Bacillus deglycans and application thereof in nitrogen removal treatment of nitrogenous aquaculture wastewater

Abstract

The invention relates to the technical field of nitrogen-containing aquaculture wastewater treatment, in particular to bacillus deglycans and application thereof in nitrogen removal treatment of nitrogen-containing aquaculture wastewater. The invention relates to a strain of bacillus subtilis glycaniformis, which is a strain DQ-1 of bacillus subtilis glycaniformis, the strain is preserved in China Center for Type Culture Collection on June 13, 2024, and the preservation number of the strain is CCTCC M 20241211. The bacterial strain of the genus has a heterotrophic nitrification-aerobic denitrification effect, and compared with other heterotrophic nitrification-aerobic denitrification bacteria, the bacterial strain shows a stronger denitrification effect in a shorter time.

Description

Technical Field

[0001] The present invention relates to the technical field of nitrogen-containing aquaculture wastewater treatment, and particularly relates to a strain of Paenibacillus glucanolyticus and its application in the denitrification treatment of nitrogen-containing aquaculture wastewater. Background Art

[0002] In aquaculture, aquaculture ponds contain a large amount of fish and shrimp feces, pathological secretions, bait decomposition products, and dead organisms. During the process of sedimentation and decomposition of these organic matters, a large amount of inorganic nitrogen products such as ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen will be formed. These inorganic nitrogen not only affect the life and health of aquaculture animals, but also directly discharged into the natural environment without treatment will cause water eutrophication and damage the ecological environment. Therefore, nitrogen-rich aquaculture wastewater needs to be denitrified and meet the standards before it can be discharged into the natural environment.

[0003] Biological denitrification is to use the metabolic activities of organisms to reduce the concentration of nitrogen-containing pollutants in water bodies. It is a widely used method for treating nitrogen-containing sewage at present, with advantages such as good treatment effect, stable and reliable treatment process, and convenient operation and management. The biological denitrification wastewater treatment technology mainly reduces the ammonia nitrogen in the wastewater to nitrogen gas through the action of functional bacteria such as ammonia-oxidizing bacteria, nitrifying bacteria, and denitrifying bacteria, so as to achieve the purpose of denitrification. During the treatment process, nitrifying bacteria and nitrite bacteria convert ammonia nitrogen into nitrate nitrogen. Nitrate nitrogen has low chemical potential energy and high stability, and is prone to nitrate nitrogen accumulation. Denitrifying bacteria act on nitrate nitrogen in water bodies, gradually reducing it to nitrite nitrogen, nitric oxide, and nitrous oxide, and finally being reduced to nitrogen gas and released into the atmosphere to complete the cycle of nitrogen elements, which is an important link in wastewater denitrification. Traditional biological denitrification is completed by the aerobic autotrophic nitrification of nitrifying bacteria and the anaerobic heterotrophic denitrification of denitrifying bacteria. This process requires two independent reaction spaces with aerobic and anaerobic conditions respectively for nitrification and denitrification to achieve the purpose of denitrification. Therefore, such a treatment method occupies a large space, has a high operating cost, and the process is relatively cumbersome.

[0004] Until the 1980s, a strain of aerobic denitrifying bacteria was first reported, and the aerobic denitrification technology was recognized and further promoted. Aerobic denitrifying bacteria can use oxygen and nitrate as electron acceptors in the presence of oxygen, and carry out nitrification and denitrification reactions simultaneously, so as to quickly remove organic nitrogen. This technology has important application value in the field of biological denitrification. After more than 30 years of research, a variety of aerobic denitrifying microbial strains have been isolated and identified, and there are significant differences in their growth characteristics and treatment capabilities during the heterotrophic nitrification-aerobic denitrification process. Therefore, screening out highly efficient strains that can carry out nitrification and denitrification simultaneously has important practical significance for the denitrification of polluted water bodies. Summary of the Invention

[0005] The object of the present invention is to provide a strain of Paenibacillus glycanilyticus, which, as a heterotrophic nitrification-aerobic denitrification bacterium, can perform denitrification treatment on nitrogen-containing aquaculture wastewater and has a better ammonia nitrogen removal effect.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0007] A strain of Paenibacillus glycanilyticus, which is the Paenibacillus glycanilyticus strain DQ-1. This strain was deposited at the China Center for Type Culture Collection on June 13, 2024, and its deposit number is: CCTCC M 20241211, and the deposit address is: Wuhan University, Wuhan, China.

[0008] The heterotrophic nitrification-aerobic denitrification bacterium Paenibacillus glycanilyticus DQ-1 of the present invention has the following biological characteristics: It is identified as a Gram-positive bacterium. The colony is white, opaque and round, with a smooth surface and viscosity, and the edge is neat and orderly. Observed under SEM electron microscopy, it is short rod-shaped, with a size of about (1.16-1.67) μm × (0.76-0.97) μm, and has flagella and spores. The suitable carbon source for the growth of the strain is glucose, the suitable C / N is 25, the suitable pH is 7.0, and the suitable growth temperature is 30°C.

[0009] A bacterial agent containing the Paenibacillus glycanilyticus described in the present invention.

[0010] Preferably, the bacterial agent further contains a pharmaceutically acceptable carrier, and its dosage form is a solution, an emulsion, a suspension, a powder, a gel, a granule or a freeze-dried preparation.

[0011] An application of the Paenibacillus glycanilyticus described in the present invention or the bacterial agent described in the present invention in the denitrification treatment of nitrogen-containing aquaculture wastewater.

[0012] Preferably, the Paenibacillus glycanilyticus performs denitrification treatment on nitrogen-containing aquaculture wastewater as a heterotrophic nitrification-aerobic denitrification bacterium.

[0013] Preferably, the specific application is: inoculating the Paenibacillus glycanilyticus into nitrogen-containing aquaculture wastewater, adding a carbon source until the C / N value is 25, and culturing at pH 7.0, 30°C and 121 rpm to remove ammonia nitrogen, nitrate nitrogen and total nitrogen in the water body; the carbon source is glucose or sucrose.

[0014] Preferably, the cultivation is carried out under the conditions of a C / N ratio of 25, a pH of 7, a temperature of 30°C and a rotation speed of 121 rpm.

[0015] The beneficial effects of the present invention are as follows: Paenibacillus glycanilyticus DQ-1 of the present invention, as a heterotrophic nitrification-aerobic denitrification bacterium, can utilize glucose and sucrose as carbon sources, and under the conditions of glucose as the optimal carbon source, C / N mass ratio of 25, pH of 7.0, temperature of 30 °C, and rotation speed of 121 rpm, the optimal denitrification nitrogen removal effect is 92.5% after 24 hours of aerobic culture; the optimal ammonia nitrogen removal effect is 93.3% after 48 hours; the optimal total nitrogen removal effect is 95.8% after 30 hours. This has important practical significance for the treatment of nitrogen-containing aquaculture wastewater. For Paenibacillus glycanilyticus, it is the first time that a strain of this genus has been found to have heterotrophic nitrification-aerobic denitrification function. Compared with other heterotrophic nitrification-aerobic denitrification bacteria, this strain shows a stronger nitrogen removal effect in a shorter time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a morphological identification diagram of the heterotrophic nitrification-aerobic denitrification bacterium DQ-1 of the present invention;

[0017] Figure 2 It is a phylogenetic tree of the 16S rDNA of the heterotrophic nitrification-aerobic denitrification bacterium DQ-1 of the present invention;

[0018] Figure 3 It is a schematic diagram of the ammonia nitrogen degradation ability of the heterotrophic nitrification-aerobic denitrification bacterium DQ-1 of the present invention;

[0019] Figure 4 It is a schematic diagram of the nitrate nitrogen degradation ability of the heterotrophic nitrification-aerobic denitrification bacterium DQ-1 of the present invention;

[0020] Figure 5 It is a schematic diagram of the total nitrogen degradation ability of the heterotrophic nitrification-aerobic denitrification bacterium DQ-1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The technical solutions of the present invention will be further specifically described below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0022] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0023] The reagents used in the following embodiments can be purchased from conventional biochemical reagent stores without special instructions.

[0024] The reagent MnSO4·4H2O in the following culture medium was purchased from Shanghai Kelin Biochemical Technology Co., Ltd., the LB culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., and the rest of the chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0025] Example 1: Screening and identification of heterotrophic nitrifying-aerobic denitrifying bacteria

[0026] Take 10 g of the sediment sample from the bottom of a certain aquaculture wastewater treatment pond in Huzhou and add it to a conical flask containing 100 mL of enrichment medium. Incubate it in a shaker at 28 °C and 120 rpm for 7 - 15 d. Then transfer it to a constant temperature incubator at 28 °C and incubate for 7 - 15 d. Take a small amount of the sample in a colorimetric tube, add Griess reagent to detect the generation of nitrite. Add 1 mL of 5 g / L (NH4)2SO4 solution to the sample showing red in the reagent result and continue enrichment culture. Check again. When the result shows dark red, pipette 1 mL of the enrichment solution into 100 mL of separation medium and continue incubating at a constant temperature. When the Griess reagent result shows dark red, use the dilution plate coating method to gradient-dilute the bacterial solution, coat it on the solid separation medium respectively, and place it in a constant temperature incubator at 28 °C for 7 d. Pick a single colony the size of a needle tip and streak it on the solid separation medium, and incubate at 28 °C in a constant temperature. Repeat the streak separation to purify the bacteria. Inoculate the isolated strain into the separation medium with a glucose concentration of 4%, incubate at 28 °C and 120 rpm for 7 d, and use the continuous flow injection method to measure the ammonia nitrogen content in the culture solution to verify the nitrification ability of the strain to ammonia nitrogen. Inoculate the isolated strain into 100 mL of denitrification medium (added with a glucose concentration of 5 g / L), incubate in a shaker at 28 °C and 120 rpm for 24 h, and then use the continuous flow injection method to measure the nitrate content in the culture solution to verify the denitrification ability of the isolated strain.

[0027] The formula of the enrichment medium (g / L): (NH4)2SO4 5 g, KH2PO4 0.7 g, MgSO4·7H2O 0.5 g, CaCl2 0.36 g, distilled water 1 L, pH 7.5; the formula of the isolation medium (g / L): (NH4)2SO4 2 g, K2HPO4 0.75 g, NaH2PO4 0.25 g, MgSO4·7H2O 0.03 g, MnSO4·4H2O 0.01 g, CaCO3 5 g, distilled water 1 L, pH 7.0. 1.8 g of agar is added to every 100 mL of the liquid isolation medium to obtain the solid isolation medium; the denitrification medium (g / L): NaNO3 0.1 g, KH2PO4 0.7 g, MgSO4·7H2O 0.5 g, CaCl2 0.36 g, distilled water 1 L, pH 7.0; the LB medium (g / L): tryptone 10 g, yeast extract 5 g, NaCl 10 g. The solid medium is prepared by adding 1.8% agar powder to the liquid medium.

[0028] The isolated strains were streaked on the LB medium plate and then cultured in a constant temperature incubator at 30 °C for 48 hours to observe their colony characteristics. The cell samples in the logarithmic growth phase were selected for Gram staining test to determine the type of their cell walls. Subsequently, a scanning electron microscope (SEM) was used to perform high-resolution imaging of the bacterial cells to observe their detailed individual morphology. According to the guidelines of the "Manual of Common Bacteriology" and the "Bergey's Manual of Systematic Bacteriology", we carried out a detailed physiological and biochemical identification of the strain DQ-1.

[0029] As Figure 1 shown, the colonies formed by the strain DQ-1 on the LB solid medium are round, with a smooth and sticky surface, presenting a white opaque appearance, and the edges are neat and orderly. It is Gram-positive; under SEM observation, it is short rod-shaped, with a size of approximately (1.16 - 1.67) μm × (0.76 - 0.97) μm, and has flagella and spores. The bacterial genome of the target strain was extracted using a genomic kit; using the universal primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO:1,

[0030] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3', SEQ ID NO:2,

[0031] Perform PCR amplification on it and verify by agarose gel electrophoresis (1.5%); perform electrophoresis detection, cut the gel for purification and sequencing, and the sequence determination is carried out by Sangon Biotech Co., Ltd. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min; denaturation for 1 min; annealing at 55°C for 1 min; extension at 72°C for 10 min; finally cool at 4°C for 10 min.

[0032] The length of the 16S rDNA sequence of the strain Paenibacillus glycanilyticus DQ-1 is 1449 bp. Upload the strain sequence results to the NCBI database and compare them with the existing bacterial 16S rDNA gene sequences in the database. The results show that this bacterium is in the same branch as Paenibacillus glycanilyticus. Combining the colony morphological characteristics and physiological and biochemical identification results of DQ1, the strain DQ-1 was finally identified as Paenibacillus glycanilyticus. Figure 2 It is the phylogenetic evolution tree of strain DQ-1.

[0033] This strain DQ-1 was named Paenibacillus glycanilyticus DQ-1 and deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M 20241211, and the address is Wuhan University, Wuhan, China.

[0034] Example 2: Identification of the nitrification / denitrification ability of strain DQ-1 in different nitrogen source media

[0035] The formula of the nitrification medium is: (NH4)2SO4 0.1 g, K2HPO4·3H2O 0.75 g, NaH2PO4·2H2O 0.25 g, NaCl 0.12 g, MnSO4·H2O 0.01 g, MgSO4·7H2O 0.01 g, FeSO4·7H2O 0.01 g, made up to 1000 mL with distilled water, and the pH value is 7.0 - 7.5;

[0036] The formula of the denitrification medium: KNO3 0.1 g, K2HPO4·3H2O 0.75 g, NaH2PO4·2H2O 0.25 g, NaCl 0.12 g, MnSO4·H2O 0.01 g, MgSO4·7H2O 0.01 g, FeSO4·7H2O 0.01 g, made up to 1000 mL with distilled water, and the pH value is 7.0 - 7.5;

[0037] Synchronous nitrification and denitrification culture medium formula: 0.1 g of (NH4)2SO4, 0.1 g of KNO3, 0.75 g of K2HPO4·3H2O, 0.25 g of NaH2PO4·2H2O, 0.12 g of NaCl, 0.01 g of MnSO4·H2O, 0.01 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, made up to 1000 mL with distilled water, pH value 7.0 - 7.5;

[0038] The treatment method is as follows:

[0039] Sample group: According to an inoculation amount of 1%, the seed liquid of strain DQ - 1 (the seed liquid is a bacterial suspension cultured in LB liquid medium at 30 °C and 121 rmp for 24 h) was respectively inoculated into the nitrification medium, denitrification medium, and synchronous nitrification and denitrification medium, and cultured in a shaker at 30 °C and 121 rmp.

[0040] Control group: Under the same conditions, sterile water was respectively inoculated into the nitrification medium, denitrification medium, and synchronous nitrification and denitrification medium according to an inoculation amount of 1%, and cultured in a shaker at 30 °C and 121 rmp.

[0041] Among them, the ammonia nitrogen content was measured by sampling every 6 h in the nitrification medium group; the nitrate nitrogen content was measured by sampling every 3 h in the denitrification medium group; the total nitrogen content was measured by sampling every 6 h in the synchronous nitrification and denitrification medium group. The results are shown in Figure 3 、 Figure 4 and Figure 5 .

[0042] It can be seen from Figure 3 that when the culture conditions are 30 °C and 121 rmp, strain DQ - 1 reduced the ammonia nitrogen concentration from 22.03 mg / L to 1.48 mg / L within 48 h, and the degradation rate was 93.3%, indicating that DQ - 1 has good degradation ability for ammonia nitrogen. It can be seen from Figure 4 that when the culture conditions are 30 °C and 121 rmp, strain DQ - 1 reduced the nitrate nitrogen concentration from 11.56 mg / L to 0.87 mg / L within 24 h, and the degradation rate was 92.5%, indicating that DQ - 1 has good degradation ability for nitrate nitrogen. It can be seen from Figure 5 that when the culture conditions are 30 °C and 121 rmp, strain DQ - 1 reduced the total nitrogen concentration from 33.59 mg / L to 1.43 mg / L within 30 h, and the degradation rate was 95.8%, indicating that DQ - 1 has good degradation ability for total nitrogen.

[0043] The above has introduced in detail a strain of Paenibacillus saccharolyticus provided by the present invention and its application in the denitrification treatment of nitrogen-containing aquaculture wastewater. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A Paenibacillus glycanilyticus strain, which is the Paenibacillus glycanilyticus strain DQ-1. This strain was deposited at the China Center for Type Culture Collection on June 13, 2024, and its deposit number is: CCTCC M 20241211.

2. A bacterial agent containing the Paenibacillus glycanilyticus described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent also contains a pharmaceutically acceptable carrier, and its dosage form is a solution, an emulsion, a suspension, a powder, a gel, a granule or a freeze-dried preparation.

4. An application of the Paenibacillus glycanilyticus described in claim 1 or the bacterial agent described in claim 2 in the denitrification treatment of nitrogen-containing aquaculture wastewater.

5. The application according to claim 4, wherein: The Paenibacillus glycanilyticus is used as a heterotrophic nitrification-aerobic denitrification bacterium to carry out denitrification treatment on nitrogen-containing aquaculture wastewater.

6. The application according to claim 1, wherein The specific application is: inoculating the Paenibacillus glycanilyticus into nitrogen-containing aquaculture wastewater, adding a carbon source until the C / N value is 25, and culturing at pH 7.0, 30 °C and 121 rpm to remove ammonia nitrogen, nitrate nitrogen and total nitrogen in the water body; the carbon source is glucose or sucrose.

7. The application according to claim 1, characterized in that: The culturing is carried out under the conditions of a C / N ratio of 25, a pH of 7, a temperature of 30 °C and a rotation speed of 121 rpm.

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