Cold-tolerant sulfur autotrophic denitrifying bacteria and application thereof

Abstract

The present application relates to the field of environmental microorganism and sewage treatment technology, and proposes a cold-resistant sulfur autotrophic denitrification bacteria and application thereof, wherein the sulfur autotrophic denitrification bacteria is Thiobacillus denitrificans SZG-SAD-004, and the preservation number is CCTCC NO:M2023992.The Thiobacillus denitrificans SZG-SAD-004 in the present application can help the sulfur autotrophic denitrification device to start quickly at low temperature, achieve normal-temperature starting efficiency, and still achieve high-efficiency denitrification at 6-15 DEG C, and has high application value due to strong low-temperature tolerance and denitrification performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental microorganisms and sewage treatment technology, and particularly relates to a cold-tolerant sulfur autotrophic denitrifying bacteria and application thereof. BACKGROUND

[0002] Traditional heterotrophic denitrification method cannot completely remove total nitrogen, and needs to frequently add carbon source, which is high in cost. As a representative process of autotrophic denitrification, high-sulfur autotrophic denitrification (SAD) has the advantages of not needing to add carbon source, high denitrification efficiency, safe operation, relatively low cost, and less residual sludge, and is considered as a potential cost-effective alternative process.

[0003] Most of the existing microorganisms are suitable for growth at a temperature of 20-35 DEG C, and the biological denitrification metabolic speed is inhibited at low temperature. The sulfur autotrophic denitrifying bacteria has poor effect at low temperature, and the denitrification efficiency is low when applied in a low-temperature environment. SUMMARY

[0004] Therefore, the present application provides a cold-tolerant sulfur autotrophic denitrifying bacteria which is low-temperature resistant and high in denitrification efficiency, and application thereof.

[0005] The technical scheme of the present application is as follows: In a first aspect, the present application provides a cold-tolerant sulfur autotrophic denitrifying bacterial strain, which is Thiobacillus denitrificans SZG-SAD-004, and the preservation number is CCTCC NO: M2023992.

[0006] In a second aspect, the present application provides a sulfur autotrophic denitrifying bacterial preparation, which comprises Thiobacillus denitrificans SZG-SAD-004.

[0007] Preferably, the preparation is a liquid, and the concentration of Thiobacillus denitrificans SZG-SAD-004 in the preparation is greater than or equal to 10 9 CFU / mL.

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

[0009] On the basis of the above technical scheme, preferably, inoculate Thiobacillus denitrificans SZG-SAD-004, and carry out sulfur autotrophic denitrification at 5-15 DEG 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 application provides application of the cold-tolerant sulfur autotrophic denitrification strain in sewage treatment.

[0012] The cold-tolerant sulfur autotrophic denitrification strain and its application have the following beneficial effects:

[0013] The Thiobacillus denitrificans SZG-SAD-004 in the application can help the sulfur autotrophic denitrification device to start quickly at low temperature, achieve normal temperature starting efficiency, and still have high denitrification efficiency at 6-15 DEG C, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0015] Figure 1 The figure is the denitrification effect of SZG-SAD-004 seed culture under different temperature culture;

[0016] Figure 2 The figure is the denitrification effect of SZG-SAD-004 seed culture under different temperature culture. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0018] I. Screening of cold-tolerant denitrifying sulfur bacteria

[0019] The denitrifying Thiobacillus in the application is screened from a mixture of sludge and water in an anaerobic tank of a sewage plant, and is obtained through liquid enrichment, device domestication and purification screening, and the specific method is as follows:

[0020] 1.1 Liquid enrichment

[0021] 5mL of the mixture of sludge and water in the anaerobic tank is taken into 30mL of enrichment culture medium, and is cultured at 28℃ until the nitrate nitrogen is consumed to 5mg / L, and then is transferred to the next generation at a proportion of 10% (volume ratio), after 5 generations of transfer, the nitrate nitrogen is completely consumed in 2d, and the enrichment liquid is obtained, and the process cycle is 17d.

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

[0023] 1.2 Bacterial domestication

[0024] The pebble cushion is placed in the bottommost part, and occupies 1 / 3 of the device, then 500g of granular sulfur autotrophic carrier containing sulfur and pyrite is added, the sulfur autotrophic denitrification device is assembled, the bacteria culture liquid (100mg / L of potassium nitrate solution containing nitrate nitrogen) is added, 10mL of the enrichment liquid (1%) is inoculated, and the device is placed at room temperature for 6-7d of bacteria culture, and then the nitrate nitrogen is measured to be less than 2mg / L, the water inlet and outlet are started, the water inlet is simulated wastewater containing 50mg / L of nitrate nitrogen, the HRT (hydraulic retention time) is 8h, and the device is operated at 28℃, when the nitrate nitrogen in the effluent is less than 5mg / L, the operation temperature is reduced, the temperature is reduced by 2℃ as a gradient, and the temperature is gradually reduced until the temperature is 6℃, and the nitrate nitrogen in the effluent is still less than 5mg / L, and the domestication is completed; 30g of the carrier particles is mechanically broken, and then is cultured in 100mL of the enrichment culture medium, the culture temperature is 10℃, when the nitrate nitrogen is consumed to 5mg / L, the bacteria culture liquid is transferred to the next generation at a proportion of 10% (volume ratio), after 2 generations of transfer, the nitrate nitrogen is completely consumed in 2d, and the domesticated enrichment liquid is obtained.

[0025] 1.3 Purification screening

[0026] The solid culture medium for coating is the enrichment liquid culture medium with the addition of 18g / L of agar, 1mL of the domesticated enrichment liquid is gradient-diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 times, respectively, is coated on the solid plates, and is cultured in an anaerobic constant-temperature incubator, the temperature is 10℃, until obvious colonies are produced, single colonies with different morphologies, sizes and colors are picked, and are repeatedly streaked and cultured, until the purified single colonies are obtained. The colony morphology and the color of the bacteria are recorded.

[0027] The single colony after purification was picked and placed in an EP tube containing 2 mL enrichment medium, and the nitrate nitrogen was measured after 72 h of static culture at 10 DEG C. The strain with a large nitrate nitrogen degradation range was sent for identification. Through molecular biology sequencing and similarity comparison with known sequences in the Genbank database, it was identified as Thiobacillus denitrificans. Finally, the Thiobacillus denitrificans SZG-SAD-004 in the present application was obtained, and the nucleotide sequence is shown as SEQ ID No. 1.

[0028] The colony morphology of the strain is colorless and transparent or white, with regular edges, and appears as short rods under an optical microscope. The strain is negative for Gram staining, and the optimum pH is 6-8.

[0029] Therefore, the Thiobacillus denitrificans SZG-SAD-004 was preserved in the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China, postal code: 430072) on June 12, 2023, and the preservation number is CCTCC No. 2023992. The strain Thiobacillus denitrificans SZG-SAD-004 was identified as active by the protection center on June 12, 2023.

[0030] 1.4 Strain activation

[0031] The glycerol freezing tube of the Thiobacillus denitrificans SZG-SAD-004 strain preserved at -80 DEG C was taken out, streaked on solid culture medium, and cultured in an anaerobic incubator for 3-5 days at 30 DEG C.

[0032] Amplification culture: a single colony on the solid culture medium was picked and inoculated in 50 mL of seed liquid medium, and cultured at 28 DEG C for 3 days. The nitrate nitrogen was measured to be below 20 mg / L. 200 mL of liquid medium was transferred at a ratio of 5%, and cultured at 28 DEG C for 2 days. The seed liquid was inoculated in a 5L fermenter at a ratio of 10%, and cultured for 5-7 days to obtain the target bacterial liquid. At this time, the effective viable cell count was not less than 10 9 CFU / mL.

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

[0034] 5L fermentor medium: 0.2 g / L ammonium chloride, 0.3 g / L magnesium chloride, 12 g / L sodium thiosulfate, 8 g / L potassium nitrate, 2 g / L potassium dihydrogen phosphate, 3 g / L sodium bicarbonate, 0.1 g / L ferrous sulfate, 0.001 g / L anhydrous calcium chloride, under the conditions of 220 rpm, 28°C, pH 7.5, and the pH adjusting solution is 100 g / L sodium carbonate.

[0035] II. Low temperature resistance test

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

[0037] After seed culture, 10% (volume ratio) inoculation amount (same OD600) was inoculated into the fermentation medium, and culture was carried out at 10°C and 28°C. It was found that the nitrate nitrogen was reduced to below 10 mg / L at 95h (see Figure 2 ). The same conclusion was obtained after 3 rounds of repeated verification.

[0038] The above experiments show that the growth performance of SZG-SAD-004 strain at 28°C is better than that at 10°C, but the low temperature performance after 28°C culture is not much different from that at 10°C. It is judged that the bacteria is cold-tolerant bacteria. Based on this conclusion, in order to save fermentation time and cost, the normal temperature fermentation method is adopted in the follow-up.

[0039] III. Application of SZG-SAD-004 strain for 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 conditions, specifically:

[0042] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, the inoculation amount is 5 mL / L (0.5%), and the cultivation is static for 4 days. When the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 150 mg / L of nitrate nitrogen, the HRT is 4 h under the condition of 5°C, the water inlet is operated to the 7th day after the water inlet, and the nitrate nitrogen concentration in the outlet water is less than 5 mg / L. After stable operation for 20 days, the nitrate nitrogen removal rate is more than 96.67%, and the highest nitrate nitrogen removal rate is 99.98%.

[0043] Example 2

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

[0045] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, the inoculation amount is 5 mL / L (0.5%), and the cultivation is static for 4 days. When the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 150 mg / L of nitrate nitrogen, the HRT is 4 h under the condition of 5°C, the water inlet is operated to the 7th day after the water inlet, and the nitrate nitrogen concentration in the outlet water is less than 5 mg / L. After stable operation for 20 days, the nitrate nitrogen removal rate is more than 96.67%, and the highest nitrate nitrogen removal rate is 99.98%.

[0046] Example 3

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

[0048] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, the inoculation amount is 5 mL / L (0.5%), and the cultivation is static for 4 days. When the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 150 mg / L of nitrate nitrogen, the HRT is 4 h under the condition of 5°C, the water inlet is operated to the 7th day after the water inlet, and the nitrate nitrogen concentration in the outlet water is less than 5 mg / L. After stable operation for 20 days, the nitrate nitrogen removal rate is more than 96.67%, and the highest nitrate nitrogen removal rate is 99.98%.

[0049] Example 4

[0050] Example 4 is the application of the SZG-SAD-004 strain for sulfur autotrophic denitrification under the condition of 15°C and the inoculation amount of 10 ml / L, specifically as follows:

[0051] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, and the inoculation amount is 10 ml / L (1%). After 3 days of static cultivation, when the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 350 mg / L of nitrate nitrogen, and the HRT is 4 h at 15°C. After running for 3 days after the water inlet, the nitrate nitrogen concentration in the effluent is less than 3 mg / L. After stable running for 21 days, the nitrate nitrogen removal rate is more than 95.92%, and the highest nitrate nitrogen removal rate is 99.88%.

[0052] Example 5

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

[0054] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, and the inoculation amount is 5 ml / L (0.5%). After 2 days of static cultivation, when the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 200 mg / L of nitrate nitrogen, and the HRT is 0.5 h at 25°C. After running for 4 days after the water inlet, the nitrate nitrogen concentration in the effluent is less than 3 mg / L. After stable running for 18 days, the nitrate nitrogen removal rate is more than 98.92%, and the highest nitrate nitrogen removal rate is 99.58%.

[0055] Example 6

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

[0057] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (cultivation liquid) containing 100 mg / L of nitrate nitrogen, inoculated with the activated SZG-SAD-004 strain, and the inoculation amount is 5 ml / L (0.5%). After 2 days of static cultivation, when the nitrate nitrogen in the cultivation liquid is reduced to less than 10 mg / L, the cultivation is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 200 mg / L of nitrate nitrogen, and the HRT is 0.5 h at 35°C. After running for 4 days after the water inlet, the nitrate nitrogen concentration in the effluent is less than 3 mg / L. After stable running for 18 days, the nitrate nitrogen removal rate is more than 98.92%, and the highest nitrate nitrogen removal rate is 99.68%.

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

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

[0060] Comparative Example 1

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

[0062] Normal-temperature enrichment liquid collection: the normal-temperature enrichment liquid is obtained by enriching the denitrifying bacterial community from sludge at normal temperature, which is also the enrichment liquid commonly used for starting the existing sulfur autotrophic denitrification process.

[0063] The sulfur autotrophic denitrification device is added with a potassium nitrate solution (bacterial culture solution) containing 100 mg / L of nitrate nitrogen, and the normal-temperature enrichment liquid is inoculated at an inoculation amount of 5 mL / L (0.5%), and the bacterial culture is incubated for 2 days. When the nitrate nitrogen in the bacterial culture solution is reduced to below 10 mg / L, the bacterial culture is successful, and the water inlet can be started. The water inlet is simulated wastewater containing 200 mg / L of nitrate nitrogen, and the HRT is 0.5 h at 25°C. After 7 days of operation after the water inlet, the nitrate nitrogen concentration in the effluent is below 3 mg / L. After 18 days of stable operation, the nitrate nitrogen removal rate is above 90.92%, and the maximum nitrate nitrogen removal rate is 98.73%.

[0064] Comparative Example 2

[0065] The difference between Comparative Example 2 and Example 3 is that the normal-temperature enrichment liquid is used for sulfur autotrophic denitrification at 15°C in Comparative Example 2.

[0066] The normal temperature enrichment liquid is added with the normal temperature enrichment liquid of the comparative example 1, the potassium nitrate solution containing nitrate nitrogen 100 mg / L (bacteria culture liquid) is added in the sulfur autotrophic denitrification device, the normal temperature enrichment liquid is inoculated, the inoculation amount is 5 mL / L (0.5%), and the bacteria culture is placed for 7 days. On the 7th day of bacteria culture, the nitrate nitrogen is still 88 mg / L, the slow water (the simulated wastewater containing nitrate 50 mg / L) is tried, the hydraulic retention time HRT is 8 h, and the operation is 11 days. The minimum residual amount of nitrate nitrogen in the effluent is still 47.23 mg / L, so it is indicated that the normal temperature enrichment liquid is difficult to start the sulfur autotrophic denitrification at low temperature.

[0067] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A sulfur-autotrophic denitrifying bacteria preparation, characterized in that: This includes Thiobacillus denitrificans SZG-SAD-004, with accession number CCTCC NO:M2023992. The formulation is a liquid, and the concentration of Thiobacillus denitrificans SZG-SAD-004 in the formulation is ≥10. 9 CFU / mL.

2. The application of the sulfur autotrophic denitrifying bacteria preparation as described in claim 1 in a sulfur autotrophic denitrification system.

3. The application of the sulfur autotrophic denitrifying bacteria preparation as described in claim 2 in a sulfur autotrophic denitrification system, characterized in that: Thiobacillus denitrificans SZG-SAD-004 was inoculated and sulfur autotrophic denitrification was carried out at 5℃-15℃.

4. The application of the sulfur autotrophic denitrifying bacteria preparation as described in claim 2 in a sulfur autotrophic denitrification system, characterized in that: The inoculation amount of Thiobacillus denitrificans SZG-SAD-004 is 5-10 mL / L.

5. The application of the sulfur autotrophic denitrifying bacteria preparation as described in claim 1 in wastewater treatment.

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