Salt-tolerant efficient denitrification strain and application thereof

Abstract

The invention discloses a salt-tolerant efficient denitrification strain and application thereof, and belongs to the technical field of microorganisms. The invention provides a salt-tolerant high-efficiency denitrification (Brucella anthropi N2-2) strain, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 20241934. The salt-tolerant efficient denitrification strain (Brucella anthropi N2-2) provided by the invention has the best denitrification effect when the salinity is 0-3 wt%, the inoculum size is 2% (v / v) and the pH value is 7.5-8.5. According to the present invention, the thallus preparation containing the strain (Brucella anthropi N2-2) is put into the culture pond, such that the contents of ammonia nitrogen, nitrite nitrogen, organic nitrogen and nitrate in the water body can be effectively reduced, the pH value of the water body can be regulated to the normal range so as to maintain the relative stability of the culture water body;

Description

Technical Field

[0001] The invention belongs to the field of microbial technology, and relates to a salt-tolerant and efficient denitrification strain and its application, and specifically relates to a salt-tolerant and efficient denitrification strain Brucella anthropoides N2-2 and its applications. Background Art

[0002] With the increasing demand for high-quality aquatic products and beautiful water ecological environment, high-density intensive aquaculture has emerged. This aquaculture model has become an important development direction of my country's aquaculture industry with its advantages of short aquaculture cycle, high yield and convenient management. Maintaining good water quality during the aquaculture process is the key to ensuring the healthy growth of aquaculture organisms and improving aquaculture efficiency. However, as the aquaculture process progresses, the excess nitrogen in the water cannot be decomposed and utilized by microorganisms in time or converted into gas for release, resulting in ammonia nitrogen (NH4 + -N), nitrite nitrogen (NO2 - -N) and nitrate nitrogen (NO3 - -N) and other contents exceed the standard, which seriously endangers the healthy growth of aquatic animals. In the breeding process, water quality is mainly maintained by water exchange, which leads to excessive consumption of water resources. Therefore, the rapid development of intensive breeding mode has also brought a series of new problems, such as aggravated water pollution and excessive consumption of water resources. Among them, ammonia nitrogen (NH4 + -N), nitrite nitrogen (NO2 - The excessive content of harmful nitrogen such as -N) has a stressful effect on aquatic animals, and the lack of efficient and practical technology for nitrogen eutrophication of water bodies are major common and key challenges that need to be urgently addressed in the current aquaculture industry.

[0003] Microorganisms can participate in the circulation of chemical elements such as nitrogen and carbon in aquaculture water and purify aquaculture water quality. For example, some microorganisms can convert excess inorganic nitrogen in water into nitrogen-containing gas, providing a high-quality water environment for the healthy growth of farmed animals. In aquaculture, efficient microbial strains or microecological preparations with specific functions are added to improve the treatment effect, but the type and quantity of microorganisms need to be selected and optimized according to the specific characteristics of the wastewater, which requires professional knowledge and experience, otherwise the expected treatment effect may not be achieved.

[0004] There are still some problems with the current biological method for treating seawater aquaculture water: on the one hand, it is difficult to treat seawater aquaculture water in a short period of time and requires a long start-up time; on the other hand, due to the high salinity of seawater aquaculture water, the efficiency of pollutant removal is not ideal and the residual pollutant concentration still cannot directly meet the discharge requirements. Summary of the invention

[0005] In view of the fact that in the prior art, the types and quantities of microorganisms need to be selected and optimized according to the specific characteristics of wastewater, and the problem that pollutants are difficult to remove due to high salinity in seawater aquaculture, the purpose of the present invention is to provide a salt-tolerant and efficient denitrification strain ( Brucella anthropoides N2-2).

[0006] The present invention provides a salt-tolerant and efficient denitrification strain ( Brucella anthropoides N2-2), the salt-tolerant and efficient denitrification strain was deposited in the China Center for Type Culture Collection on September 6, 2024, with the deposit number CCTCC NO: M20241934.

[0007] The salt-tolerant and efficient denitrification strain has a nucleotide sequence as shown in SEQ ID No.1.

[0008] The colony of the salt-tolerant and efficient denitrification strain of the present invention is smooth and oval, full and raised, light yellow, with neat edges, smooth, moist and shiny.

[0009] The present invention also discloses a bacterial preparation, comprising the above-mentioned salt-tolerant and efficient denitrification strain.

[0010] The bacterial preparation comprises the salt-tolerant and efficient denitrification strain and a microbial culture, wherein the salt-tolerant and efficient denitrification strain serves as an active ingredient of the bacterial preparation.

[0011] The present invention also discloses a method for preparing the bacterial preparation as described above, comprising the following steps: (1) Inoculate the salt-tolerant and efficient denitrification strain into liquid LB medium and culture for 20-26 h as seed solution; (2) Expanding the seed solution for culture; (3) Mixing the bacterial liquid obtained by the expanded culture with a nutrient agent, packing into barrels, sealing them, and refrigerating them to prepare a bacterial preparation.

[0012] Furthermore, in step (3), the components of the nutrient agent include: 450-550 L of water, 4-6 kg of peptone, 2-3 kg of yeast powder, and 14-16 kg of sodium chloride; the pH of the nutrient agent is 7-8.5.

[0013] Furthermore, in the step (3), the bacterial liquid and the nutrient agent are mixed in a volume ratio of 1:1.

[0014] The present invention also discloses an application of the bacterial preparation as described above in purifying aquaculture wastewater.

[0015] Furthermore, the bacterial preparation is used to purify aquaculture wastewater by directly putting the bacterial preparation into the aquaculture pond.

[0016] Furthermore, the bacterial preparation is administered once every five days.

[0017] The present invention adopts a microbial cultivation method to screen out a salt-tolerant and efficient denitrification strain ( Brucella anthropoides N2-2), the denitrification effect is best when the salinity is 0-3 wt%, the inoculum amount is 2% (v / v), and the pH is 7.5-8.5. Brucella anthropoides N2-2) bacterial preparation is put into the breeding pond, which can effectively reduce the content of ammonia nitrogen, nitrite, organic nitrogen and nitrate in the water, and at the same time can adjust the pH of the water to a normal range, maintaining the relative stability of the breeding water. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony morphology diagram of the salt-tolerant and efficient denitrification strain of the present invention inoculated on a nitrification solid culture medium; Figure 2 NH4 in the salinity test of the salt-tolerant and efficient denitrification strain of the present invention + -N degradation result diagram; Figure 3 It is a bacterial growth curve diagram of the salt-tolerant and efficient denitrification strain in a salinity test of the present invention; Figure 4 This is a result diagram showing the change of ammonia nitrogen content in the aquaculture water of the experimental group and the control group in Example 5 of the present invention over time; Figure 5 This is a result diagram showing the change of nitrite nitrogen content in the aquaculture water of the experimental group and the control group in Example 5 of the present invention over time; Figure 6 This is a result diagram showing the change of total nitrogen content in the aquaculture water of the experimental group and the control group in Example 5 of the present invention over time; Figure 7 This is a result diagram showing the pH of the aquaculture water of the experimental group and the control group of Example 5 of the present invention changing over time; Figure 8 The growth curve of strains N2-2 and C-4-2 in Example 6 of the present invention in aquaculture tail water; Fig. 9 This is a comparison chart of the shrimp sizes of the experimental group and the control group in Example 5 of the present invention, which were cultured for 55 days. DETAILED DESCRIPTION

[0019] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Culture medium: (1) LB medium: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 30 g / L.

[0021] (2) Nitration medium (NM medium): sodium acetate 5 g / L, (NH4)2SO4 10 mg / L, Na2HPO4 0.5 g / L, KH2PO4 0.5 g / L, MgSO4•7H2O 0.4 g / L, NaCl 30 g / L, pH 7.5-8.5.

[0022] (3) Nitration solid culture medium: Add 2 wt% agar to the nitrification culture medium.

[0023] Instruments: clean bench, biochemical incubator, UV-visible spectrophotometer, vertical high-pressure steam sterilizer, gas bath constant temperature oscillator, water quality rapid tester, etc.

[0024] The total nitrogen concentration was determined according to HJ 636-2012 "Alkaline potassium persulfate digestion ultraviolet spectrophotometry" using a UV-visible spectrophotometer. The ammonia nitrogen concentration was tested using a water quality rapid tester.

[0025] Example 1 Screening of salt-tolerant denitrification strains: 1. Take 5 g of aquaculture water sludge and add it to sterile water. After shaking for 30 minutes, let it stand. Take the supernatant and inoculate it into LB medium for enrichment. Cultivate it at 30℃ and 180 rpm / min with shaking until it becomes turbid.

[0026] 2. Take 10 mL of the enriched solution and inoculate it into freshly sterilized LB medium for further culture. Repeat the enrichment 4 times.

[0027] 3. Take 1 mL of the enriched culture medium for gradient dilution, and take 10 -4 , 10 -5 , 10 -6 200 μL of bacterial solution was spread on nitrification solid culture medium and inverted in triplicate in a 30°C biochemical incubator for culture; single colonies with different morphologies were selected and streaked several times using the three-line method to obtain pure strains. Pure strains were selected and inoculated into NM medium in a 96-well plate. After shaking and culturing at 30°C for 24 h, nitrite colorimetric agent and ammonium colorimetric agent were added for color development reaction. The strain with the best denitrification effect was selected according to the depth of color (the lighter the color, the better the denitrification effect), and the strain was expanded and cultured, and the glycerol tube was stored in a -80°C refrigerator.

[0028] 4. The selected strain was named N2-2, and the culture conditions such as carbon source, pH, and inoculation amount were optimized using the single factor analysis method. The specific steps were as follows: (1) Carbon source: Prepare NM culture medium with four different carbon sources, namely sodium succinate, sodium acetate, glucose, and sodium acetate. After sterilization, inoculate the strain and culture it in a shaker at 30°C and 180 rpm / min for 48 h. After centrifugation of the bacterial solution every 6 h, dilute it appropriately to detect the NH4 + -N concentration. (2) pH: Prepare NM culture medium with pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, inoculate the strain after sterilization, and culture in a shaker at 30°C and 180 rpm / min for 24 h. Centrifuge the bacterial solution every 6 h and dilute it appropriately to detect NH4 + -N concentration. (3) Inoculation amount: Add 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), and 5% (v / v) bacterial solution to NM culture medium, sterilize and inoculate the bacterial solution, culture in a shaker at 30°C and 180 rpm / min for 24 h, centrifuge the bacterial solution every 6 h, and dilute appropriately to detect NH4 in different culture media. + -N concentration. According to the above experiments, it was found that the denitrification effect was best when the inoculation amount was 2% (v / v) and the pH was 7.5-8.5.

[0029] Example 2 Identification of salt-tolerant and denitrifying strains: (1) Morphological characteristics The strain N2-2 was cultured on LB medium plates at 30°C for 24 h. Figure 1 The colonies shown are smooth oval, 2-3 mm in diameter, plump and convex, light yellow in color, with neat edges, smooth, moist and shiny. The colonies increase in size over time, and after a few days, the diameter can reach more than 5 mm, and the Gram staining reaction is negative.

[0030] (2) Genetic identification of strain N-2 Extract bacterial genomic DNA and amplify using universal primers: The forward primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', The reverse primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′; The RCR conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 30 cycles, extension at 72°C for 10 min, and insulation at 12°C; The 16SrDNA sequence of the strain was determined, and the result of the determination was SEQ ID No.1:

[0031] The results were compared with the 16SrDNA gene sequence of the model strain in the NCBI database, and the salt-tolerant and efficient denitrification strain was identified as Brucella anthropoides N2-2.

[0032] Example 3 Salinity test: The strain N2-2 was inoculated into LB medium for activation for 24 h, and the bacterial solution was inoculated into 100 mL of NM medium at a 2% (v / v) inoculation rate. The concentrations of NaCl were controlled to be 0, 10, 20, 30, 40, and 50 g / L, respectively. The initial NH4 + -N concentration is about 20 mg / L, culture for 48 h, take 10 mL sample and measure OD 600 The supernatant was taken to determine the NH4 before and after cultivation. + -N concentration, NH4 + -N removal rate and growth curves Figure 2-Figure 3 shown.

[0033] like Figure 2-Figure 3 As shown in the figure, the strain N2-2 degraded in NM medium for 48 h. The results showed that when the concentration of NaCl was between 0 and 30 g / L, the strain grew well, and the OD 600 Maintained at around 1.4, and NH4 + The removal rates of -N were all greater than 90%, and the concentrations of 0g / L, 10g / L, 20g / L, and 30g / L were 95.78%, 91.46%, 91.87%, and 91.67%, respectively. When the concentration of NaCl was 40g / L, the growth of the strain was inhibited, and OD 600 When the concentration dropped to 1.0, the removal rate dropped to 63.84%, while when the concentration increased to 50 g / L, the OD 600 Further decreasing to 0.65, the removal rate also dropped to only 8.89%.

[0034] In summary, the optimal denitrification salinity of strain N2-2 is 0-30 g / L. Strain N2-2 has certain salt tolerance and can grow and denitrify in salt-free and certain salt concentration environments. It has a wide range of applications in actual aquaculture wastewater treatment.

[0035] Example 4 Preparation of bacterial preparations: The salt-tolerant and efficient denitrification strain was inoculated into liquid LB medium, cultured at 30°C and 180 rpm / min in a shaking incubator for 24 hours, and then expanded in a fermenter with an inoculation volume of 2% (v / v) and a liquid LB medium. The bacterial agent and nutrient agent cultured in the fermenter were mixed in a ratio of 1:1, barreled, sealed, and refrigerated at 2-8°C.

[0036] The nutrients for expanding the culture in the fermenter were prepared according to the following proportions: 5 kg of peptone, 2.5 kg of yeast powder, 15 kg of sodium chloride in 500 L of water, pH 8.2, and sterilized by wet heat at 121°C for 20 min.

[0037] Example 5 Application in marine aquaculture of Penaeus vannamei: The prepared bacterial preparation was sprayed into the culture pond at a dosage ratio of 2% (v / v) (2000 mL / mu), and the bacterial preparation was added once every 5 days. Samples were taken 3 days after the addition, and water quality was tested. The results were as follows: Figure 4-Figure 7 As shown, CK is the control group (no bacterial preparation added), treatment 1, treatment 2 and treatment 3 are three repeated experimental groups with added bacterial preparations.

[0038] like Figure 4 As shown in the figure, compared with the control group, experimental groups 1-3 can effectively reduce the ammonia nitrogen content in the aquaculture water. Figure 5 As shown in the figure, compared with the control group, the nitrite content in the aquaculture water in the experimental groups 1-3 was significantly reduced. Figure 6 As shown in Figure 2, the total nitrogen content in the aquaculture water in experimental groups 1-3 decreased compared with the control group. Figure 7 As shown in the figure, the pH of the aquaculture water in experimental groups 1-3 was always between 8-8.5, while the pH of the control group was between 7.5-9. In addition, the addition of bacterial agents during the aquaculture process can increase the growth rate of white shrimp to a certain extent, such as Fig. 9 As shown, the size of the shrimps in the experimental group was significantly larger than that in the control group. After about 55 days of cultivation, the size of the shrimps in the control group was 50 per jin, while the size of the shrimps in the experimental group was 35 per jin.

[0039] In summary, under the premise of a certain dosage, the addition of bacterial preparations has a positive effect on the water body for breeding of whiteleg shrimp. First, in terms of regulating water quality, compared with the control group, the experimental group with the addition of bacterial preparations can effectively reduce the ammonia nitrogen content in the water by more than 39%, the nitrite content by more than 35%, and the total nitrogen content by 17.6%. At the same time, the pH of the water body can be adjusted to a normal range. The pH range of the control group is greater than that of the experimental groups and will exceed the safety threshold. In addition, the addition of bacterial preparations during the breeding process can increase the growth rate of whiteleg shrimp to a certain extent, shorten the breeding cycle, and improve economic benefits.

[0040] Example 6 The initial ammonia nitrogen concentration in the aquaculture tail water was kept constant at 20-30 mg / L, and the carbon source concentration in the aquaculture tail water was adjusted to make the C / N ratio 4. The salt-tolerant denitrifying bacteria were activated and cultured in LB medium for 24 h, and the bacterial liquid of strain N2-2 and salt-intolerant denitrifying bacteria (C-4-2) were inoculated into the seawater aquaculture tail water at a 2% inoculation rate, respectively, and placed at 30°C for stirring and culture, and their growth curves were measured.

[0041] like Figure 8 As shown, the salt-tolerant and efficient nitrogen removal strains within 60 h Brucella anthropoides The N2-2 growth curve shows that the salt-tolerant and efficient denitrifying bacteria grow slowly within 12 h, but the OD 600 The value reached the maximum value of 1.489 after 36 h of cultivation. The exponential growth phase of strain N2-1 was 0-36 h, and the growth entered a stable phase at 36-60 h. However, the growth (delayed phase time and number of bacteria) of the control bacteria C-4-2 in the tail water of marine aquaculture was significantly inhibited.

[0042] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A salt-tolerant and efficient denitrification strain ( Brucella anthropoides N2-2), where The salt-tolerant and efficient denitrification strain was deposited in the China Center for Type Culture Collection on September 6, 2024, with the deposit number being CCTCC NO: M 20241934.

2. The salt-tolerant and efficient denitrification strain according to claim 1, characterized in that: The salt-tolerant and efficient denitrification strain has a nucleotide sequence as shown in SEQ ID No.

1.

3. A salt-tolerant and efficient denitrification strain according to claim 1, characterized in that: The colony of the salt-tolerant and efficient denitrification strain is smooth and oval, full and raised, light yellow, with neat edges, smooth, moist and shiny.

4. A bacterial preparation, characterized in that It includes the salt-tolerant and efficient denitrification strain as described in claim 1.

5. A method for preparing a bacterial preparation as claimed in claim 4, characterized in that: The following steps are involved: (1) inoculating the salt-tolerant and efficient denitrification strain into liquid LB medium and culturing for 20-26 hours to serve as seed solution; (2) Expanding the seed solution for culture; (3) Mixing the bacterial liquid obtained by the expanded culture with a nutrient agent, packing into barrels, sealing them, and refrigerating them to prepare a bacterial preparation.

6. The method for preparing a bacterial preparation according to claim 5, characterized in that: In step (3), the components of the nutrient agent include: 450-550 L of water, 4-6 kg of peptone, 2-3 kg of yeast powder, and 14-16 kg of sodium chloride; the pH of the nutrient agent is 7-8.

5.

7. The method for preparing a bacterial preparation according to claim 6, characterized in that: In the step (3), the bacterial liquid and the nutrient agent are mixed in a volume ratio of 1:

1.

8. Use of the bacterial preparation as claimed in claim 4 in purifying aquaculture wastewater.

9. The use of the bacterial preparation according to claim 8 in purifying aquaculture wastewater, characterized in that: The bacterial preparation is directly put into the culture pond.

10. The use of the bacterial preparation according to claim 9 in purifying aquaculture wastewater, characterized in that: The bacterial preparation is administered once every five days.

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