Cold-resistant sulfur autotrophic denitrifying bacterium and application thereof

Abstract

The invention relates to the technical field of environmental microorganisms and sewage treatment, and provides a cold-resistant sulfur autotrophic denitrifying bacterium and application thereof, the sulfur autotrophic denitrifying bacterium is thiobacillus denitrificans SZG-SAD-004, and the preservation number of the sulfur autotrophic denitrifying bacterium is CCTCC (China Center for Type Culture Collection) NO: M2023992. The Thiobacillus denitrificans SZG-SAD-004 disclosed by the invention can help a sulfur autotrophic denitrification device to be quickly started at a low temperature, the normal-temperature starting efficiency is achieved, meanwhile, efficient denitrification can still be performed at the temperature of 6-15 DEG C, the low-temperature tolerance performance and the denitrification performance are strong, and the Thiobacillus denitrificans SZG-SAD-004 has a very high application value.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms and sewage treatment, and in particular to cold-resistant sulfur autotrophic denitrifying bacteria and applications thereof. Background Art

[0002] Traditional heterotrophic denitrification methods do not completely remove total nitrogen, and require frequent addition of carbon sources, which is costly. High-sulfur autotrophic denitrification (SAD) is a representative process of autotrophic denitrification. In the autotrophic denitrification system, sulfur autotrophic denitrifying bacteria use inorganic sulfur as an electron donor for denitrification. It does not require the addition of carbon sources, has high denitrification efficiency, is safe to operate, has relatively low costs, and has a small amount of residual sludge. It is considered to be a highly cost-effective alternative process.

[0003] The most suitable growth temperature for the existing microorganisms is 20℃-35℃. The biological denitrification metabolism rate is inhibited at low temperatures. Sulfur autotrophic denitrifying bacteria are not effective at low temperatures. When the application environment temperature is low, the denitrification efficiency is low in the face of high-concentration wastewater. Therefore, it is necessary to develop a low-temperature resistant sulfur autotrophic denitrifying bacteria. Summary of the invention

[0004] In view of this, the present invention proposes a cold-resistant sulfur autotrophic denitrifying bacterium that is resistant to low temperatures and has high denitrification efficiency and an application thereof.

[0005] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a cold-resistant sulfur autotrophic denitrification strain, which is Thiobacillus denitrificans SZG-SAD-004, with a deposit number of CCTCC NO: M2023992.

[0006] In a second aspect, the present invention provides a sulfur autotrophic denitrifying bacteria preparation, including Thiobacillus denitrificans SZG-SAD-004.

[0007] On the basis of the above technical solution, preferably, the preparation is a liquid, and the concentration of Thiobacillus denitrificans SZG-SAD-004 in the preparation is ≥10 9 CFU / mL.

[0008] In a third aspect, the present invention provides an application of a cold-resistant sulfur autotrophic denitrifying strain in a sulfur autotrophic denitrification system.

[0009] On the basis of the above technical scheme, preferably, Thiobacillus denitrificans SZG-SAD-004 is inoculated to carry out sulfur autotrophic denitrification at 5°C-15°C.

[0010] On the basis of the above technical scheme, preferably, the inoculation amount of Thiobacillus denitrificans SZG-SAD-004 is 5-10 mL / L.

[0011] In a fourth aspect, the present invention provides an application of a cold-resistant sulfur autotrophic denitrifying strain in sewage treatment.

[0012] The cold-resistant sulfur autotrophic denitrifying bacteria and the application thereof of the present invention have the following beneficial effects compared with the prior art:

[0013] The Thiobacillus denitrificans SZG-SAD-004 in the present invention can help the sulfur autotrophic denitrification device to start quickly at low temperature and achieve normal temperature start-up efficiency. At the same time, it can still efficiently denitrify at 6-15°C, has strong low temperature tolerance and denitrification performance, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a diagram showing the denitrification effect of SZG-SAD-004 strain seed culture at different temperatures;

[0016] Figure 2 This is a diagram of the denitrification effect of SZG-SAD-004 bacteria during fermentation culture at different temperatures. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] 1. Screening of cold-resistant Thiobacillus denitrificans

[0019] The denitrifying Thiobacillus of the present invention is obtained by screening the mud-water mixture in the anaerobic pool of a sewage plant through liquid enrichment, device domestication, purification and screening. The specific method is as follows:

[0020] 1.1 Liquid enrichment

[0021] Take 5mL of anaerobic pond mud and water mixture in 30mL of enrichment culture medium, culture at 28℃ until nitrate nitrogen is consumed to 5mg / L, transfer to the next generation at a volume ratio of 10%. After 5 generations of transfer, nitrate nitrogen is completely consumed in 2 days and enrichment solution is obtained. The cycle of this process is 17 days.

[0022] Enrichment medium: 0.1g / L ammonium chloride, 0.2g / L magnesium chloride, 3g / L sodium thiosulfate, 2g / L potassium nitrate, 1g / L potassium dihydrogen phosphate, 1.25g / L sodium bicarbonate, 0.02g / L ferrous sulfate, 0.001g / L anhydrous calcium chloride.

[0023] 1.2 Bacterial strain training

[0024] Put the pebble cushion layer at the bottom, occupying 1 / 3 of the device, then add 500g of granular sulfur autotrophic carrier containing sulfur and pyrite, assemble the sulfur autotrophic denitrification device, add culture solution (potassium nitrate solution containing 100mg / L nitrate nitrogen), inoculate 10mL enrichment solution (1%), let the bacteria stand at room temperature for 6-7d, and measure the nitrate nitrogen to be less than 2mg / L, start water inlet and outlet, the inlet water is simulated wastewater containing 50mg / L nitrate nitrogen, HRT (hydraulic retention time) is 8h at 28℃ The device was operated. When the nitrate-nitrogen content in the effluent was less than 5 mg / L, the operating temperature was lowered by 2°C until the temperature reached 6°C and the nitrate-nitrogen content in the effluent was still less than 5 mg / L, and the acclimation was completed. 30 g of carrier particles were taken, mechanically crushed and cultured in 100 mL of enrichment medium at a culture temperature of 10°C. When the nitrate-nitrogen content was consumed to 5 mg / L, the particles were transferred to the next generation at a ratio of 10% (volume ratio). After two generations of transfer, the nitrate-nitrogen content was completely consumed in 2 days, and the acclimated enrichment solution was obtained.

[0025] 1.3 Purification screening

[0026] The solid medium for coating was enriched medium with 18 g / L agar added. 1 mL of the acclimated enriched medium was taken and gradiently diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 times, spread them on solid plates, invert and culture them in an anaerobic constant temperature incubator at 10°C until obvious colonies are produced, pick single colonies with different shapes, sizes and colors, and culture them by streaking multiple times until purified single colonies are obtained. Record the colony morphology and bacterial color results and record them.

[0027] The purified single colony was picked and placed in an EP tube containing 2 mL of enrichment medium, and the nitrate nitrogen was determined by static culture at 10° C. for 72 h. The bacterial species corresponding to the large nitrate nitrogen degradation range were sent for identification. Through molecular biological sequencing and similarity comparison with known sequences in the Genbank database, it was analyzed and identified as denitrifying Thiobacillus denitrificans, and finally the denitrifying Thiobacillus denitrificans SZG-SAD-004 in the present invention was obtained, and the nucleotide sequence is shown in SEQ ID No. 1.

[0028] The colony morphology of this bacterium: the colonies are colorless, transparent or white, with regular edges, short rod-shaped under an optical microscope, Gram staining is negative, and the most suitable pH is 6-8.

[0029] Therefore, Thiobacillus denitrificans SZG-SAD-004 was deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China, Postal Code: 430072) on June 12, 2023, with the deposit number: CCTCC No. 2023992, and the protection center identified the strain Thiobacillus denitrificans SZG-SAD-004 as being in an active state on June 12, 2023.

[0030] 1.4 Bacteria Activation

[0031] Take out the glycerol cryopreservation tube of Thiobacillus denitrificans SZG-SAD-004 strain stored at -80°C, streak it on the solid culture medium, and culture it upside down in an anaerobic incubator at 30°C for 3-5 days.

[0032] Amplification culture: Pick a single colony on the solid culture medium and inoculate it into 50mL seed liquid culture medium, incubate at 28℃ for 3 days, determine the nitrate nitrogen is below 20mg / L, transfer to 200mL liquid culture medium at a ratio of 5%, incubate at 28℃ for 2 days, inoculate a 5L fermenter as seed liquid, inoculation ratio is 10%, incubate for 5-7 days to obtain the target bacterial liquid, at this time the effective viable count is not less than 10 9 CFU / mL.

[0033] Seed liquid culture medium: 0.4g / L ammonium chloride, 0.5g / L magnesium chloride, 6g / L sodium thiosulfate, 4g / L potassium nitrate, 2g / L potassium dihydrogen phosphate, 3g / L sodium bicarbonate, 0.05g / L ferrous sulfate, 0.001g / L anhydrous calcium chloride. On this basis, solid culture medium is supplemented with 18g / L agar.

[0034] 5L fermentation tank culture medium: 0.2g / L ammonium chloride, 0.3g / L magnesium chloride, 12g / L sodium thiosulfate, 8g / L potassium nitrate, 2g / L potassium dihydrogen phosphate, 3g / L sodium bicarbonate, 0.1g / L ferrous sulfate, 0.001g / L anhydrous calcium chloride, the conditions are rotation speed 220rpm, temperature 28℃, pH 7.5, and the pH regulating liquid is 100g / L sodium carbonate.

[0035] 2. Low temperature resistance test

[0036] A single colony of Thiobacillus denitrificans SZG-SAD-004 (hereinafter referred to as SZG-SAD-004) was selected and placed in 50 mL of seed liquid culture medium and cultured at 10°C and 28°C at the same time. After 7 days of culture at 10°C, the nitrate nitrogen in the solution decreased to below 10 mg / L, and after 5 days of culture at 28°C, the nitrate nitrogen in the solution decreased to below 10 mg / L (see Figure 1 ) to obtain seed solutions cultured at different temperatures.

[0037] After the seed culture was completed, the inoculum was inoculated into the fermentation medium at 10% (volume ratio) (same OD600) and cultured at 10°C and 28°C. It was found that the nitrate nitrogen was reduced to below 10 mg / L within 95 h (see Figure 2 ), repeated verification for 3 rounds, and the conclusion was the same.

[0038] The above experiments show that the growth performance of SZG-SAD-004 at 28°C is better than that at 10°C, but the low-temperature performance after cultivation at 28°C is not much different from that at 10°C, so it is judged that the bacteria is a cold-resistant bacteria. Based on this conclusion, in order to save fermentation time and cost, the room temperature fermentation method was adopted in the subsequent process.

[0039] 3. Application of SZG-SAD-004 strain in sulfur autotrophic denitrification under different temperature conditions

[0040] Example 1

[0041] Example 1 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification under 5°C, specifically:

[0042] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation amount of 5 mL / L (0.5%), and the culture was allowed to stand for 4 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 150 mg / L of nitrate nitrogen. The HRT was 4h at 5°C. After the influent, the system was operated for 7 days, and the nitrate nitrogen concentration in the effluent was below 5 mg / L. After 20 days of stable operation, the nitrate nitrogen removal rate was above 96.67%, and the highest nitrate nitrogen removal rate was 99.98%.

[0043] Example 2

[0044] Example 2 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification under 10°C conditions, specifically:

[0045] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation amount of 5 mL / L (0.5%), and the culture was allowed to stand for 4 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 150 mg / L of nitrate nitrogen. The HRT was 1h at 10°C. After the influent, the system was operated for 6 days, and the nitrate nitrogen concentration in the effluent was below 3 mg / L. After 26 days of stable operation, the nitrate nitrogen removal rate was above 91.42%, and the highest nitrate nitrogen removal rate was 98.81%.

[0046] Example 3

[0047] Example 3 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification under the conditions of 15°C and an inoculation volume of 5 ml / L, specifically:

[0048] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation amount of 5 mL / L (0.5%), and the culture was allowed to stand for 4 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 150 mg / L of nitrate nitrogen. At 15°C, the HRT was 1h. After the influent, the nitrate nitrogen concentration in the effluent was below 3 mg / L on the 5th day. After 21 days of stable operation, the nitrate nitrogen removal rate was above 98%, and the highest nitrate nitrogen removal rate was 99.85%.

[0049] Example 4

[0050] Example 4 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification under the conditions of 15°C and an inoculum volume of 10 ml / L, specifically:

[0051] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation amount of 10 ml / L (1%), and the culture was allowed to stand for 3 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 350 mg / L of nitrate nitrogen. The HRT was 4h at 15°C. After the influent, the nitrate nitrogen concentration in the effluent was below 3 mg / L after running for 3 days. After 21 days of stable operation, the nitrate nitrogen removal rate was above 95.92%, and the highest nitrate nitrogen removal rate was 99.88%.

[0052] Example 5

[0053] Example 5 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification at 25°C, specifically:

[0054] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation volume of 5 ml / L (0.5%), and the culture was allowed to stand for 2 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 200 mg / L of nitrate nitrogen. The HRT was 0.5h at 25°C. After the influent, the system was operated for 4 days, and the nitrate nitrogen concentration in the effluent was below 3 mg / L. After 18 days of stable operation, the nitrate nitrogen removal rate was above 98.92%, and the highest nitrate nitrogen removal rate was 99.58%.

[0055] Example 6

[0056] Example 6 is the application of SZG-SAD-004 strain for sulfur autotrophic denitrification under 35°C, specifically:

[0057] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the activated SZG-SAD-004 strain was inoculated with an inoculation volume of 5 ml / L (0.5%), and the culture was allowed to stand for 2 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 200 mg / L of nitrate nitrogen. The HRT was 0.5h at 35°C. After the influent, the system was operated for 4 days, and the nitrate nitrogen concentration in the effluent was below 3 mg / L. After 18 days of stable operation, the nitrate nitrogen removal rate was above 98.92%, and the highest nitrate nitrogen removal rate was 99.68%.

[0058] It can be seen from Examples 1-6 that the SZG-SAD-004 strain can still grow normally at 5°C, and in the sulfur autotrophic denitrification device, it still has a high efficiency in denitrification at low temperature: after inoculation with 5 ml / L (0.5%) of bacterial agent, the system can be operated at 5°C for 4 hours. After 7 days of operation, the nitrate nitrogen can be reduced from 150 mg / L to below 5 mg / L. After stable operation for 20 days, the nitrate nitrogen removal rate is above 96.67%, with a maximum of 99.98%.

[0059] In actual application, the temperature at the site is generally 10-15°C. At HRT 1h, the nitrate nitrogen can be reduced from 150mg / L to below 3mg / L after 5-6d of operation. After stable operation for 21d, the nitrate removal rate is above 91.42%, and the highest is 99.85% (see Example 3). At HRT 4h, the nitrate nitrogen can be reduced from 350mg / L to below 3mg / L. After stable operation for 21d, the nitrate removal rate is above 95.92%, and the highest is 99.88% (see Example 4).

[0060] Comparative Example 1

[0061] The difference between Comparative Example 1 and Example 5 is that the room temperature enrichment solution is used for sulfur autotrophic denitrification at 25°C.

[0062] Collection of room temperature enriched liquid: Enrichment of denitrifying bacteria from sludge at room temperature to obtain room temperature enriched liquid, which is also the enriched liquid commonly used for starting the existing sulfur autotrophic denitrification process.

[0063] A potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the room temperature enrichment solution was inoculated with an inoculation volume of 5 mL / L (0.5%), and the bacteria were cultured for 2 days. When the nitrate nitrogen in the culture solution dropped below 10 mg / L, the culture was successful and the water intake could be started. The influent was simulated wastewater containing 200 mg / L of nitrate nitrogen. The HRT was 0.5h at 25°C. After the water intake, it was operated for 7 days, and the nitrate nitrogen concentration in the effluent was below 3 mg / L. After 18 days of stable operation, the nitrate nitrogen removal rate was above 90.92%, and the highest nitrate nitrogen removal rate was 98.73%.

[0064] Comparative Example 2

[0065] The difference between Comparative Example 2 and Example 3 is that Comparative Example 2 is the application of room temperature enrichment liquid for sulfur autotrophic denitrification at 15°C.

[0066] Normal temperature enrichment solution The same normal temperature enrichment solution as that of comparative example 1, a potassium nitrate solution (culture solution) containing 100 mg / L of nitrate nitrogen was added to the sulfur autotrophic denitrification device, and the normal temperature enrichment solution was inoculated with an inoculation amount of 5 mL / L (0.5%), and the bacteria were cultured for 7 days. On the 7th day of culture, the nitrate nitrogen was still 88 mg / L, and water was slowly introduced (simulated wastewater containing 50 mg / L of nitrate), the hydraulic retention time HRT of the device was 8h, and it was operated for 11 days, and the minimum residual nitrate nitrogen in the effluent was still 47.23 mg / L, which showed that it was difficult to start sulfur autotrophic denitrification with normal temperature enrichment solution at low temperature.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cold-resistant sulfur autotrophic denitrifying strain, characterized in that: The strain is Thiobacillus denitrificans SZG-SAD-004, and its deposit number is CCTCC NO: M2023992.

2. A sulfur autotrophic denitrifying bacteria preparation, characterized in that: The invention comprises the Thiobacillus denitrificans SZG-SAD-004 as described in claim 1.

3. A sulfur autotrophic denitrifying bacteria preparation as claimed in claim 2, characterized in that: The preparation is a liquid, and the concentration of Thiobacillus denitrificans SZG-SAD-004 in the preparation is ≥10 9 CFU / mL.

4. Use of the cold-resistant sulfur autotrophic denitrifying strain according to claim 1 in a sulfur autotrophic denitrification system.

5. The use of a cold-resistant sulfur autotrophic denitrifying strain in a sulfur autotrophic denitrification system as claimed in claim 4, characterized in that: Thiobacillus denitrificans SZG-SAD-004 was inoculated and sulfur autotrophic denitrification was carried out at 5°C-15°C.

6. The use of a cold-resistant sulfur autotrophic denitrifying strain in a sulfur autotrophic denitrification system as claimed in claim 5, characterized in that: The inoculation amount of Thiobacillus denitrificans SZG-SAD-004 is 5-10 mL / L.

7. Use of the cold-resistant sulfur autotrophic denitrifying strain according to claim 1 in sewage treatment.

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