A strain of heterotrophic nitrification-aerobic denitrification Pseudomonas flavus with wide temperature range and its application

Abstract

The present invention belongs to the field of environmental microbial technology, and specifically relates to a wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas lurida and its application. The strain is Pseudomonas lurida WS43, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 22, 2021, with a deposit number of CGMCC No.22944. Under 2-40°C conditions, the strain WS43 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 a strong tolerance to low temperatures, and has great potential in the application of 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, and 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 environmental microorganisms, and specifically relates to a wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas flavus and application thereof. Background Art

[0002] Over the past half century, with the rapid development of global industry and agriculture and the acceleration of urbanization, nitrogen pollution in various types of water bodies in ecosystems has occurred frequently, posing a continuous threat to the quality of ecosystem environment and human health. According to monitoring, the concentration of ammonia nitrogen and nitrate nitrogen in my country's surface and nearshore water bodies exceeds the standard, which is not optimistic. Effectively removing inorganic nitrogen pollution in water bodies is of great significance to improving the quality of the ecological environment.

[0003] At present, the technologies used for sewage denitrification mainly include chemical, physical and biological methods. Among them, biological denitrification technology represented by microbial denitrification has the advantages of wide application range and environmental friendliness. Microbial denitrification technology mainly uses microbial autotrophic nitrification and anaerobic denitrification metabolic pathways to achieve the conversion and removal of inorganic nitrogen pollutants (ammonia nitrogen, nitrate nitrogen, nitrite nitrogen) in sewage. Common biological denitrification processes include A 2 O, SBR, etc. The efficient denitrification microorganisms in the sludge are the key to ensure the effect of biological denitrification technology.

[0004] Most areas in northern my country have long winters, and water temperatures are often below 15°C. Low temperatures significantly inhibit the denitrification activity of microorganisms, which in turn leads to poor effluent quality at sewage treatment plants and difficulty in meeting effluent standards. The optimum temperature for 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), and the resources of nitrifying and denitrifying denitrifying microbial strains that can withstand low temperature conditions (below 20°C) are still relatively scarce.

[0005] In the past decade or so, screening of low-temperature-resistant and efficient denitrifying bacteria from natural environments has received much attention, and related research has also made certain progress. For example, CN110964663A discloses the denitrification performance and potential application of a low-temperature resistant strain Acinetobacter johnsonii BC-15 (CGMCC No. 18736) at 10-30°C; CN103773723A discloses the application of a strain of Pseudomonas stutzeri (Pseudomonas stutzeri; CGMCC No. 8291) DN-3 in removing nitrite nitrogen from water at 15-20°C; CN103275908A discloses the ability of a strain of Pseudomonas fluorescens (Pseudomonas fluorescens, CGMCC No. 7476) WSW-1001 to degrade ammonia nitrogen, nitrate nitrogen and nitrite nitrogen at 8°C; CN113308410A discloses a strain of Zebellella denitrificans LBSW21-01 (CGMCC No.22730) has a denitrification capacity at 15°C; CN107988125A discloses a cold-resistant nitrifying bacterium, Pseudomonas mohnii.M-8 (CGMCC No.14936), which has a good denitrification capacity at 2°C and under suitable nutritional conditions.

[0006] Compared with the complex environment of nitrogen-polluted water bodies and the requirements for denitrification processes, most of the existing denitrification strains can only exhibit denitrification activity in a narrow temperature range, and there is still a severe shortage of bacterial resources that can exert denitrification capabilities in a wider temperature range. Summary of the invention

[0007] In view of the current situation that the biological denitrification technology for sewage has poor effect at low temperature and the resources of low-temperature bacteria are insufficient, the present invention aims to provide a light yellow Pseudomonas lurida WS43 and its application in removing inorganic nitrogen pollution in water under wide temperature range conditions.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas lurida, the strain is Pseudomonas lurida WS43, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2021, with the deposit number CGMCC No.22944.

[0010] The invention discloses an application of wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas flavus, and the application of the strain to remove inorganic nitrogen pollutants in water under wide temperature range conditions.

[0011] The strain removes one or more of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water at 2-40°C.

[0012] The dissolved oxygen concentration in the water is 2-9 mg / L, and the nitrogen removed is one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, and the total concentration is not higher than 500 mg / L. The ammonia nitrogen concentration in the sewage is 0.1-200 mg / L, the nitrate nitrogen concentration is 0.1-500 mg / L, and the nitrite concentration is 0.01-60 mg / L.

[0013] A bacterial agent for removing inorganic nitrogen pollutants in water, the bacterial agent containing the strain.

[0014] The bacterial agent is required to activate the strain.

[0015] The bacterial agent is used to remove inorganic nitrogen pollutants in water under a wide temperature range.

[0016] 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 WS43 in the water is ≥10 3 Pieces / mL.

[0017] The dissolved oxygen concentration in the water to be treated is above 3 mg / L, the C / N ratio in the water is 3-30, the pH of the water is 6-9, the water temperature is 2-40°C, the salinity (in terms of NaCl concentration) is 0.1-30 g / L, and the hydraulic retention time is more than 3 days; the inorganic nitrogen pollutants in the water can be removed.

[0018] The activation of the strain is as follows: the strain WS43 is inoculated into a culture medium, the rotation speed is 90-250 rpm, the temperature is 20-35° C., and the activation time is 10-72 hours; wherein the culture medium components are: NaCl 0.1-20.0 g; peptone 0.1-12.0 g; yeast extract 0.2-6.0 g; distilled water 1000 mL, and the pH is adjusted to 7.0-8.0.

[0019] Beneficial effects of the present invention:

[0020] (1) The Pseudomonas lurida strain WS43 of the present invention can tolerate low temperature conditions well and can effectively remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water at 2-40°C;

[0021] (2) The strain of the present invention maintains good denitrification activity in a wide temperature range of 2-40°C, and can be used for sewage denitrification treatment in geographical areas in northern my country where the annual average temperature difference varies greatly;

[0022] (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

[0023] 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.

[0024] The pale yellow Pseudomonas strain WS43 of the present invention can effectively remove nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage under a wide temperature range of 2-40°C. The strain has strong tolerance to low temperatures and has metabolic activities of heterotrophic nitrification and aerobic denitrification, and has potential value in the application of nitrogen pollution control in water bodies in cold regions of my country.

[0025] The strain WS43 of the present invention was isolated and screened from a river sediment in Inner Mongolia, and was preliminarily identified as Pseudomonas aureus by 16S rRNA gene sequence comparison. The strain has an obvious degradation and removal effect on inorganic nitrogen pollution under a wide temperature condition.

[0026] 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:

[0027] 1) River sediments: collected from a river in Inner Mongolia Autonomous Region.

[0028] 2) Culture medium:

[0029] 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.

[0030] 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.

[0031] Trace element solution (g / L): EDTA 0.35; ZnSO4·7H2O 0.2; CuSO4·5H2O 0.1; MnSO4·7H2O0.1; Co(NO3)2·7H2O 0.09; H3BO3 0.1; Na2MoO4 0.1.

[0032] 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.

[0033] 3) Main experimental instruments:

[0034] Constant temperature biochemical incubator, constant temperature shaker, clean bench, pressure steam sterilizer, micro UV spectrophotometer, PCR instrument, electrophoresis instrument, etc.

[0035] Example 1

[0036] (I) Screening, preliminary identification and preservation of strain WS43

[0037] Take 20 g of the sludge and transfer it into a 250 mL conical flask pre-filled with 100 mL of denitrifying bacteria enrichment medium in a sterile clean bench, and then shake and culture it on a constant temperature shaker at 5 °C and 160 rpm for 5 days;

[0038] 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 5℃ for more than 5 days;

[0039] After the colonies appeared on the plate culture medium, the plate streak separation method was used to pick the colonies, and the streak separation culture process was repeated until the colonies had a single morphology. After microscopic examination and denitrification effect determination, the strain with the highest inorganic nitrogen removal rate was selected and numbered as WS43. WS43 was inoculated on a solid slant culture medium and stored at 4°C. It was used for strain identification and denitrification performance evaluation within a one-month temporary storage period.

[0040] The genomic DNA of strain WS43 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 WS43 and Pseudomonas lurida reached 99%, indicating that the strain was most closely related to Pseudomonas lurida. The 16S rRNA gene sequence of strain WS43 is:

[0041]

[0042] The strain WS43 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.22944.

[0043] Example 2: Evaluation of denitrification effect of strain WS43 at 2°C

[0044] (I) Activation of strains. Take a slant of WS43 strains and inoculate it into a 500 mL conical flask containing 150 mL of denitrifying bacteria enrichment medium on a sterile workbench, then place it in a constant temperature shaker at 30°C and 180 rpm for 48 hours of activation.

[0045] (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 an inoculation ratio of 2%, and culture it at a constant temperature of 2°C and 180rpm for 5 days. The control group was inoculated with an equal volume of WS43 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 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 1.

[0046] Table 1. Denitrification effect of strain WS43 at 2℃

[0047]

[0048] As shown in Table 1, after 5 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS43 strain in the treatment group at 2℃ were 25.4%, 33.6% and 42.1% respectively, while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.09%, 0.12% and 0.07% respectively. This result shows that WS43 has a good denitrification ability at the low temperature of 2℃.

[0049] Example 3: Evaluation of denitrification effect of strain WS43 at 5°C

[0050] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0051] (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 5°C and 180rpm for 5 days. The control group was inoculated with an equal volume of WS43 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 2.

[0052] Table 2. Denitrification effect of strain WS43 at 5℃

[0053]

[0054] As shown in Table 2, after 5 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS43 strain in the treatment group at 5℃ were 45.8%, 52.9% and 49.8% 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%, 0.15% and 0.10% respectively. The experimental results show that WS43 has a good denitrification ability at 5℃.

[0055] Example 4: Evaluation of denitrification effect of strain WS43 at 10°C

[0056] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0057] (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 10°C and 180rpm for 6 days. The control group was inoculated with an equal volume of WS43 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 3.

[0058] Table 3. Denitrification effect of strain WS43 at 10℃

[0059]

[0060] As shown in Table 3, after 6 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS43 strain in the treatment group under low temperature conditions of 10℃ were 75.1%, 82.4% and 69.1%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.13%, 0.14% and 0.05%, respectively. The experimental results show that WS43 has a good denitrification ability under low temperature conditions of 10℃.

[0061] Example 5: Evaluation of denitrification effect of strain WS43 at 20°C

[0062] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0063] (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 20°C and 180rpm for 5 days. The control group was inoculated with an equal volume of WS43 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 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.

[0064] Table 4. Denitrification effect of strain WS43 at 20℃

[0065]

[0066] As shown in Table 4, after 5 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS43 strain in the treatment group at 20°C were 91.5%, 93.8% and 89.7%, 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%, 0.24% and 0.15%, respectively. The experimental results show that WS43 has a good denitrification ability at 20°C.

[0067] Example 6: Evaluation of denitrification effect of strain WS43 at 35°C

[0068] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0069] (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 180rpm for 5 days. The control group was inoculated with an equal volume of WS43 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 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 5.

[0070] Table 5. Denitrification effect of strain WS43 at 35°C

[0071]

[0072] As shown in Table 5, after 5 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS43 strain in the treatment group at 35°C were 98.5%, 99.1% and 96.6%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.12%, 0.16% and 0.11%, respectively. The experimental results show that WS43 has excellent denitrification ability at 35°C.

Claims

1. A wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas flavus strain, characterized by: The strain is Pseudomonas lurida WS43, 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.22944.

2. An application of the wide temperature range heterotrophic nitrification-aerobic denitrification Pseudomonas flavus according to claim 1, characterized in that: The strain is used to remove inorganic nitrogen pollutants in water under a wide temperature range of 2-40°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 2-40°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 of 2-40°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 WS43 in the water is ≥10 3 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 water to be treated is above 3 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 2-40°C, the salinity is 0.1-30 g / L in terms of NaCl concentration, and the hydraulic retention time is more than 3 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 WS43 is inoculated into a culture medium, the rotation speed is 90-250 rpm, the temperature is 20-35° C., and the activation time is 10-72 hours; wherein the culture medium components are: NaCl 0.1-20.0 g; peptone 0.1-12.0 g; yeast extract 0.2-6.0 g; distilled water 1000 mL, and the pH is adjusted to 7.0-8.0.

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