bacterium, and provide the application of the bacterium in sewage treatment.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A short-range denitrifying bacterium, Pseudomonas stutzeri WH-01. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on December 29, 2022, and its classification was named Stutzerimonas stutzeri WH-01, the deposit number is CCTCC NO: M 20222100.

[0007] The above-mentioned Pseudomonas stutzeri can grow rapidly under aerobic conditions to prepare a large amount of bacterial agent, which is suitable for short-range denitrification of municipal sewage, industrial sewage, etc.

[0008] Compared with the prior art, the present invention has the following advantages and beneficial effects: there is no reported short-range denitrifying Pseudomonas stutzeri, while the Pseudomonas stutzeri involved in the present invention has a short-range denitrification efficiency of more than 95% in sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the growth of WH-01 bacteria under aerobic conditions.

[0010] Figure 2 This is the accumulation of nitrite nitrogen by WH-01 bacteria in culture medium under anaerobic conditions.

[0011] Figure 3 This is the accumulation of nitrite nitrogen in the steel pipe factory wastewater under anoxic conditions in the control group.

[0012] Figure 4 This is the accumulation of nitrite nitrogen in the steel pipe factory wastewater under anoxic conditions in the experimental group. DETAILED DESCRIPTION

[0013] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0014] The culture medium used in the following examples was prepared as follows:

[0015] For basal culture medium, add the following ingredients to 1 L of distilled water: 0.5 g of CaCl₂⋅2H₂O, 0.4 g of FeSO₄⋅7H₂O, 1 g of K₂HPO₄, 0.05 g of MgSO₄⋅7H₂O, 2 g of NaCl, 1.5 g of NaHCO₃, 0.4 g of NH₄Cl, 0.25 g of sodium succinate, and 1 mL of trace element solution. For solid culture medium, add 15 g / L of agarose to the basal culture medium.

[0016] Fermentation medium: Add the following ingredients to 1 L of distilled water: 0.5 g of CaCl₂⋅2H₂O, 0.4 g of FeSO₄⋅7H₂O, 1 g of K₂HPO₄, 0.05 g of MgSO₄⋅7H₂O, 2 g of NaCl, 1.5 g of NaHCO₃, 1.15 g of NH₄Cl, 1.5 g of glucose, and 1 mL of trace element solution. The solid medium consists of basal medium supplemented with 15 g / L of agarose.

[0017] Anoxic short-range denitrification medium: 0.324 g / L KNO3 replaces NH4Cl in the basic medium, and 1 g / L sodium acetate is added.

[0018] Trace element solution: add the following ingredients to every 1 L of distilled water: CoCl2⋅6H2O 0.375 g, CuSO4⋅5H2O 0.075 g, EDTA 0.5 g, H3BO4 0.014 g, MnCl2⋅2H2O 0.3 g, NaMoO4⋅2H2O 0.22 g, ZnSO4⋅7H2O 0.3 g. Example 1

[0019] 10 mL of activated sludge from the secondary sedimentation tank of a domestic sewage treatment system in Wuhan, Hubei Province, was placed in 100 mL of basal culture medium. After continuous enrichment culture at 12°C for 60 days, three rounds of plate streaking were performed. Single colonies with obvious differences in colony morphology were selected and incubated in 5 mL of basal culture medium. After shaking culture at 12°C and 140 rpm for 5 days, genomic DNA of the isolated strains was extracted using the Genomic Rapid Bacterial Genomic DNA Isolation Kit (Shanghai Sangon). 16S rDNA was amplified by PCR and sequenced for molecular biological identification. The upstream and downstream primers used for PCR amplification were:

[0020] Upstream primer 27F: 5′-AGA-GTTTGATCATGGCTCAG-3′ (SEQ ID NO. 1).

[0021] Downstream primer 1492R: 5′-TAC-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO. 2).

[0022] The purification and sequencing of the PCR products were completed by Shanghai Bioengineering, and the sequencing results are shown in SEQ ID NO. 3. The 16SrDNA sequencing results showed that the isolated strain was Pseudomonas stutzeri (formerly known as Pseudomonas stutzeri , now known as Stutzerimonas stutzeri ), and named it Pseudomonas stutzeri WH-01. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on December 29, 2022, and its classification was named Stutzerimonas stutzeri WH-01, the deposit number is CCTCC NO: M 20222100. Example 2

[0023] Preparation of inoculum: inoculate 1 mL of bacterial solution into basal culture medium and culture at 15°C and 140 rpm for 5 days to obtain the inoculum solution.

[0024] Figure 1 The implementation method is as follows: the pH of the fermentation medium is set to 5, 6, 7, 8 and 9 respectively, the inoculum is added at a ratio of 1%, and the culture is carried out at 35°C and 140 r / min. The OD value is measured every 8 hours. 600 To determine the best growth.

[0025] according to Figure 1 : At the stable stage, the OD of the initial pH 6~8 group 600 The OD values ​​of the pH 5 and pH 9 groups were significantly higher than those of the pH 5 and pH 9 groups. 600, indicating that the initial pH 6~8 is most suitable for the growth of the bacteria. Moreover, the initial OD 600 0.01, and it only takes 24 hours for the pH 6-8 group to reach its peak value (OD 600 More than 1.0), that is, the biomass increased by more than 100 times within 24 hours, indicating that the bacteria grew rapidly and was convenient for the fermentation production of the inoculant. Example 3

[0026] Preparation of inoculum: Set the pH of the fermentation medium to 8, add inoculum at a ratio of 1%, and culture at 35°C and 140 rpm for 1 day until the OD 600 The concentration is about 1.0, and the inoculated bacterial solution is obtained after centrifugation to wash away the ammonia nitrogen.

[0027] Figure 2 The implementation method is: add 10% inoculum to 100mL anoxic short-range denitrification culture medium, culture at 35℃ and 140r / min for 48h, and sample and detect NO2 every 24h. - -N, NO3 - -N and TN.

[0028] according to Figure 2 : Under anoxic conditions, within 24 hours, the accumulation of nitrite nitrogen reached about 270 mg / L, accounting for 81% of the reduction of nitrate nitrogen. At this time, the residual nitrate nitrogen still exceeded 28 mg / L, indicating that the WH-01 bacterial agent can accumulate a large amount of nitrite nitrogen during the anoxic process (i.e. short-term denitrification). Example 4

[0029] The preparation method of the inoculum solution is the same as that in Example 3.

[0030] Figure 3 、 4 The implementation method is as follows: Take wastewater from a steel pipe factory (its nitrogen is mainly nitric nitrogen, with a concentration of about 1500mg / L). Take 100ml of wastewater and filter it with a 0.22μm filter membrane, dilute it to 500ml with pure water, add 0.5g of K2HPO4, and use 1500mg / L sodium acetate as the only carbon source. Set up a blank control group and an experimental group inoculated with 10% inoculum. Sampling is carried out every 24 hours at 35℃ and 140r / min. The NO2 - -N, NO3 - -N and TN values.

[0031] Depend on Figure 3 It can be seen that although the nitrate nitrogen concentration of the control group (without WH-01 inoculant) decreased by about 25 mg / L within 96 hours, the accumulation of nitrite nitrogen was only about 3 mg / L. In contrast, by 72 hours, WH-01 inoculant had removed about 250 mg / L of nitrate nitrogen ( Figure 4), and accumulated approximately 240 mg / L of nitrite nitrogen. This nitrite nitrogen accumulation accounted for 96% of the nitrate nitrogen reduction. Correspondingly, the total nitrogen removal was only about 5 mg / L. These results indicate that WH-01 can significantly accumulate nitrite nitrogen in wastewater (i.e., short-term denitrification).

[0032] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A short-range denitrifying Pseudomonas stutzeri ( Stutzerimonas stutzeri )WH-01, the accession number is CCTCCNO: M 20222100.

2. Use of the Pseudomonas stutzeri according to claim 1 in sewage treatment, wherein the sewage is nitric nitrogen-containing sewage.

3. The use according to claim 2, characterized in that Able to accumulate nitrite under anaerobic conditions.

4. The use according to claim 2 or 3, characterized in that The short-cut denitrification efficiency of Pseudomonas stutzeri in sewage exceeds 95%.

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