Enrichment culture method of salt-tolerant nitrite oxidizing bacteria

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an enrichment culture method of salt-tolerant nitrite oxidizing bacteria, which comprises the following steps: (1) enrichment culture of the nitrite oxidizing bacteria: inoculating the nitrite oxidizing bacteria into an SBR (sequencing batch reactor) device, then adding an enrichment nutrient solution, controlling the pH value through sodium nitrite, and controlling the dissolved oxygen concentration through the aeration rate; (2) performing salt tolerance domestication on the nitrite oxidizing flora: performing salt tolerance domestication on the nitrite oxidizing flora subjected to enrichment culture by adopting a method of gradually increasing the salt concentration. The salt-tolerant nitrite oxidizing bacterium has the beneficial effects that the problem of nitrite accumulation in aquaculture can be effectively solved, the water quality improvement efficiency is high, and the duration time is long; according to the enrichment culture method of the salt-tolerant nitrite oxidizing bacteria, an organic carbon source does not need to be additionally arranged, the water treatment cost is greatly reduced, and the economic benefit is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for enriching and culturing salt-tolerant nitrite oxidizing bacteria. Background Art

[0002] Nitrite is produced during marine aquaculture, which harms the growth and reproduction of fish, shrimp and other organisms. It is extremely important to control the concentration of nitrite. Chemical and physical methods improve water quality quickly, but are costly and short-lived, and will bring new pollution to the water quality. They can only temporarily slow down the pollution in the aquaculture water. An effective way to reduce nitrite nitrogen in water bodies is to utilize the effects of beneficial microorganisms. The most thorough way in microbial treatment is to use nitrite-oxidizing bacteria to convert nitrite nitrogen into nitrate nitrogen. However, the salinity of seawater in marine aquaculture is relatively high, generally around 3.5%. Nitrite-oxidizing bacteria are generally not salt-tolerant and cannot survive in seawater, and cannot reduce nitrite. Therefore, a method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria is provided. Summary of the Invention

[0003] In view of the above problems, the present invention provides a method for enriching and culturing salt-tolerant nitrite oxidizing bacteria.

[0004] The present invention relates to a method for enriching and culturing salt-tolerant nitrite oxidizing bacteria, comprising the following steps: (1) Enrichment culture of nitrite-oxidizing bacteria: Nitrite oxidizing bacteria were inoculated into the SBR device, and then enriched nutrient solution was added, the pH value was controlled by sodium nitrite, and the dissolved oxygen concentration was controlled by aeration; (2) Salt tolerance acclimation of nitrite oxidizing bacteria: The salt tolerance of the enriched nitrite oxidizing bacteria was acclimated by gradually increasing the salt concentration.

[0005] Preferably, in step (1), the concentration of the nitrite oxidizing bacteria inoculated into the SBR device is 5-15%.

[0006] Preferably, in step (1), the enriched nutrient solution comprises 0.5 g / L NaHCO3, 0.2 g / L CaCl2·2H2O, 0.5 g / L NaCl, 0.2 g / L KH2PO4, 0.6 g / L K2HPO4, 0.1 g / L MgSO4· 7H2O, 0.014g / L H3BO3, 0.099g / L MnCl2·4H2O, 0.02g / L NiCl2·6H2O, 0.022g / L Na2MoO4·2H2O, 0.025g / L CuSO4·5H2O, 0.02g / L ZnSO4·7H2O, 0.02g / L EDTA-2Na, 0.015g / L COCl2·6H2O and 0.05g / L FeSO4·7H2O.

[0007] Preferably, in step (1), a peristaltic pump is used to add the sodium nitrite solution, the sodium nitrite concentration in the SBR device system is maintained at 10 to 25 mg / L, and the pH value is controlled at 7.3 to 8.5.

[0008] Preferably, in step (1), the concentration of dissolved oxygen is controlled at 4 to 6.5 mg / L.

[0009] Preferably, in step (1), the culture temperature is 26-30° C. and the culture is carried out for 30-50 days.

[0010] Preferably, in step (1), one operation cycle is 24 hours. In each operation cycle, water is introduced for 20 minutes, aeration is performed for 20 hours, sedimentation is performed for 2 hours, drainage is performed for 1 hour, and the remaining time is an idle period.

[0011] Preferably, in step (2), in the initial stage of acclimation, the concentration of sodium chloride in the simulated seawater is added at 0.5%, and in the subsequent acclimation process, the concentration of sodium chloride in the simulated seawater is increased in a gradient from 0.5% to 1%, and the final concentration is 4.5%.

[0012] Preferably, in step (2), the acclimation period for each salinity is 20 to 40 days.

[0013] Preferably, in step (2), one operation cycle is 24 hours. In each operation cycle, water is introduced for 20 minutes, aeration is performed for 20 hours, sedimentation is performed for 2 hours, drainage is performed for 1 hour, and the remaining time is an idle period.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are: (1) The salt-tolerant nitrite oxidizing bacteria can effectively reduce the problem of nitrite accumulation in aquaculture and improve water quality with high efficiency and long duration; (2) This method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria does not require the addition of an external organic carbon source, which greatly reduces the cost of water treatment and improves economic benefits; (3) The salt-tolerant nitrite oxidizing bacteria are highly active in seawater, thus fundamentally restoring the microecological balance in marine aquaculture. DETAILED DESCRIPTION

[0015] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below with reference to the embodiments.

[0016] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0017] Example 1 A method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria comprises the following steps: (1) Enrichment culture of nitrite-oxidizing bacteria: 10% nitrite oxidizing bacteria were inoculated into the SBR device, and then enriched nutrient solution was added, including: 0.5 g / L NaHCO3, 0.2 g / L CaCl2·2H2O, 0.5 g / L NaCl, 0.2 g / L KH2PO4, 0.6 g / L K2HPO4, 0.1 g / L MgSO4·7H2O, 0.014 g / L H3BO3, 0.099 g / L MnCl2·4H2O, 0.02 g / L NiCl2·6H2O, 0.022 g / L Na2MoO4·2H2O, 0.025 g / L CuSO4·5H2O, 0.02 g / L ZnSO4·7H2O, 0.02 g / L EDTA-2Na, 0.015 g / L COC l2·6H2O and 0.05g / L FeSO4·7H2O; sodium nitrite solution was added using a peristaltic pump, the sodium nitrite concentration of the SBR device system was controlled at 15mg / L, the dissolved oxygen concentration was controlled at 5.5mg / L, the pH value was controlled at 8.0, the culture temperature was 28℃, and one operation cycle was 24h, including: water inlet 20min, aeration 20h, sedimentation 2h, drainage 1h, and the remaining time was idle period, and the culture was 30d.

[0018] (2) Salt tolerance acclimation of nitrite oxidizing bacteria: During the initial acclimation phase, sodium chloride was added to the simulated seawater at a concentration of 0.5%. During subsequent acclimation, the sodium chloride concentration in the simulated seawater was increased in a 1% gradient to a final concentration of 4.5%. The acclimation period for one salinity was 25 days, and the SBR reactor operated for 24 hours, consisting of 20 minutes of water inlet, 20 hours of aeration, 2 hours of sedimentation, and 1 hour of drainage, with the remainder of the time being idle. After acclimation, the bacteria formed small, dense flocs, and could remove over 98% of 25 mg / L of nitrite nitrogen (sedimentation ratio 14%) within one hour.

[0019] Example 2 A method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria comprises the following steps: (1) Enrichment culture of nitrite-oxidizing bacteria: The difference from Example 1 is that 5% nitrite oxidizing bacteria were inoculated into the SBR device, the sodium nitrite concentration of the SBR device system was maintained at 10 mg / L, the dissolved oxygen was 4.5 mg / L, the pH was 7.3, the temperature was 26°C, and the culture was carried out for 40 days. Other conditions were the same as in Example 1.

[0020] (2) Salt tolerance acclimation of nitrite oxidizing bacteria: The difference from Example 1 is that the sodium chloride concentration in the simulated seawater is increased in a gradient of 0.5%, the acclimation period for one salinity is 30 days, and other conditions are the same as in Example 1. After the acclimation, the bacteria are in the form of small flocs, the bacterial particles are small and dense, and 20 mg / L of nitrite nitrogen (sedimentation ratio 14%) can be removed by more than 98% within 1 hour.

[0021] Example 3 A method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria comprises the following steps: (1) Enrichment culture of nitrite-oxidizing bacteria: The difference from Example 1 is that 15% nitrite oxidizing bacteria were inoculated into the SBR device, the sodium nitrite concentration of the SBR device system was controlled at 20 mg / L, the dissolved oxygen concentration was controlled at 5 mg / L, the pH was 7.6, and the culture was carried out for 50 days. Other conditions were the same as in Example 1.

[0022] (2) Salt tolerance acclimation of nitrite oxidizing bacteria: The difference from Example 1 is that the acclimation period for one salinity is 40 days, and other conditions are the same as those in Example 1. After the acclimation, the bacteria are in the form of small flocs, the bacterial particles are small and dense, and 30 mg / L of nitrite nitrogen (sedimentation ratio 14%) can be removed by more than 98% within 1 hour.

[0023] Test example The salt-tolerant nitrite oxidizing bacteria of Example 1 were tested for nitrite removal, and the test method was as follows: Take two 1L beakers and add 1L of marine aquaculture wastewater to each beaker; no salt-tolerant nitrite oxidizing bacteria were added to one beaker as a blank control, and 0.1mL of salt-tolerant nitrite oxidizing bacteria (SV30: 14%) was added to the other beaker. Then, both beakers were aerated, the dissolved oxygen concentration was controlled at 5mg / L, the temperature was 28℃, and the pH value was controlled at 8.3. Samples were taken at regular intervals, and the nitrite nitrogen content was detected by spectrophotometry. The results are shown in Table 1.

[0024] Table 1. Results of changes in nitrite nitrogen concentration over time As shown in Table 1, the salt-tolerant nitrite oxidizing bacteria of Example 1 can reduce the nitrite concentration in marine aquaculture wastewater from 20 mg / L to 0.05 mg / L in 142 hours, which proves that the domesticated salt-tolerant nitrite oxidizing bacteria of the present invention can efficiently remove nitrite in marine aquaculture.

[0025] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification or equivalent change made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for enriching and culturing salt-tolerant nitrite-oxidizing bacteria, characterized in that: The following steps are involved: (1) Enrichment culture of nitrite-oxidizing bacteria: Nitrite oxidizing bacteria were inoculated into the SBR device, and then enriched nutrient solution was added, the pH value was controlled by sodium nitrite, and the dissolved oxygen concentration was controlled by aeration; (2) Salt tolerance acclimation of nitrite oxidizing bacteria: The salt tolerance of the enriched nitrite oxidizing bacteria was acclimated by gradually increasing the salt concentration.

2. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein In the step (1), the concentration of the nitrite oxidizing bacteria inoculated into the SBR device is 5-15%.

3. The enrichment and culture method for salt-tolerant nitrite oxidizing bacteria according to claim 1, characterized in that: In the step (1), the enriched nutrient solution includes 0.5 g / L NaHCO3, 0.2 g / L CaCl2·2H2O, 0.5 g / L NaCl, 0.2 g / L KH2PO4, 0.6 g / L K2HPO4, 0.1 g / L MgSO4·7H2O, 0.014 g / L H3BO3, 0.099 g / L MnCl2·4 H2O, 0.02g / L NiCl2·6H2O, 0.022g / L Na2MoO4·2H2O, 0.025g / L CuSO4·5H2O, 0.02g / L ZnSO4·7H2O, 0.02g / L EDTA-2Na, 0.015g / L COCl2·6H2O and 0.05g / L FeSO4·7H2O.

4. The enrichment and culture method for salt-tolerant nitrite oxidizing bacteria according to claim 1, characterized in that: In the step (1), a sodium nitrite solution is added using a peristaltic pump, and the sodium nitrite concentration in the SBR device system is maintained at 10 to 25 mg / L, and the pH value is controlled at 7.3 to 8.

5.

5. The enrichment and cultivation method for salt-tolerant nitrite oxidizing bacteria according to claim 1, characterized in that: In the step (1), the concentration of dissolved oxygen is controlled at 4 to 6.5 mg / L. In the step (1), the culture temperature is 26 to 30° C., and the culture is carried out for 30 to 50 days.

6. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein: In the step (1), one operation cycle is 24 hours. In each operation cycle, water is added for 20 minutes, aeration is performed for 20 hours, sedimentation is performed for 2 hours, drainage is performed for 1 hour, and the remaining time is an idle period.

7. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein: In step (2), in the initial stage of acclimation, the concentration of sodium chloride in the simulated seawater is added at 0.5%. In the subsequent acclimation process, the concentration of sodium chloride in the simulated seawater is increased in a gradient from 0.5% to 1%, and the final concentration is 4.5%.

8. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein: In the step (2), the acclimation period for each salinity is 20 to 40 days.

9. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein: In step (2), one operation cycle is 24 hours. In each operation cycle, water is added for 20 minutes, aeration is performed for 20 hours, sedimentation is performed for 2 hours, drainage is performed for 1 hour, and the remaining time is an idle period.

10. The method for enriching and culturing salt-tolerant nitrite oxidizing bacteria according to claim 1, wherein: In the step (3), the concentration of dissolved oxygen is controlled at 4-6 mg / L, the temperature is controlled at 25-30° C., and the pH value is controlled at 7.6-8.3.

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