A strain of Pseudomonas marginale and its application

Abstract

The present invention belongs to the technical field of biological denitrification of sewage, and specifically relates to a strain of Pseudomonas marginalis and its application. Pseudomonas marginalis (Pseudomonas marginalis), characterized in that the strain is WS45, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on July 22, 2021, with a deposit number of CGMCC No.22943. Under 1-40°C conditions, the strain WS45 has heterotrophic nitrification-aerobic denitrification metabolic capabilities, and has a good removal effect on nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage. The strain has good adaptability to low temperatures and can be used for inorganic nitrogen pollution control in various water bodies such as medium and high concentration domestic sewage, livestock and poultry breeding wastewater or industrial organic wastewater in cold areas of my country. It has the advantages of being environmentally friendly and having a wide applicable temperature range.

Description

Technical Field

[0001] The invention belongs to the technical field of biological denitrification of sewage, and specifically relates to a strain of Pseudomonas marginale and application thereof. Background Art

[0002] Inorganic nitrogen pollution in water bodies frequently occurs in many countries around the world, and has posed a potential threat to the quality of the ecosystem environment and human health. Evidence shows that the concentration of ammonia nitrogen and nitrate nitrogen in my country's surface and coastal waters exceeds the standard, which is also worthy of attention. How to economically and effectively remove inorganic nitrogen pollution in water bodies is an important part of improving the quality of the ecological environment and ensuring the health of the ecosystem.

[0003] The technologies for denitrification of sewage mainly include chemical, physical and biological methods. Microbial denitrification technology is widely valued because of its wide range of applications, environmental friendliness and economic feasibility. The core of microbial denitrification technology is the efficient operation of microbial autotrophic nitrification, anaerobic denitrification metabolism and other processes. These metabolic pathways can promote the degradation of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in sewage. After years of development, there are relatively mature biological denitrification processes such as A 2 O, SBR, etc. are used in engineering practice of sewage denitrification. In the process of microbial denitrification, maintaining the metabolic activity of denitrification microorganisms in sludge under complex and changing environmental conditions is the key to maintaining the denitrification effect.

[0004] Many studies have shown that low temperature is one of the main factors affecting the effect of biological denitrification. Since the optimum temperature of most denitrifying microbial strains or denitrifying process technologies such as nitrifying bacteria and denitrifying bacteria is mostly in the medium temperature range (20-35°C), the resources of nitrifying and denitrifying denitrifying microbial strains that can tolerate low temperature conditions (below 20°C) are relatively scarce. On the other hand, most parts of northern my country have long winters and water temperatures are often below 15°C. Under such low temperature conditions, the metabolic activity of denitrifying microorganisms in sludge is significantly inhibited, and the effluent quality of sewage treatment plants is difficult to meet standards. There is a realistic technical demand for low-temperature denitrifying microbial strain resources.

[0005] In recent years, screening for low-temperature resistant denitrifying bacteria has received increasing attention and has made some progress. However, due to the often complex and changeable environmental conditions of nitrogen-polluted water bodies (such as water temperature, salinity, inorganic nitrogen pollution concentration, etc.), existing mesophilic denitrifying strains cannot exhibit efficient denitrification activity under low temperature conditions, and their denitrification effects are often unsatisfactory. Summary of the invention

[0006] In view of the current situation that the biological denitrification technology for sewage has poor effect under complex environmental conditions, especially at low temperatures, and there is a lack of low-temperature resistant denitrification bacteria resources, the present invention aims to provide a strain of Pseudomonas marginalis WS45 and its application in removing inorganic nitrogen pollutants from water bodies.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A strain of Pseudomonas marginalis, Pseudomonas marginalis (Pseudomonas marginalis), characterized in that the strain is WS45, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 22, 2021, with the deposit number CGMCC No.22943.

[0009] The application of the strain is to remove inorganic nitrogen pollutants in water under the condition of 1-40°C.

[0010] The strain removes one or more of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water at 1-40°C.

[0011] The nitrogen removed is one or more of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, and the total concentration is not higher than 600 mg / L. The ammonia nitrogen concentration in the sewage is 0.1-250 mg / L, the nitrate nitrogen concentration is 0.1-600 mg / L, and the nitrite concentration is 0.1-80 mg / L.

[0012] A bacterial agent for removing inorganic nitrogen pollutants in water, the bacterial agent containing the strain.

[0013] The bacterial agent is required to activate the strain.

[0014] The application of the bacterial agent is to remove inorganic nitrogen pollutants in water under a wide temperature range.

[0015] A method for removing inorganic nitrogen pollutants from water, wherein the activated strain or the bacterial agent is added to the water environment to be treated at an inoculation rate of 0.1-20% (v / v), thereby removing inorganic nitrogen pollutants from the water; wherein the cell concentration of strain WS45 in the water is ≥10 4 Pieces / mL.

[0016] The dissolved oxygen concentration in the water to be treated is above 5 mg / L, the C / N ratio in the water is 3-30, the pH of the water is 6-9, the water temperature is 1-40°C, the salinity (in terms of NaCl concentration) is 0.2-30 g / L, and the hydraulic retention time is above 5 days; the inorganic nitrogen pollutants in the water can be removed.

[0017] The activation of the strain is as follows: the strain WS45 is inoculated into a culture medium, with a rotation speed of 120-220 rpm, 15-35° C., and activation for 10-72 hours; wherein the culture medium components are: NaCl 0.2-30.0; peptone 0.5-20.0; yeast extract 0.5-10.0; distilled water 1000 mL, and the pH is adjusted to 7.0-8.0.

[0018] Beneficial effects of the present invention:

[0019] (1) The Pseudomonas marginalis strain WS45 of the present invention can tolerate low temperature conditions well and can effectively remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water at 1-40°C;

[0020] (2) The strain of the present invention maintains good denitrification activity in a wide temperature range of 1-40°C, and can be used for sewage denitrification treatment in geographical areas in northern my country where the annual average temperature difference varies greatly;

[0021] (3) The culture medium components required for the activation and expansion of the strain of the present invention are simple, and the bacterial liquid preparation process is relatively easy, which is conducive to industrial production and subsequent applications. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiment, and the embodiment provided is only to set forth the present invention, but is not intended to limit the scope of the present invention. The embodiment provided below can be used as a reference for further optimization and improvement by those skilled in the art, and is not intended to limit the present invention in any way. Contents of the present invention are not limited to the following examples.

[0023] The Pseudomonas marginale strain WS45 of the present invention has the ability to effectively remove nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage under the condition of 1-40°C, can tolerate low temperature, and has the metabolic activity of heterotrophic nitrification and aerobic denitrification, and has good potential application value in the treatment of nitrogen pollution in water bodies in cold regions of my country.

[0024] The strain WS45 of the present invention was isolated and screened from the activated sludge of a domestic sewage treatment plant in Heilongjiang Province, and was preliminarily identified as Pseudomonas marginale through 16SrRNA gene sequence comparison. The strain has an obvious degradation and removal effect on inorganic nitrogen pollution under a wide temperature condition.

[0025] The experimental methods in the following examples, unless otherwise specified, are all conventional or standard methods, and can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Materials required in the following examples:

[0026] 1) Activated sludge: collected from the activated sludge pool of a sewage treatment plant in a city in Heilongjiang.

[0027] 2) Culture medium:

[0028] Denitrifying bacteria enrichment medium (g·L -1 ): NaCl 1.0; peptone 12.0; yeast extract 6.0; distilled water 1L; pH 7.0~8.0.

[0029] Denitrification medium (g·L -1 ): CH3COONa 0.6; NH4Cl 0.2, KNO3 0.4, NaNO2 0.2, KH2PO4 0.05; CaCl2·2H2O 0.15; MgSO4·7H2O 0.06; trace element solution 1mL; distilled water 1L; pH 7.2-8.0; solid culture medium added with 2% agar.

[0030] Trace element liquid (g / L): EDTA0.35; ZnSO4·7H2O 0.2; CuSO4·5H2O 0.1; MnSO4·7H2O0.1; Co(NO3)2·7H2O 0.09; H3BO3 0.1; Na2MoO4 0.1.

[0031] After the above culture medium is prepared, it needs to be sterilized at 103.4kPa and 121℃ for 20-25min, and then used after the culture medium has cooled down.

[0032] 3) Main experimental instruments:

[0033] Constant temperature biochemical incubator, constant temperature shaker, clean bench, pressure steam sterilizer, micro UV spectrophotometer, PCR instrument, electrophoresis instrument, etc.

[0034] Example 1

[0035] (I) Screening, preliminary identification and preservation of strain WS45

[0036] Take 30g of activated sludge and transfer it into a 500mL conical flask pre-filled with 100mL of denitrifying bacteria enrichment medium in a sterile clean bench, and then shake and culture it on a constant temperature shaker at 2℃ and 180rpm for 15 days;

[0037] Take an appropriate amount of bacterial liquid after shaking culture, dilute it 10 times in a gradient manner, and then spread it on a solid denitrification medium plate, and then culture it at 2℃ for more than 10 days;

[0038] After the colonies appear on the plate culture medium, the plate streak separation method is used to pick the colonies, and the streak separation culture process is repeated until the colonies have a single morphology. After microscopic examination and denitrification effect determination, the strain with the highest inorganic nitrogen removal rate is selected and numbered as WS45. WS45 is inoculated on a solid slant culture medium and stored at 4°C. It is used for strain identification and denitrification performance evaluation within a one-month temporary storage period.

[0039] The genomic DNA of strain WS45 was extracted using a bacterial genomic DNA extraction kit according to the instructions. After PCR detection and DNA quality assessment, it was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for 16S rRNA gene sequencing. The measured gene sequence was compared with the NCBI database. The results showed that the sequence identity of WS45 and Pseudomonas marginalis (DSM 13124) reached 99%, indicating that the strain was most closely related to Pseudomonas marginalis. The 16S rRNA gene sequence of strain WS45 is:

[0040] WS45

[0041] Assembly of 2sequences into 1contig(s)

[0042]

[0043] The strain WS45 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.22943.

[0044] Example 2: Evaluation of denitrification effect of strain WS45 at 1°C

[0045] (I) Activation of strains. Take the WS45 strain slant and inoculate it into a 500 mL conical flask containing 150 mL of denitrifying bacteria enrichment medium on a sterile workbench, and then place it in a constant temperature shaker at 25°C and 180 rpm for 48 hours.

[0046] (II) Evaluation of denitrification performance of strains. The experiment was divided into two groups, namely the inoculated group and the control group. Inoculated group: Take an appropriate amount of activated bacterial solution, inoculate it into the denitrification medium at a volume ratio of 2%, and culture it at a constant temperature of 1°C and 180rpm for 6 days. The control group was inoculated with an equal volume of WS45 bacterial solution sterilized by high-temperature steam, and the other conditions were exactly the same as those of the inoculated group. Each treatment was set up with 3 parallels. After the 6-day culture period, filter the water sample in the bottle with a 0.22μm filter membrane, and take the filtrate to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 1.

[0047] Table 1. Denitrification effect of strain WS45 at 1℃

[0048]

[0049] As shown in Table 1, after 6 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS45 strain in the treatment group at 1°C were 30.2%, 35.7% and 24.4%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.04%, 0.02% and 0.05%, respectively. The results of this experiment show that the strain WS45 has a good denitrification ability at a low temperature of 1°C.

[0050] Example 3: Evaluation of denitrification effect of strain WS45 at 5°C

[0051] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0052] (II) Evaluation of denitrification performance of strains. The experiment was divided into an inoculated bacteria group and a control group. The brief process of the inoculated bacteria group was as follows: take an appropriate amount of activated bacterial solution, inoculate it into the denitrification medium at a 2% inoculation ratio, and culture it at a constant temperature of 5°C and 180rpm for 6 days. The control group was inoculated with an equal volume of WS45 bacterial solution sterilized by high-temperature steam, and the other conditions were exactly the same as those of the inoculated bacteria group. Each treatment was set up with 3 parallels. After the 6-day culture period, the water sample in the bottle was filtered with a 0.22μm filter membrane, and the filtrate was taken to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 2.

[0053] Table 2. Denitrification effect of strain WS45 at 5℃

[0054]

[0055] As shown in Table 2, after 6 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS45 strain in the treatment group at 5℃ were 52.5%, 61.4% and 45.2% respectively, while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.01%, 0.10% and 0.12% respectively. The experimental results show that WS45 has a good denitrification ability at 5℃.

[0056] Example 4: Evaluation of denitrification effect of strain WS45 at 10°C

[0057] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0058] (II) Evaluation of denitrification performance of strains. The experiment was divided into an inoculated bacteria group and a control group. The brief process of the inoculated bacteria group was as follows: take an appropriate amount of activated bacterial liquid, inoculate it into the denitrification medium at a 2% inoculation ratio, and culture it at a constant temperature of 10°C and 180 rpm for 6 days. The control group was inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the other conditions were exactly the same as those of the inoculated bacteria group. Each treatment was set up with 3 parallels. After the 6-day culture period, the water sample in the bottle was filtered with a 0.22μm filter membrane, and the filtrate was taken to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 3.

[0059] Table 3. Denitrification effect of strain WS45 at 10℃

[0060]

[0061] As shown in Table 3, after 6 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS45 strain in the treatment group under the low temperature condition of 10℃ were 77.2%, 84.5% and 68.7% respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.10%, 0.10% and 0.11% respectively. The experimental results show that WS45 has a good denitrification ability under the low temperature condition of 10℃.

[0062] Example 5: Evaluation of denitrification effect of strain WS45 at 20°C

[0063] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0064] (II) Evaluation of denitrification performance of strains. The experiment was divided into an inoculated bacteria group and a control group. The brief process of the inoculated bacteria group was as follows: take an appropriate amount of activated bacterial solution, inoculate it into the denitrification medium at a 1% inoculation ratio, and culture it at a constant temperature of 20°C and 180 rpm for 5 days. The control group was inoculated with an equal volume of WS45 bacterial solution sterilized by high-temperature steam, and the other conditions were exactly the same as those of the inoculated bacteria group. Each treatment was set up with 3 parallels. After the 5-day culture period, the water sample in the bottle was filtered with a 0.22μm filter membrane, and the filtrate was taken to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 4.

[0065] Table 4. Denitrification effect of strain WS45 at 20℃

[0066]

[0067] As shown in Table 4, after 5 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS45 strain in the treatment group at 20℃ were 92.4%, 93.6% and 90.4%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.11%, 0.14% and 0.10%, respectively. The experimental results show that WS45 has a good denitrification ability at 20℃.

[0068] Example 6: Evaluation of denitrification effect of strain WS45 at 35°C

[0069] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0070] (II) Evaluation of denitrification performance of strains. The experiment was divided into an inoculated bacteria group and a control group. The brief process of the inoculated bacteria group was as follows: take an appropriate amount of activated bacterial liquid, inoculate it into the denitrification medium at a 1% inoculation ratio, and culture it at a constant temperature of 35°C and 180 rpm for 3 days. The control group was inoculated with an equal volume of WS45 bacterial liquid sterilized by high-temperature steam, and the other conditions were exactly the same as those of the inoculated bacteria group. Each treatment was set up with 3 parallels. After the 3-day culture period, the water sample in the bottle was filtered with a 0.22μm filter membrane, and the filtrate was taken to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The results are shown in Table 5.

[0071] Table 5. Denitrification effect of strain WS45 at 35°C

[0072]

[0073] As shown in Table 5, after 3 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS45 strain in the treatment group at 35°C were 99.1%, 99.4% and 97.2%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.24%, 0.06% and 0.01%, respectively. The experimental results show that WS45 has excellent denitrification ability at 35°C.

Claims

1. A strain of Pseudomonas marginale, characterized in that: The strain is Pseudomonas marginalis WS45, which was deposited in the General Microbiology Center of China Culture Collection Administration on July 22, 2021, with the deposit number CGMCC No.22943.

2. The use of the strain according to claim 1, characterized in that: The strain is used for removing inorganic nitrogen pollutants in water under the condition of 1-40°C.

3. The use according to claim 2, characterized in that: The strain removes one or more of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water at 1-40°C.

4. A bacterial agent for removing inorganic nitrogen pollutants in water, characterized in that: The bacterial agent contains the strain described in claim 1.

5. The bacterial agent according to claim 4, characterized in that: The bacterial agent is required to activate the strain described in claim 1.

6. The use of the bacterial agent according to claim 4, characterized in that: The bacterial agent is used to remove inorganic nitrogen pollutants in water under a wide temperature range; the temperature in the wide temperature range is 1-40°C.

7. A method for removing inorganic nitrogen pollutants from water, characterized in that: The activated strain of claim 1 or the bacterial agent of claim 4 is added to the water environment to be treated at a v / v inoculation amount of 0.1-20%, thereby removing inorganic nitrogen pollutants in the water; wherein the cell concentration of strain WS45 in the water is ≥10 4 Pieces / mL.

8. The method for removing inorganic nitrogen pollutants from water according to claim 7, characterized in that: The dissolved oxygen concentration in the polluted water to be treated is above 5 mg / L, the C / N ratio in the water is 3-30, the pH value of the water is 6-9, the water temperature is 1-40°C, the salinity is 0.2-30 g / L in terms of NaCl concentration, and the hydraulic retention time is above 5 days; the inorganic nitrogen pollutants in the water can be removed.

9. The method for removing inorganic nitrogen pollutants from water according to claim 7, characterized in that: The activation of the strain is as follows: the strain WS45 is inoculated into the culture medium, the rotation speed is 120-220 rpm, the temperature is 15-35° C., and the activation time is 10-72 hours; wherein the culture medium components are: NaCl 0.2-30.0 g·L -1 ; Peptone 0.5-20.0 g·L -1 ; Yeast extract 0.5-10.0g·L -1 ; 1000mL of distilled water, adjust the pH to 7.0-8.0.

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