A heat-resistant short-term denitrifying bacterium and its application in sewage treatment

Abstract

The present invention relates to the field of microbial wastewater treatment, and more specifically to a short-range denitrifying bacterium and its application in wastewater treatment. Halalkalibacterium halodurans HBCW-DN-HT01, deposited under CCTCC No. M2024664. This strain can grow rapidly under aerobic conditions, enabling the large-scale production of a bioagent suitable for short-range denitrification of industrial wastewater in summer or at high temperatures.

Description

Technical Field

[0001] The present invention relates to the field of sewage treatment by microorganisms, and in particular to a heat-resistant 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. However, with increasing wastewater discharge standards, conventional biological denitrification technologies are no longer sufficient for treating wastewater in extreme environments. This is particularly true when treating wastewater at high temperatures (>40°C), where these technologies face technical bottlenecks such as low biological activity, slow reaction rates, and poor treatment results.

[0003] Because many industrial wastewaters reach temperatures exceeding 45°C, previous research focused on controlled cooling followed by conventional biological denitrification. However, this requires the use of cooling equipment and can lead to corrosion. Directly applying thermophilic bacteria (whose metabolic rate is 3-10 times that of mesophilic bacteria) would shorten wastewater treatment time, improve efficiency, and reduce equipment investment and wear. Therefore, screening and isolating bacteria capable of efficient, short-range denitrification even under high-temperature conditions is of great significance in wastewater and waste gas treatment. 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 high-temperature resistant 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 thermostable short-range denitrifying bacterium, named HBCW-DN-HT01, belongs to the genus: Halophilic Bacillus. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on April 11, 2024, and its classification is named Halalkalibacterium halodurans HBCW-DN-HT01, the deposit number is CCTCC NO: M2024664.

[0007] The above-mentioned HBCW-DN-HT01 can grow rapidly under aerobic conditions to prepare a large amount of bacterial agent, and the bacterial agent is suitable for anoxic denitrification of high-temperature industrial wastewater, etc.

[0008] Compared with the existing technology, the present invention has the following advantages and beneficial effects: currently there are no reported short-term denitrifying bacteria that can tolerate high temperatures of 45°C in China, while the halophilic Bacillus HBCW-DN-HT01 involved in the present invention has a short-term denitrification efficiency of more than 80% in high-temperature sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the growth of HBCW-DN-HT01 bacteria under aerobic conditions.

[0010] Figure 2 This is the nitrate nitrogen removal by HBCW-DN-HT01 bacteria in culture medium under anaerobic and different temperature conditions.

[0011] Figure 3 It is the nitrate nitrogen removal and nitrite nitrogen accumulation of HBCW-DN-HT01 bacteria under the anoxic conditions of physical and chemical wastewater. DETAILED DESCRIPTION

[0012] 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.

[0013] The culture medium used in the following examples was prepared as follows:

[0014] 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, 0.8 g of KNO₃, 1.6 g of sodium acetate; and 1 mL of trace element solution. For solid culture medium, add 15 g / L of agarose to the basal culture medium.

[0015] 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

[0016] 10 mL of activated sludge from a biochemical leachate treatment system in Shenzhen was placed in 100 mL of basal culture medium. After continuous enrichment culture at 45°C for 3-5 days, three rounds of plate streaking were performed. Single colonies with obvious differences in colony morphology were picked and incubated in 5 mL of basal culture medium. After shaking culture at 45°C and 140 rpm for 2-3 days, genomic DNA of the isolated strains was extracted using the Genomic Rapid Bacterial Genomic DNA Isolation Kit (Shanghai Biotech). 16S rDNA was amplified by PCR and sequenced for molecular biological identification. The upstream and downstream primers used for PCR amplification were:

[0017] Upstream primer 27F: 5′-AGA-GTTTGATCATGGCTCAG-3′ (SEQ ID NO. 1).

[0018] Downstream primer 1492R: 5′-TAC-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO. 2).

[0019] The purification and sequencing of the PCR product were completed by Shanghai Bioengineering, and the sequencing result is shown in SEQ ID NO.3. The 16SrDNA sequencing results showed that the isolated strain was halophilic Bacillus ( Halalkalibacterium halodurans) , and named it HBCW-DN-HT01. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on April 11, 2024, and its classification was named Halalkalibacterium halodurans HBCW-DN-HT01, the deposit number is CCTCC NO: M2024664. Example

[0020] Preparation of inoculum: Inoculate 1 mL of bacterial solution into the basal culture medium and culture at 45°C and 120 rpm for 2-3 days to obtain the inoculum solution.

[0021] Figure 1 The implementation method is: set the initial nitrate nitrogen concentration of the basal culture medium to 40 mg / L, 80 mg / L, 160 mg / L, 320 mg / L and 640 mg / L respectively, add inoculum at a ratio of 1%, culture at 45 ° C and 140 r / min, and take samples after 24 hours to measure the reduction in nitrate nitrogen.

[0022] according to Figure 1 By the end, the nitrate nitrogen removal rates of the groups with initial nitrate nitrogen of 40-320 mg / L were significantly higher than those of the group with initial nitrate nitrogen of 640 mg / L. In terms of the absolute value of nitrate nitrogen reduction, it shows that the initial nitrate nitrogen of 320 mg / L is most suitable for the growth of the bacteria. Example

[0023] Preparation of inoculum: Set the pH of the basal culture medium to 8, add inoculum at a ratio of 1%, culture at 45°C and 120 rpm for about 2-3 days, and centrifuge to wash away nitrate and nitrogen to obtain the inoculum solution.

[0024] Figure 2 The implementation method is as follows: add 5% inoculum to 100 mL of basal culture medium, culture under anoxic conditions, 15℃, 30℃ and 45℃, 120 r / min, and take samples to detect NO2 - -N, NO3 - -N.

[0025] according to Figure 2 Under anoxic conditions, at 45°C, the accumulation of nitrite nitrogen reached about 110 mg / L, accounting for 80% of the nitrate nitrogen reduction, which was significantly higher than the nitrite nitrogen accumulation and nitrate nitrogen removal in other temperature groups. This shows that 45°C is the optimal condition for HBCW-DN-HT01 bacterial agent during the anoxic process, and it can accumulate nitrite nitrogen in large quantities (i.e., short-term denitrification). Example

[0026] The preparation method of the inoculum solution is the same as that in Example 3.

[0027] Figure 3 The implementation method is as follows: take a certain factory's physical and chemical wastewater (its nitrogen is mainly nitric nitrogen, with a concentration of about 1600mg / L), take 100ml of wastewater, dilute it to 500ml with culture medium, and use 1600mg / L sodium acetate as the only carbon source. Set up a blank control group and a short-range denitrifying bacteria WH-01 control group, and the experimental group is inoculated with 5% inoculum. Sampling is carried out at 45℃ and 120r / min, and NO2 - -N and NO3 - -N value.

[0028] Depend on Figure 3 As can be seen, the blank control group showed no accumulation of nitrite at the end of the treatment, with nitrate decreasing by only 3.5 mg / L. While the WH-01 control group saw a decrease in nitrate, it also showed no accumulation of nitrite, which is not conducive to short-term denitrification. In contrast, the experimental group, using the HBCW-DN-HT01 inoculant, removed approximately 108 mg / L of nitrate and accumulated approximately 85 mg / L of nitrite. This nitrite accumulation accounted for 79% of the nitrate reduction. These results demonstrate that the HBCW-DN-HT01 inoculant significantly accumulates nitrite in wastewater (i.e., short-term denitrification).

[0029] 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 thermostable short-range denitrifying bacterium, which is classified and named Halalkalibacterium halodurans HBCW-DN-HT01, the deposit number is CCTCC NO: M2024664.

2. Use of the high-temperature resistant short-range denitrifying bacteria according to claim 1 in sewage treatment, wherein the sewage is sewage containing nitric nitrogen.

3. The use according to claim 2, characterized in that The sewage has a water temperature of 45°C.

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