Heterotrophic nitrification and aerobic denitrification halomonas resistant to low temperature and high salt and application of heterotrophic nitrification and aerobic denitrification halomonas

Abstract

The invention discloses a heterotrophic nitrification and aerobic denitrification halomonas sp. ZC-1 with low temperature resistance and high salt, and the preservation number of the heterotrophic nitrification and aerobic denitrification halomonas sp. ZC-1 is CCTCC (China Center for Type Culture Collection) M 20241695. The strain ZC-1 screened by the invention can simultaneously tolerate the influence of low temperature and high salt, and still can efficiently remove ammonia nitrogen under double stress conditions, especially under the environment of the temperature of 10 DEG C and the salinity of 10% w / v, the ammonia nitrogen removal rate reaches 94% or above, the TN removal rate reaches 93% or above, and salt-containing wastewater in northern China in winter can be treated in a targeted manner.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a low-temperature and high-salt resistant heterotrophic nitrifying aerobic denitrifying halomonas and application of the bacterium. Background Art

[0002] In recent years, with the rapid economic development, the nitrogen compounds produced by human activities have greatly exceeded the natural circulation capacity, resulting in increasingly serious nitrogen pollution. More and more studies have shown that the consequences of nitrogen pollution will be worse than the impact of carbon dioxide on the environment. It will not only cause global warming, but also affect human health, biodiversity and ozone levels, posing a threat to the entire ecosystem. For example, nitrates discharged from farms can enter drinking water and cause methemoglobinemia; nitrogen oxides produced by burning fossil fuels can form ground ozone, which is harmful to the ecosystem and human health, and can also combine with sulfur dioxide and water vapor in the air to form nitrogen and sulfur acid rain; large amounts of nitrates and ammonia discharged into rivers, lakes and oceans will cause eutrophication, causing algae, plankton, plants, aquatic organisms and fish to die or even become extinct, seriously affecting the ecology.

[0003] The treatment of nitrogen pollution is generally divided into physical and chemical methods and biological methods. Compared with physical and chemical methods, biological methods have the significant advantages of low operating costs and no secondary pollution, and are often widely used in sewage treatment. Traditional biological denitrification technology is completed through the combination of autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria, that is, under aerobic conditions, ammonia nitrogen undergoes nitrification reaction under the action of autotrophic bacteria, and under anaerobic conditions and heterotrophic bacteria, denitrification reaction is carried out to convert nitrogen into gas. However, the growth rate and oxygen demand of autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria are different. Different reactors usually need to be set up during the application process, resulting in disadvantages such as large footprint, high infrastructure and operation and maintenance costs.

[0004] Heterotrophic nitrifying aerobic denitrifying bacteria are bacteria that can complete nitrification and denitrification reactions under the same conditions. Compared with traditional denitrifying bacteria, they have a faster growth rate and higher denitrification efficiency. Therefore, they have received extensive attention and research in recent years. At present, there have been reports that heterotrophic nitrifying aerobic denitrifying bacteria are used for the treatment of ammonia nitrogen wastewater. However, due to the complex characteristics of industrial wastewater and the adaptability requirements of denitrifying bacteria, some specific wastewaters, such as mustard wastewater in northern my country in winter, will significantly reduce the denitrification ability of bacteria due to their low temperature and high osmotic pressure environment. Therefore, when treating them, they often need to be heated or desalted first, which increases the operating costs and the complexity of the treatment process. Therefore, the development of heterotrophic nitrifying aerobic denitrifying bacteria that can tolerate the dual stress of low temperature and high osmotic pressure and maintain efficient denitrification removal rate will be of great significance for the treatment of saline wastewater. Summary of the invention

[0005] Based on the above-mentioned needs of the prior art in the art, the present invention provides a strain of heterotrophic nitrifying aerobic denitrifying Halomonas sp. ZC-1 which has nitrification and denitrification functions and is also resistant to low temperature and high salt, as well as the bacterial agent product and application.

[0006] The low temperature and high salt resistant heterotrophic nitrifying aerobic denitrifying Halomonas sp. ZC-1 of the present invention has a preservation number of CCTCC M 20241695.

[0007] As a limitation of the above technical solution, the 16SrDNA sequence of Halomonas sp. ZC-1 is shown as SEQ ID No.1.

[0008] The present invention provides a bacterial agent made from the above-mentioned Halomonas sp. ZC-1, which can be mainly prepared from the culture solution of Halomonas sp. ZC-1 or by adding auxiliary materials, or can be prepared by compounding the culture solution of Halomonas sp. ZC-1 with the culture solution of other bacterial species, or by adding auxiliary materials.

[0009] At the same time, the present invention also provides an application of Halomonas sp. ZC-1, which is to use the above Halomonas sp. ZC-1 or the above bacterial agent for the treatment of ammonia nitrogen wastewater.

[0010] As a limitation of the above technical solution, the carbon source used for strain culture is selected from sodium succinate, sodium citrate, and anhydrous sodium acetate, preferably sodium succinate.

[0011] As a limitation of the above technical solution, the carbon-nitrogen ratio required for strain culture is 10-30, preferably 20.

[0012] As a limitation of the above technical scheme, it is used for the treatment of saline ammonia nitrogen wastewater, the salt content in the ammonia nitrogen wastewater is NaCl concentration of 1% to 13% w / v, and the applicable temperature of the treatment process is 10 to 40°C.

[0013] As a limitation of the above technical solution, it is used for the treatment of high-salt ammonia nitrogen wastewater under low temperature conditions, high salt refers to the NaCl concentration in the wastewater as high as 10% to 13% w / v, and low temperature refers to the temperature as low as 10°C.

[0014] As a limitation of the above technical solution, under the dual stress of low temperature 10°C and high salt concentration 10% w / v, the ammonia nitrogen removal rate of the Halomonas sp. ZC-1 reaches more than 94% in 4 days, the maximum removal rate in 2 to 4 days reaches 3.36 mg / L / h, and the TN removal rate in 6 days reaches more than 93%.

[0015] In addition, the present invention also provides a treatment method, including using the above-mentioned Halomonas Halomonas sp. ZC-1 to perform nitrification and denitrification treatment on the sample to be treated, and the sample to be treated includes wastewater, waste liquid, waste residue, etc. contaminated with ammonia nitrogen.

[0016] The beneficial effect of the present invention is that the Halomonas sp. ZC-1 disclosed in the present invention can effectively remove ammonia nitrogen and total nitrogen under the dual stress conditions of low temperature and high salt (high osmotic pressure), and the bacterial body grows fast and has a high removal rate, and is particularly suitable for the degradation of ammonia nitrogen in high-salt wastewater in cold areas. The present invention breaks through the single tolerance limitation of existing heterotrophic nitrifying aerobic denitrifying bacteria strains to low temperature and high salt environments, and can still maintain high denitrification efficiency under dual stress conditions, solves the problem that existing heterotrophic nitrifying aerobic denitrifying bacteria are inhibited by the dual stress of temperature and salinity during the denitrification process, and is conducive to application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 , is the phylogenetic tree and Gram staining picture of Halomonas sp.ZC-1;

[0018] Figure 2 , is a curve diagram of the degradation effect of Halomonas sp. ZC-1 on ammonia nitrogen and total nitrogen under different conditions;

[0019] Figure 3 , is a curve diagram of the nitrogen removal effect of Halomonas sp. ZC-1 under the dual stress of low temperature and high salt;

[0020] Figure 4 , is the growth curve of Halomonas in 10℃, 10% NaCl environment. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. 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.

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0023] The test materials used in the present invention are as follows:

[0024] Enrichment medium: peptone 10g / L, beef extract 3g / L, NaCl 20g / L, pH 7.0~7.4.

[0025] Nitrification medium: (NH4)2SO4 0.942g / L, NaCl 20g / L, C4H4Na2O4 2.531g / L, C / N is 10, pH is 7.0-7.4, and Visconazole salt solution 50mL / L.

[0026] Denitrification culture medium: KNO3 1.44g / L, NaCl 20g / L, C4H4Na2O4 2.531g / L, C / N is 10, pH is 7.0-7.4, and Viscount salt solution 50mL / L.

[0027] Viscose salt solution: 6.55g K2HPO4·3H2O, 2.5g MgSO4·7H2O, 2.5g NaCl, 0.05g FeSO4·7H2O, 0.05g MnSO4·4H2O per liter of water.

[0028] Solid culture medium: Add 20 g / L agar powder to the nitrification medium.

[0029] Example 1: Bacteria enrichment and purification

[0030] 1) Enrichment culture: 2 mL of wastewater sample from a chemical company in Shijiazhuang, Hebei, China was inoculated into 100 mL of enrichment medium, and cultured at 28°C and 170 rpm for 3 days to obtain an initial bacterial solution. The initial bacterial solution was then inoculated into 100 mL of enrichment medium at an inoculum rate of 5% (v / v) and cultured at 28°C and 170 rpm for 3 days to obtain an enriched bacterial solution.

[0031] 2) Subculture: Take 5 mL of enriched bacterial solution and inoculate it into 100 mL of nitrification medium. Culture it at 10°C and 170 rpm for 3 days. Subculture it 5 times continuously. Detect the OD value of the culture medium every 24 hours during the culture process. 600 NH4 + -N and TN to obtain a mixed bacterial community with stable denitrification effect.

[0032] 3) Pure bacteria isolation: dilute the bacterial solution after continuous subculture with sterile water to 10 -1 ~10 -7 The gradient dilutions were evenly spread on the solid culture medium and cultured in a biochemical incubator at 30°C for 3 days. After that, single colonies were streaked on consecutive plates until there were no obvious abnormal colonies on the plate.

[0033] Example 2: Identification and preservation of bacterial strains

[0034] Chengdu Bio-Qingke Company was commissioned to complete the 16SrDNA sequencing of the purified strain, and its 16S rRNA gene sequence is as follows (SEQ ID No.1):

[0035]

[0036] The 16S rDNA sequence results were compared by Blast in NCBI, and the bacterial species with higher similarity to the target gene sequence were selected, and the phylogenetic tree was constructed using MEGA 11.0 software.

[0037] Identification results: The strain is a Gram-negative bacterium, with a smooth and convex surface, moist and shiny, opaque, light yellow colonies, neat edges, and round colonies. The strain is closely related to Halomonas.Soap Lake by blast analysis of the gene sequence in Genbank, with a similarity of 99.30%. Figure 1 As shown, the phylogenetic tree constructed by MEGA11.0 was determined to be the genus Halomonas.

[0038] The purified single colonies were inoculated into nitrification medium and denitrification medium respectively to verify the heterotrophic nitrification and aerobic denitrification functions. The nitrogen balance of ZC-1 bacteria in different culture media was analyzed by ultrasonic cell disruptor. The calculation method is as follows. The results are shown in Table 1.

[0039] TN1 is the total nitrogen in the supernatant after centrifugation in the culture medium;

[0040] TN2 is the total nitrogen in the supernatant after centrifugation of cells disrupted by ultrasonic cell disruptor;

[0041] Intracellular nitrogen = TN2-TN1;

[0042] Nitrogen assimilation rate: Δ intracellular nitrogen / (0d)TN2 (assimilation is used for bacterial growth)

[0043] Nitrogen loss: [(0d)TN2-(3d)TN2] / (0d)TN2 (mainly due to conversion into gaseous form by dissimilation).

[0044] Table 1. Nitrogen balance analysis of degradation effect of ZC-1 bacteria in nitrification medium and denitrification medium for 3 days

[0045]

[0046]

[0047] As shown in Table 1, under aerobic conditions, ZC-1 bacteria + The removal of -N and KNO3 mainly includes assimilation and dissimilation. When ammonium sulfate is the only nitrogen source, nitrite nitrogen accumulates in the culture medium, indicating that there is NH4 + -N is converted into NO2 through nitrification - -N; When potassium nitrate was used as the only nitrogen source, nitrite and nitrate nitrogen accumulated in the culture medium, indicating that KNO3 was converted into NO2 through denitrification.- -N and NH4 + -N.

[0048] Strain preservation: The isolated and purified strain was deposited in the China Center for Type Culture Collection with the deposit number CCTCCM 20241695. The address is Wuhan University, Wuhan City, Hubei Province, China. The deposit date is July 26, 2024. The classification name is Halomonas sp. ZC-1.

[0049] Example 3: Degradation effect of Halomonas sp. ZC-1 on ammonia nitrogen and TN under low temperature conditions

[0050] The effects of different factors on the denitrification effect of strain ZC-1 were evaluated, and the carbon source, C / N ratio, culture speed, temperature and other conditions of the nitrification culture medium were optimized.

[0051] Culture conditions: nitrification medium (initial nitrogen was configured to 200 mg / L with ammonium sulfate as the sole nitrogen source, sodium succinate was used as the sole carbon source, C / N was set to 10, i.e., COD concentration was 2000 mg / L, and COD concentration was calculated according to the oxidation reaction chemical formula of different single carbon sources to obtain the mass to be weighed); inoculation amount 5% (v / v); rotation speed was set to 170 rpm; temperature was set to 28°C; culture was performed for 3 days.

[0052] At the end of the culture, samples were taken and ammonia nitrogen and total nitrogen were determined. Ammonia nitrogen was determined by Nessler's reagent spectrophotometry, and total nitrogen was determined by alkaline potassium persulfate method.

[0053] (1) Experimental study on the effect of carbon source in nitrification medium. Only the carbon source of nitrification medium was changed, that is, sodium succinate (2.531 g / L), anhydrous sodium acetate (5.125 g / L), sodium citrate (2.336 g / L), glucose (1.877 g / L), and sucrose (1.781 g / L) were used as the single carbon source for microorganisms, respectively. The initial COD concentration was kept at 2000 mg / L, and other components and culture conditions were kept unchanged. The best carbon source was screened, such as Figure 2 As shown in A, the best carbon source is sodium succinate.

[0054] (2) Experimental study on the effect of C / N ratio in nitrification medium. Only the C / N ratio of nitrification medium was changed, that is, sodium succinate was used as the sole carbon source. 0.1266, 0.2531, 0.3797, 0.5062, 0.6328, and 0.7593 g of sodium succinate were added to 100 mL of medium, corresponding to C / N ratios of 5, 10, 15, 20, 25, and 30, respectively. Other components and culture conditions remained unchanged. The best C / N ratio was selected, such as Figure 2 As shown in B, the optimal C / N is 20.

[0055] (3) Experimental culture speed effect: Sodium succinate was used as the sole carbon source, C / N was set to 20 (i.e., the amount of sodium succinate added to 100 mL of culture medium was 0.5062 g), other components of the nitrification medium remained unchanged, and the shaking speed was adjusted to 130 rpm, 150 rpm, 170 rpm, 190 rpm, and 210 rpm, respectively. Other culture conditions remained unchanged, and the optimal speed was screened out, such as Figure 2 As shown in C, the optimal speed is 190 rpm.

[0056] (4) The effect of experimental culture temperature. Sodium succinate was used as the sole carbon source, the C / N ratio was set to 20, the other components of the nitrification medium remained unchanged, the shaking speed was 190 rpm, and different culture temperatures were set at 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 40°C, and 45°C. Other culture conditions remained unchanged. The lowest tolerable temperature of ZC-1 bacteria was investigated. Figure 2 As shown in D, the lowest tolerable temperature is 10℃.

[0057] The denitrification effect of Halomonas sp. ZC-1 under low temperature conditions is as follows Figure 2 D. At a temperature of 10°C (carbon source: sodium succinate; C / N: 20; rotation speed: 190rpm), ZC-1 bacteria can remove more than 97% of ammonia nitrogen in 3 days, and the initial ammonia nitrogen concentration is reduced from 200mg / L to 4.88mg / L, and the removal rate reaches 2.71mg / L / h, indicating that ZC-1 bacteria has good prospects for sewage treatment under low temperature conditions.

[0058] Example 4: Effect of Halomonas sp. ZC-1 on ammonia nitrogen degradation under dual stress conditions

[0059] Halomonas sp.ZC-1 showed excellent denitrification effect at 10℃. Taking 10℃ as one of the stress conditions and salinity as another stress condition, the degradation effect of Halomonas sp.ZC-1 on ammonia nitrogen under different NaCl concentrations was tested.

[0060] Different culture media with NaCl concentrations of 0%, 1%, 3%, 5%, 7%, 10%, 13%, and 15% (w / v) were prepared (the culture medium used an optimized nitrification medium, that is, the amount of sodium succinate added was 0.5062 g, the C / N ratio was 20, and other components and amounts remained unchanged), and cultured for 6 days at a culture speed of 190 rpm, a culture temperature of 10°C, and an inoculation amount of 5% (v / v). A blank culture medium without any bacteria was set as a control. During the culture period, samples were taken every 24 hours to determine ammonia nitrogen, total nitrogen, and OD indicating bacterial growth. 600 Values, measured ammonia nitrogen, total nitrogen degradation data and OD 600The results are shown in Tables 2 to 4.

[0061] Table 2. Effect of ZC-1 bacteria on ammonia nitrogen removal in nitrification medium with different salinities at 10°C

[0062]

[0063] Table 3. TN removal effect of ZC-1 bacteria on nitrification medium with different salinities at 10℃

[0064]

[0065]

[0066] Table 4. OD of ZC-1 bacteria in nitrification medium at different salinities at 10℃ 600

[0067]

[0068] From the results in Tables 2 to 4 and Figure 3 As shown, the nitrogen removal effect of Halomonas sp. ZC-1 under the dual stress conditions of 10°C low temperature and 10% salinity (i.e., high osmotic pressure) is as follows: the ammonia nitrogen removal rate can reach more than 94.0% on the fourth day, the maximum removal rate is 3.36 mg / L / h in 2-4 days, and the TN removal rate reaches more than 93% after 6 days; even under 13% salinity, the removal rate of ammonia nitrogen (initial ammonia nitrogen was measured to be 212.17 mg / L) can reach 97.8% in 6 days, and the TN removal rate is 93.9% at this time. If the salinity continues to increase, the ZC-1 bacteria lose their growth activity.

[0069] From the above, it can be seen that the Halomonas sp. ZC-1 screened by the present invention has heterotrophic nitrification and aerobic denitrification functions, can not only tolerate low temperature and high salt extreme environments, but also show efficient denitrification capabilities under the dual stress conditions of low temperature and high salt. The strong tolerance and efficient denitrification capabilities of Halomonas sp. ZC-1 are expected to make new breakthroughs in the treatment and application of low temperature and high salt wastewater.

Claims

1. A low temperature and high salt resistant heterotrophic nitrifying aerobic denitrifying Halomonas sp. ZC-1, characterized by: Its accession number is CCTCC M 20241695.

2. The low temperature and high salt resistant heterotrophic nitrifying aerobic denitrifying Halomonas sp. ZC-1 according to claim 1, characterized in that: The 16SrDNA sequence of Halomonas sp. ZC-1 is shown in SEQ ID No.

1.

3. A bacterial agent, characterized in that: It includes the Halomonas sp. ZC-1 described in claim 1.

4. An application of Halomonas sp. ZC-1, characterized in that: The Halomonas sp. ZC-1 described in claim 1 or the bacterial agent described in claim 4 is used for treating ammonia nitrogen wastewater.

5. The use of Halomonas sp. ZC-1 according to claim 4, characterized in that: The carbon source used for strain culture is selected from sodium succinate, sodium citrate, anhydrous sodium acetate, and sodium succinate is preferred.

6. The use of Halomonas sp. ZC-1 according to claim 4, characterized in that: The carbon-nitrogen ratio required for strain cultivation is 10-30, preferably 20.

7. The use of Halomonas sp. ZC-1 according to claim 4, characterized in that: It is used for the treatment of saline ammonia nitrogen wastewater, where the salt content in the ammonia nitrogen wastewater is NaCl concentration of 1% to 13% w / v, and the applicable temperature for the treatment process is 10 to 40°C.

8. The use of Halomonas sp. ZC-1 according to claim 7, characterized in that: Used for the treatment of high-salt ammonia nitrogen wastewater under low temperature conditions. High salt refers to the NaCl concentration in the wastewater as high as 10% to 13% w / v, and low temperature refers to the temperature as low as 10°C.

9. The use of Halomonas sp. ZC-1 according to claim 8, characterized in that: Under the dual stress of low temperature 10°C and high salt concentration 10% w / v, the ammonia nitrogen removal rate of the Halomonas sp. ZC-1 reached more than 94% in 4 days, the maximum removal rate reached 3.36 mg / L / h in 2 to 4 days, and the TN removal rate reached more than 93% in 6 days.

10. A treatment method, characterized in that: The method comprises using the Halomonas sp. ZC-1 described in claim 1 or the bacterial agent described in claim 3 to perform nitrification and denitrification treatment on the sample to be treated.

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