CN114774320B - Denitrifying microorganism and application thereof - Google Patents
Denitrifying microorganism and application thereof Download PDFInfo
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- CN114774320B CN114774320B CN202210454924.3A CN202210454924A CN114774320B CN 114774320 B CN114774320 B CN 114774320B CN 202210454924 A CN202210454924 A CN 202210454924A CN 114774320 B CN114774320 B CN 114774320B
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- pseudomonas
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- removal rate
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- 244000005700 microbiome Species 0.000 title claims abstract description 21
- 241000589516 Pseudomonas Species 0.000 claims abstract description 37
- 239000010865 sewage Substances 0.000 claims abstract description 32
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 6
- 239000002351 wastewater Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 33
- 241000894006 Bacteria Species 0.000 abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 38
- 239000001963 growth medium Substances 0.000 description 18
- 239000002609 medium Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000012266 salt solution Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003911 water pollution Methods 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The invention discloses a denitrifying microorganism and application thereof, wherein the denitrifying microorganism is Pseudomonas sp (Pseudomonas sp.) Y-5 with a preservation number of CCTCC No: m2022392, the preservation time is 2022, 04 and 07. The invention provides the Pseudomonas Y-5, which is inoculated to NO-containing bacteria 3 In the sewage of-N, the NO in the sewage can be efficiently removed 3 -N and TN; inoculating Pseudomonas Y-5 into NO-containing strain 2 In the sewage of-N, the NO in the sewage can be efficiently removed 2 -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 3 In the sewage of-N, NH in the sewage can be efficiently removed 4 + ‑N、NO 3 -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 2 In the sewage of-N, NH in the sewage can be efficiently removed 4 + ‑N、NO 2 -N and TN; when the pseudomonas Y-5 is used for denitrifying the nitrogen-containing sewage, the nitrification and the denitrification are synchronously carried out, so that the treatment cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a denitrification microorganism and application thereof.
Background
Water pollution is one of reasons for water resource shortage, and causes of water pollution include nitrogen pollution, and treatment of nitrogen-polluted water is a necessary measure for promoting sustainable development of environment. Therefore, how to efficiently remove nitrogen in the nitrogen-containing wastewater is a problem to be solved.
Disclosure of Invention
The invention mainly aims to provide a denitrifying microorganism and application thereof, and aims to provide a denitrifying microorganism for efficiently removing nitrogen in nitrogenous wastewater.
In order to achieve the above object, the present invention provides a denitrifying microorganism, which is Pseudomonas sp Y-5 with a preservation number of CCTCC No: m2022392, the preservation time is 2022, 04 and 07.
The invention also provides an application of the denitrifying microorganism in treating nitrogen-containing wastewater.
The invention provides the Pseudomonas Y-5, which is inoculated to NO-containing bacteria 3 - In the sewage of-N, the NO in the sewage can be efficiently removed 3 - -N and TN; inoculating Pseudomonas Y-5 into NO-containing strain 2 - In the sewage of-N, the NO in the sewage can be efficiently removed 2 - -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 3 - In the sewage of-N, NH in the sewage can be efficiently removed 4 + -N、NO 3 - -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 2 - In the sewage of-N, NH in the sewage can be efficiently removed 4 + -N、NO 2 - -N and TN; when the pseudomonas Y-5 is used for denitrifying the nitrogen-containing sewage, the nitrification and the denitrification are synchronously carried out, so that the treatment cost is greatly reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a front view of colonies of denitrifying microorganisms provided by the present invention;
FIG. 2 is a reverse side view of colonies of denitrifying microorganisms provided by the present invention;
FIG. 3 is a scanning electron microscope image of the denitrified microorganism provided by the invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
The specific conditions were not specified in the examples, and the examples were conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Water pollution is one of reasons for water resource shortage, and causes of water pollution include nitrogen pollution, and treatment of nitrogen-polluted water is a necessary measure for promoting sustainable development of environment. Therefore, how to efficiently remove nitrogen in the nitrogen-containing wastewater is a problem to be solved.
In view of this, the present invention proposes a denitrifying microorganism which is Pseudomonas sp Y-5, which has been deposited at China center for type culture Collection, accession number: the preservation number of the Chinese university of Wuhan is CCTCC No: m2022392.
The 16S rRNA gene sequence of the denitrifying microorganism was identified as follows:
extracting genome DNA of denitrifying microorganism, and the extraction steps are carried out according to the description of DNA extraction kit. The V3-V4 hypervariable region of the strain 16S rRNA gene was amplified by PCR reaction, and the general primers F341 (5 '-CTACGGGMSGCAGCAG-3') and R518 (5'-ATTACCGCGGCTGC TGG-3') were selected, and the PCR amplification system was as shown in Table 1. Then, sequencing was performed, and the sequencing work was performed by Shanghai, inc. (Wuhan sequencing division, china). The sequence was registered in genbank to obtain the sequence No. MZ514091, which was identified as Pseudomonas, and thus the denitrifying microorganism was named Pseudomonas sp.Y-5.
TABLE 1 PCR amplification System
The invention provides the Pseudomonas Y-5, which is inoculated to NO-containing bacteria 3 - In the sewage of-N, the NO in the sewage can be efficiently removed 3 - -N and TN; inoculating Pseudomonas Y-5 into NO-containing strain 2 - In the sewage of-N, the NO in the sewage can be efficiently removed 2 - -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 3 - In the sewage of-N, NH in the sewage can be efficiently removed 4 + -N、NO 3 - -N and TN; inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 2 - In the sewage of-N, NH in the sewage can be efficiently removed 4 + -N、NO 2 - -N and TN; when the pseudomonas Y-5 is used for denitrifying the nitrogen-containing sewage, the nitrification and the denitrification are synchronously carried out, so that the nitrogen-containing sewage is greatly treatedThe processing cost is reduced.
Specifically, pseudomonas Y-5 was inoculated to NO-containing bacteria 3 - The average denitrification rate in the sewage of-N is 4.39mg/L/h for 24h, NO 3 - The maximum removal rate of N is 8.80mg/L/h, NO 3 - The removal of N was 99.34% and of TN was 88.95%, NH was not produced 4 + -N。
Inoculating Pseudomonas Y-5 into NO-containing strain 2 - 24h, NO in the N-contaminated water 2 - The average removal rate of N was 4.23mg/L/h, NO 2 - The removal of N was 95.81% and TN was 94.44% with no accumulation of ammonia and nitrate.
Inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 3 - NH at 72h,24h in N-contaminated water 4 + The removal rate of the-N is 97.80 percent, and NH is carried out within 24 hours 4 + -an average removal rate of N of 4.35mg/L/h; NO at 48h 3 - The removal rate of the-N is 100 percent, NO is removed within 48 hours 3 - -an average removal rate of N of 2.32mg/L/h; NO at 54h 2 - -N is completely removed; the TN removal rate at 60h was 93.01%, and the TN removal rate at 60h was 3.54mg/L/h.
Inoculating Pseudomonas Y-5 into NH-containing strain 4 + -N and NO 2 - NH in 72h,24h in-N sewage 4 + An average removal rate of 4.46mg/L/h for NH at 60h 4 + -N removal rate of 96.29%; NO at 60h 2 - The N is completely removed, NO is contained in 60 hours 2 - The average removal rate of N is 2.14mg/L/h, NO NO 3 - -accumulation of N; the TN removal rate at 60h was 94.26%, and the TN average removal rate within 60h was 3.77mg/L/h.
The invention also provides an application of the denitrifying microorganism in treating nitrogen-containing wastewater.
The pseudomonas Y-5 has high-efficiency denitrification capability, and can remarkably improve the wastewater treatment efficiency when being applied to the treatment of the nitrogenous wastewater to remove the nitrogen in the wastewater.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
EXAMPLE 1 screening separation of Pseudomonas Y-5
(1) Preparing a culture medium:
1# medium (per liter): 0.5g (NH) 4 ) 2 SO 4 、5g CH 3 COONa, 50mL of Vickers salt solution, and adjusting the pH value of the culture medium to 7.0.
1# solid plate: 1.2% (m/v) agar powder was added to the 1# medium.
Both the culture medium # 1 and the solid plate # 1 were sterilized in a steam autoclave at 115℃for 30min before use.
(2) Screening and separating strains: sampling from activated sludge near a sewage treatment plant of Wuhan in China, inoculating 1g of fresh sludge sample into a 250mL conical flask filled with 100mL of 1# culture medium, and culturing in a constant temperature shaking table (30 ℃ C., 150 r/min) for 4 days. The homogenized culture was then re-transferred to fresh 100mL of 1# medium at 3% (v/v) inoculation ratio every 4 days, and continued until 1# medium NH was obtained 4 + The removal rate of N reaches more than 90%, and then the culture is diluted (diluted to 10 -5 、10 -6 、10 -7 ) And spread on a plate containing No. 1 solid, and cultured in a constant temperature incubator at 30 ℃. After three days, single colonies were formed, and single colonies were picked from the solid plates into test tubes containing 5mL of 1# medium and cultured in a constant temperature shaker at 30℃at 150r/min for 24 hours. Selection of NH from these tubes 4 + The strain with high removal efficiency of N and TN is Pseudomonas Y-5.
Detecting the nitrogen removal capacity of Pseudomonas Y-5 and NH within 24 hours 4 + The N removal rate is 97.26 percent, and the average removal rate reaches 4.30mg/L/h; TN removal rate is up to 99.02%, and nitrite and nitrate accumulation does not occur in the culture medium.
EXAMPLE 2 characterization of the morphology of the Pseudomonas Y-5 Strain
The colony of Pseudomonas Y-5 was observed, and as shown in FIGS. 1 and 2, the colony was convex, circular, opaque, regular in edge and smooth in surface.
The Pseudomonas Y-5 is observed by a microscope, and as shown in FIG. 3, the Pseudomonas Y-5 is rod-shaped and has a length of 1-1.2 μm.
Example 3 KNO 3 Nitrogen removal capability detection under nitrogen source conditions
(1) Preparing a culture medium:
2# medium (per liter): 0.762g KNO 3 、5g CH 3 COONa, 50mL of Vickers salt solution, and adjusting the pH value of the culture medium to 7.0.
The No. 2 culture medium is sterilized in a steam autoclave at 115 ℃ for 30min before use.
(2) A fresh culture of Pseudomonas Y-5 was inoculated in 3% (v/v) to 2# medium and cultured in a constant temperature shaker at 30℃and 150r/min for 24 hours.
Detecting the nitrogen removal capacity of Pseudomonas Y-5, wherein the average denitrification rate within 24 hours is 4.39mg/L/h, NO 3 - The maximum removal rate of N was 8.80mg/L/h. TN in the culture medium can be effectively removed, and NO is generated after Y-5 inoculation for 24 hours 3 - The removal of N was 99.34%, TN was 88.95% and NH was not detected 4 + -N。
EXAMPLE 4 NaNO 2 Nitrogen removal capability detection under nitrogen source conditions
(1) Preparing a culture medium:
3# medium (per liter): 0.52g NaNO 2 、5g CH 3 COONa, 50mL of Vickers salt solution, and adjusting the pH value of the culture medium to 7.0.
The culture medium 3# is sterilized in a steam autoclave at 115℃for 30min before use.
(2) Fresh Pseudomonas Y-5 cultures were inoculated in 3% (v/v) to 3# medium and incubated at 30℃in a 150r/min constant temperature shaker for 24h.
Detecting the nitrogen removal capacity of Pseudomonas Y-5 and NO at 24 hours 2 - The removal rate of-N is 95.81%, NO 2 - The average removal rate of N was 4.23mg/L/h. Y-5 TN removal 24h after inoculationThe rate was 94.44%, while removing NO 2 - Neither ammonia nor nitrate accumulated during N.
Example 5 (NH) 4 ) 2 SO 4 And KNO 3 Nitrogen removal capability detection under nitrogen source conditions
(1) Preparing a culture medium:
medium # 4 (per liter): 10g CH 3 COONa, 50mL Vickers salt solution, 0.5g (NH) 4 ) 2 SO 4 、0.762g KNO 3 The pH of the medium was adjusted to 7.0.
The culture medium No. 4 is sterilized in a steam autoclave at 115℃for 30min before use.
(2) Fresh Pseudomonas Y-5 cultures were inoculated in 3% (v/v) to medium # 4 and incubated at 30℃in a 150r/min constant temperature shaker for 72h.
Detecting the nitrogen removal capacity of Pseudomonas Y-5 and NH within 24 hours 4 + -N removal of 97.80%, NH 4 + -an average removal rate of N of 4.35mg/L/h; NO at 48h 3 - -N removal rate 100% with an average removal rate of 2.32mg/L/h; NO when cultured for 54h 2 - N can be completely removed; after 60 hours the TN removal rate was 93.01% and the average removal rate was 3.54mg/L/h.
Example 6 (NH) 4 ) 2 SO 4 And NaNO 2 Nitrogen removal capability detection under nitrogen source conditions
(1) Preparing a culture medium:
culture medium 5# (per liter): 10g CH 3 COONa, 50mL Vickers salt solution, 0.5g (NH) 4 ) 2 SO 4 、0.52g NaNO 2 The pH of the medium was adjusted to 7.0.
The No. 5 culture medium is sterilized in a steam autoclave at 115 ℃ for 30min before use.
(2) A fresh culture of Pseudomonas Y-5 was inoculated in 3% (v/v) to 5# medium and cultured in a constant temperature shaker at 30℃and 150r/min for 72 hours.
Detecting the nitrogen removal ability of Pseudomonas Y-5, NH in the culture Medium 4 + -NAverage removal rate within 24h was 4.46mg/L/h, NH after 60h 4 + -N removal up to 96.29%; NO (NO) 2 - N can be completely removed at 60h, NO 2 - The average removal rate of N was 2.14mg/L/h, NO NO 3 - -accumulation of N; the TN removal effect is obvious, the TN removal rate is 94.26% at 60h, and the average removal rate reaches 3.77mg/L/h.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (2)
1. A denitrifying microorganism, characterized in that the denitrifying microorganism is Pseudomonas sp, Y-5, with a preservation number of CCTCC No: m2022392, the preservation time is 2022, 04 and 07;
when the pseudomonas Y-5 is used for denitrifying the nitrogen-containing sewage, the nitrification and the denitrification are synchronously carried out.
2. Use of the denitrifying microorganism according to claim 1 for treating nitrogen-containing wastewater.
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|---|---|---|---|---|
| JP2005034783A (en) * | 2003-07-17 | 2005-02-10 | New Industry Research Organization | Nitrification/denitrification method for removing nh4+ and no3- simultaneously |
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| CN111909867A (en) * | 2020-07-21 | 2020-11-10 | 广东石油化工学院 | Heterotrophic nitrification-aerobic denitrification bacterium and culture method and application thereof |
| CN111961609A (en) * | 2020-07-21 | 2020-11-20 | 广东石油化工学院 | Heterotrophic nitrification-aerobic denitrification bacterium agent and application thereof |
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- 2022-04-27 CN CN202210454924.3A patent/CN114774320B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005034783A (en) * | 2003-07-17 | 2005-02-10 | New Industry Research Organization | Nitrification/denitrification method for removing nh4+ and no3- simultaneously |
| CN102899263A (en) * | 2012-06-18 | 2013-01-30 | 山东大学 | Facultative anaerobic denitrifying bacteria having complete denitrification enzyme systems and use thereof |
| CN111909867A (en) * | 2020-07-21 | 2020-11-10 | 广东石油化工学院 | Heterotrophic nitrification-aerobic denitrification bacterium and culture method and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| Li C等.Removal of nitrogen by heterotrophic nitrification-aerobic denitrification of a phosphate accumulating bacterium Pseudomonas stutzeri YG-24.Bioresour Technol.2015,第182卷18-25. * |
| 张笑迎等.一株异养硝化细菌的分离鉴定及硝化性能分析.武汉轻工大学学报.2022,第41卷(第1期),36-42+76. * |
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