A low temperature tolerant heterotrophic nitrification-aerobic denitrification phage and its application

Abstract

The present invention belongs to the technical field of biological denitrification of sewage, and specifically relates to a strain of low-temperature resistant heterotrophic nitrification-aerobic denitrification Variovorax paradoxus and its application. The strain is Variovorax paradoxus WS47, which was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on July 22, 2021, with a deposit number of CGMCC No.22942. Under the conditions of 1-40°C, the strain WS47 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 low-temperature resistant heterotrophic nitrification-aerobic denitrification bacterium and application thereof. Background Art

[0002] The excessive concentration of inorganic nitrogen (ammonia nitrogen, nitrate nitrogen, nitrite nitrogen) in various water bodies has caused damage to the ecosystem and water environment quality and even human health in many countries in the world. According to statistics, in the past 10 years, the excessive concentration of ammonia nitrogen and nitrate nitrogen in my country's surface rivers and near-shore waters has been repeatedly monitored, which deserves attention. The use of advanced technology to economically and effectively control inorganic nitrogen pollution in water bodies is crucial to improving the quality of the ecological environment.

[0003] Commonly used technologies for sewage denitrification mainly include chemical, physical and (micro)biological methods. Microbial denitrification technology is widely valued due to its advantages such as environmental friendliness and economic efficiency. Microbial denitrification technology mainly relies on the efficient metabolic activities of microorganisms in sludge such as autotrophic nitrification and anaerobic denitrification, thereby promoting the degradation and removal of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in sewage under aerobic or anaerobic conditions. At present, the more mature biological denitrification processes such as A 2 O, SBR, etc. have been widely used in sewage denitrification practices around the world. How to maintain the denitrification activity of denitrification microorganisms, especially to exert more efficient metabolic activity under complex and changeable water environment conditions, is the key to the stable operation of biological denitrification technology.

[0004] Low temperature conditions are one of the important factors that reduce the efficiency of biological denitrification. At present, most denitrifying microorganisms such as nitrifying bacteria and denitrifying bacteria are mesophilic (20-35°C) bacteria, and there is a lack of microbial strains that can tolerate low temperature conditions (below 20°C). The winter in Northeast, Northwest, and North my country is long, and the temperature of surface water bodies is often below 15°C for a long time. Under such low temperature conditions, the denitrification activity of microorganisms in sludge is often significantly inhibited, making it difficult for the effluent water quality to meet the standards. Therefore, it is of great significance and practical need to further expand the resources of low-temperature resistant denitrifying microbial strains.

[0005] In recent years, research on screening low-temperature resistant denitrification bacteria has also made some progress. However, compared with the complex and changeable environmental conditions of nitrogen-polluted water bodies (such as water temperature, salinity, inorganic nitrogen pollution concentration, etc.), the diversity and types of screened low-temperature resistant denitrification strains are still obviously insufficient and cannot meet the needs of low-temperature resistant denitrification technology in various application scenarios. 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 Variovoraxparadoxus WS47 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 low-temperature-resistant heterotrophic nitrification-aerobic denitrifying Variovoraxparadoxus, the strain is Variovoraxparadoxus WS47, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2021, with the deposit number CGMCC No.22942.

[0009] The invention discloses the application of low-temperature resistant heterotrophic nitrification-aerobic denitrification phagophore, and the application of the strain in removing 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] A bacterial agent for removing inorganic nitrogen pollutants in water, the bacterial agent containing the strain.

[0012] The bacterial agent is required to activate the strain.

[0013] The application of the bacterial agent is to remove inorganic nitrogen pollutants in water under a wide temperature range.

[0014] 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 WS47 in the water is ≥10 4 Pieces / mL.

[0015] 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.

[0016] The activation of the strain is as follows: the strain WS47 is inoculated into a culture medium, with a rotation speed of 120-220 rpm, 15-35° C., and activated 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.

[0017] The ammonia nitrogen concentration in the sewage is 0.1-300 mg / L, the nitrate nitrogen concentration is 0.1-600 mg / L, and the nitrite concentration is 0.1-100 mg / L.

[0018] Beneficial effects of the present invention:

[0019] (1) The Variovorax paradoxus strain WS47 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 required for the activation and expansion of the strain of the present invention has simple components, 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 present invention discloses a strain of Phage WS47, which has the ability to effectively remove nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in sewage under the condition of 1-40°C, has good low temperature tolerance, and has the metabolic activity of heterotrophic nitrification and aerobic denitrification, and can be used for the treatment of inorganic nitrogen-contaminated water bodies in cold regions of my country.

[0024] The strain WS47 of the present invention was isolated and screened from the activated sludge of a domestic sewage treatment plant in Inner Mongolia, and was preliminarily identified as a phage of the phage WS47 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 Inner Mongolia.

[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 0.8; NH4Cl 0.25, KNO3 0.45, NaNO2 0.25, 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 supplemented 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 WS47

[0036] Take 30g of activated sludge, 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 1℃ and 180rpm for 20 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 1℃ for more than 20 days;

[0038] 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 WS47. WS47 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.

[0039] The genomic DNA of strain WS47 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 WS47 and Variovorax paradoxus (DSM30034) reached 99%, indicating that the strain was most closely related to Variovorax paradoxus. The 16S rRNA gene sequence of strain WS47 is:

[0040] WS47

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

[0042] AACATGCAAGTCGAACGGCAGCGCGGGAGCAATCCTGGCGGCGAGTGGCGAACGGGTGAG 60

[0043] TAATACATCGGAACGTGCCCAATCGTGGGGGATAACGCAGCGAAAGCTGTGCTAATACCG 120

[0044] CATACGATCTACGGATGAAAGCAGGGGATCGCAAGACCTTGCGCGAATGGAGCGGCCGAT 180

[0045] GGCAGATTAGGTAGTTGGTGAGGTAAAGGCTCACCAAGCCTTCGATCTGTAGCTGGTCTG 240

[0046] AGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG 300

[0047] TGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGCAGGATGAAGG 360

[0048] CCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAACGGCCTTTTCTAATAAAGAGGGCT 420

[0049] AATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG 480

[0050] TAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTAATGTAAG 540

[0051] ACAGTTGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGTGACTGCATTGCTGGAGT 600

[0052] ACGGCAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAAC 660

[0053] ACCGATGGCGAAGGCAATCCCCTGGGCCTGTACTGACGCTCATGCACGAAAGCGTGGGGA 720

[0054] GCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGG 780

[0055] AATTCACTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCG 840

[0056] CAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTA 900

[0057] ATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGTACGGAATTCGCCAGAGATG 960

[0058] GCTTAGTGCTCGAAAGAGAACCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC 1020

[0059] GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTCA 1080

[0060] GTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAG 1140

[0061] TCCTCATGGCCCTTATAGGTGGGGCTACACGTCATACAATGGCTGGTACAAAGGGTTG 1200

[0062] CCAACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCA 1260

[0063] ACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGTCACGGTGAATAC 1320

[0064] GTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTCTGCCAGAAGTAG 1380

[0065] TTAGCTTAACCGCAAGGAGGGC

[0066] The strain WS47 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.22942.

[0067] Example 2: Evaluation of denitrification effect of strain WS47 at 1°C

[0068] (I) Activation of strains: Take a slant of WS47 strains 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 20°C and 180 rpm for activation for 72 hours.

[0069] (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 10 days. The control group was inoculated with an equal volume of WS47 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 10-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.

[0070] Table 1. Denitrification effect of strain WS47 at 1°C

[0071]

[0072] As shown in Table 1, after 10 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS47 strain in the treatment group under the low temperature condition of 1℃ were 40.4%, 38.2% and 25.1% respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.06%, 0.08% and 0.02% respectively. The results of this experiment show that the strain WS47 has a good denitrification ability under the low temperature condition of 1℃.

[0073] Example 3: Evaluation of denitrification effect of strain WS47 at 5°C

[0074] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0075] (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 10 days. The control group was inoculated with an equal volume of WS47 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 10-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.

[0076] Table 2. Denitrification effect of strain WS47 at 5℃

[0077]

[0078] As shown in Table 2, after 6 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS47 in the treatment group at 5℃ were 56.7%, 62.5% and 48.4% respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.07%, 0.12% and 0.10% respectively. The experimental results show that WS47 has a good denitrification ability at 5℃.

[0079] Example 4: Evaluation of denitrification effect of strain WS47 at 10°C

[0080] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0081] (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 10°C and 180rpm for 10 days. The control group was inoculated with an equal volume of WS47 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 10-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.

[0082] Table 3. Denitrification effect of strain WS47 at 10℃

[0083]

[0084] As shown in Table 3, after 10 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS47 strain in the treatment group under low temperature conditions of 10℃ were 82.3%, 85.6% and 72.8% respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.14%, 0.05% and 0.13% respectively. The experimental results show that WS47 has a good denitrification ability under low temperature conditions of 10℃.

[0085] Example 5: Evaluation of denitrification effect of strain WS47 at 20°C

[0086] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0087] (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 180rpm for 3 days. The control group was inoculated with an equal volume of WS47 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 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 4.

[0088] Table 4. Denitrification effect of strain WS47 at 20℃

[0089]

[0090] As shown in Table 4, after 3 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS47 strain in the treatment group at 20°C were 93.8%, 95.4% and 91.0%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.21%, 0.03% and 0.11%, respectively. The experimental results show that WS47 has a good denitrification ability at 20°C.

[0091] Example 6: Evaluation of denitrification effect of strain WS47 at 35°C

[0092] (I) Activation of bacterial strains. See Example 2 for activation of bacterial strains.

[0093] (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 2 days. The control group was inoculated with an equal volume of WS47 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 2-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.

[0094] Table 5. Denitrification effect of strain WS47 at 35°C

[0095]

[0096] As shown in Table 5, after 2 days, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by WS47 strain in the treatment group at 35°C were 99.5%, 99.7% and 97.6%, respectively; while there was almost no denitrification effect in the control group, and the removal rates of the three inorganic nitrogen were only 0.34%, 0.15% and 0.09%, respectively. The experimental results show that WS47 has excellent denitrification ability at 35°C.

Claims

1. A low-temperature resistant heterotrophic nitrification-aerobic denitrification bacterium, characterized by: The strain is a phage Variovorax paradoxus ) WS47, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2021, with the deposit number CGMCC No. 22942.

2. The use of the low-temperature resistant heterotrophic nitrification-aerobic denitrification bacterium 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 of the low-temperature resistant heterotrophic nitrification-aerobic denitrification bacterium 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: Application of the bacterial agent to remove inorganic nitrogen pollutants in water under wide temperature range conditions; 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 rate of 0.1-20%, thereby removing inorganic nitrogen pollutants in the water; wherein the cell concentration of strain WS47 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 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 more than 5 days; the removal of inorganic nitrogen pollutants in the water can be achieved.

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 WS47 is inoculated into a culture medium, and the culture medium is activated for 10-72 h at a speed of 120-220 rpm and a temperature of 15-35° C.; wherein the culture medium comprises: NaCl 0.2-30.0 g·L -1 ; Peptone 0.5-20.0 g·L -1 ; Yeast extract 0.5-10.0 g·L -1 ; 1000 mL of distilled water, adjust the pH to 7.0-8.0.

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