CN111454857B - Nitrifying bacteria and application thereof - Google Patents
Nitrifying bacteria and application thereof Download PDFInfo
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- CN111454857B CN111454857B CN202010016397.9A CN202010016397A CN111454857B CN 111454857 B CN111454857 B CN 111454857B CN 202010016397 A CN202010016397 A CN 202010016397A CN 111454857 B CN111454857 B CN 111454857B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
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- 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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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Abstract
The invention particularly relates to a high-efficiency nitrifying bacterium Bacillus toyonensis TNH01 and application thereof. Wherein the cell preservation: the efficient nitrifying bacterium Bacillus toyonensis TNH01 is obtained by self screening of an inventor, and the preservation number is as follows: CCTCC NO:2019737, preservation date: and in 2019, 9, 23 and the preservation unit is CCTCC. The application of the high-efficiency nitrifying bacteria is the application in removing ammonia nitrogen from the culture wastewater. The bacterium can effectively remove ammoniacal nitrogen in the aquaculture water body and obviously improve the water quality.
Description
Technical Field
The invention relates to the technical field of microorganism and environmental science, in particular to a nitrifying bacterium Bacillus sp.TNH01 and application thereof.
Background
The eutrophication of water body is a pollution phenomenon caused by the change of the ecological balance of the whole water body due to the excessive content of plant nutrient substances such as nitrogen and phosphorus in water bodies such as lakes, rivers, reservoirs and the like, and becomes one of the leading causes of water environmental pollution and water body ecological function degradation in China. The root cause of water eutrophication is the increase of nutrients, mainly the input of nitrogen, phosphorus and other nutrient elements, wherein nitrogen is the most serious pollutant. The intensive production also brings new problems while improving the economic benefit, such as a series of environmental problems of the drastic increase of the discharge amount of the culture sewage and the like.
At present, the method for purifying the culture water quality at home and abroad generally adopts a physical method, a chemical method or a biological method, but the methods have a plurality of disadvantages. For example, physical methods are time-consuming and labor-consuming, and have high cost; the chemical water quality modifier can cause secondary pollution to the environment, and some chemical water quality modifiers can also cause harm to human bodies, so that the chemical water quality modifier is forbidden to be used at present. In recent years, microorganisms have been widely used due to their advantages of rapid growth and propagation, large specific surface area, rapid absorption and transformation, strong adaptability, many varieties, wide distribution, etc. Compared with the traditional physical and chemical methods, the microecological technology has the advantages of low cost, high yield, no secondary pollution and the like, and is a current research hotspot. The traditional biological removal of nitrogen in water treatment is mainly completed through two processes of nitrification and denitrification, namely, ammonia nitrogen is converted into nitrate nitrogen by autotrophic nitrifying bacteria under aerobic conditions, and then the nitrate nitrogen is reduced into nitrogen by heterotrophic denitrifying bacteria under anoxic conditions and discharged out of a water body. However, the biotransformation of ammonia nitrogen in aquaculture water still needs to be studied deeply, and the key point is to screen strains which have high-efficiency degradation effect, high temperature resistance, wide salt tolerance range, easy culture and short growth period.
Disclosure of Invention
The first purpose of the invention is to provide a nitrifying bacterium Bacillus sp.
The second purpose of the invention is to provide an application of nitrifying bacteria Bacillus sp.TNH01 in removing ammonia nitrogen from aquaculture wastewater.
Wherein the cell preservation: the nitrifying bacterium Bacillus sp.TNH01 is obtained by self-screening of an inventor, and the preservation number is as follows: CCTCC NO, M2019737, preservation date: 23.9.2019, the preservation unit is CCTCC, and the address is located in the Wuhan university school of eight paths 299 # in the Wuhan city Wuchang area of Hubei province.
The nitrifying bacterium Bacillus sp.TNH01 strain disclosed by the invention has a nearly circular colony, a faint yellow colony, irregular edges, a wet and rough surface, a slight protrusion and opaqueness on a nutrient agar plate, and the diameter of the colony is 0.5-3.5 mm. The strain is uniformly turbid in an LB liquid culture medium, and the number of the strains can be as high as 108cfu/g or more. Under aerobic conditions, 100mg/L ammonia can be removed by the strain TNH01 within 24 h.
The invention has the advantages that: 1. the culture is convenient, and the growth speed is high; 2. the number of the viable bacteria strains after the fermentation is cultured is high; 3. the bacterium can effectively remove ammoniacal nitrogen in the aquaculture water body and obviously improve the water quality.
Detailed Description
Example one, acquisition of TNH 01:
1. and (3) separating and purifying bacteria:
taking 10g of bamboo forest soil, adding 100ml of simulated sewage: 0.27g NH4CL, 2.7g sodium succinate, 5ml Vickers' salt solution (K2HP045g/L, MgS042.5g/L, NaC12.5g/L, FeSO4.7H200.05g/L, MnSO4.4H200.05g/L, the rest is deionized water). Sampling every 24h, carrying out sample application analysis, and qualitatively detecting the degradation condition of ammonia nitrogen. When the ammonia nitrogen is completely degraded, taking trace amount for separation and purification.
Qualitative detection of ammonia nitrogen: and (3) spotting with a Na reagent, wherein if a yellow reaction occurs, the culture solution contains ammonia nitrogen, and the darker the color is, the higher the concentration is.
2. And (3) separating and purifying bacteria:
diluted and spread on the surface of LB solid culture medium (peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, agar 18g/L, water for the rest; pH7.0, sterilization at 121 ℃ for 20 minutes) for continuous streak culture, colonies with different forms are selected, and after repeated several times, single isolated colonies can be formed, thus obtaining pure culture strains.
3. Bacterial strain rescreening
The primary screened strains were plated on nitrifying Medium (2.7g NH)4CL, 27g of sodium succinate, 50ml of Vickers salt solution and 1000ml of deionized water) to obtain a strain with better denitrification effect, which is named as TN 01.
4. Preservation with glycerol
In the sterilized seed tube, l.0ml of the bacterial culture solution and 0.5ml of 50% (volume concentration) glycerol were added, and after mixing, they were stored in a refrigerator at-80 ℃.
5. Strain identification
(1) Morphological characteristics
The suspension was uniformly turbid in the liquid medium with a slight amount of precipitate, and the precipitate was dispersed by gentle shaking. The diameter of a colony SC221-M on the nutrient agar plate is 2.5-3.0mm, and the colony is round, beige, smooth and opaque in surface, glossy, slightly protruded in middle, neat in edge and free of halo.
(2)16S rDNA sequence analysis
The isolated and screened strain SC228 was subjected to further 16S rDNA sequence analysis and alignment. The primer sequences are as follows: upstream primer (F27): 5'-AGAGTTTGATCCTGGCTCAG-3', upstream primer (R1492): 5'-TACGGCTACCTTGTTACGACTT-3' are provided. And (3) carrying out PCR amplification by taking the total DNA of the strains obtained by screening as a template, wherein a PCR reaction system (25) is as follows: 13 μ L of master mix enzyme, 1.0 μ L of each of the upstream and downstream primers, 1.0 μ L of template, ddH2O9 mu L; the reaction conditions are PCR reaction conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 58 deg.C for 30sec, extension at 72 deg.C for 1min, 40 cycles, and final preservation at 72 deg.C for 10min and 4 deg.C. The objective fragment was cloned and sequenced according to the conventional method (Sambrook, et al, 2001), and the result of the sequencing was compared with the 16S rDNA sequence (accession No.: OKO52292.1) registered in GenBank, whereby the gene homology reached 100%. According to Bergey' S Manual of definitive Bacteriology (Holt, J.G., Gibbons, N.E.,1994) and the Manual of identification of common bacterial systems (Dongxu bead and Zeitmiaying et al, 2001), strain TNH01 was determined to be Bacillus sp by the analysis of the behavioral characteristics and the 16S rDNA sequence.
The strain TNH01 is preserved under the preservation name: bacillus sp.tnh01, depository: china center for type culture Collection, collection address: wuhan university in Wuhan, China; the preservation date is as follows: 23/9/2019, accession number: CCTCC NO: 2019737.
Example two:
the purification effect of Bacillus sp.TNH01 on simulated culture wastewater is as follows:
50ml of simulated wastewater is respectively filled into 100ml conical flasks, wherein C/N is 10, and ammonia nitrogen is 100 mg/L. To the simulated wastewater according to the ratio of 1 multiplied by 108cfu/m3TNH01 was added to the final concentration of (4), and the control group was three replicates each, incubated at 28 ℃ and 150rpm for 24h without addition of microbial inoculum. Method for measuring ammonia Nitrogen (NH) by using Nassler reagent photometry4 +-N). After 24h, it was detected: experimental group, ammonia Nitrogen (NH)4 +-N) at a concentration of 0.28 mg/L; control group: ammonia Nitrogen (NH)4 +-N) was 99.38 mg/L.
Therefore, the TNH01 has the effects of obviously reducing ammonia nitrogen in water and improving water quality, and is suitable for being used as a water quality purifying agent in the treatment of aquaculture wastewater.
Comparative example 1, the currently available Bacillus subtilis (specifically shown in Table 1) was substituted for Bacillus subtilis SC228 of the present invention at a concentration of 1X 108cfu/m3The detection time is still 24 hours; the rest is equivalent to example 2. The results obtained are shown in Table 1 in comparison with the present invention.
TABLE 1
| NH4 +-N | |
| Blank example | 99.38mg/L |
| TNH01 of the invention | 0.28mg/L |
| Another strain TNH11 obtained in the invention process | 70.52mg/L |
| Another strain TNH18 obtained in the invention process | 65.15mg./L |
| Another strain TNH13 obtained in the invention process | 82.31mg/L |
| GDMCC 60528 | 19.25mg/L |
The foregoing list is only illustrative of several embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. A nitrifying bacterium, characterized by: the preservation name is: bacillus toyonensis TNH01 with the following deposition number: CCTCC NO: 2019737.
2. Use of the nitrifying bacteria of claim 1 wherein: is applied to removing ammonia nitrogen from aquaculture wastewater.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107794239A (en) * | 2017-11-15 | 2018-03-13 | 云南省烟草农业科学研究院 | A kind of Japan's Bacillus strain and bacterial preparation process and application |
| CN109825450A (en) * | 2019-01-17 | 2019-05-31 | 广东博沃特生物科技有限公司 | One plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its application |
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| CN104017757B (en) * | 2014-06-03 | 2016-05-18 | 江南大学 | The one plant height effect end, changed bacillus and the composite base made changes microbial inoculum and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107794239A (en) * | 2017-11-15 | 2018-03-13 | 云南省烟草农业科学研究院 | A kind of Japan's Bacillus strain and bacterial preparation process and application |
| CN109825450A (en) * | 2019-01-17 | 2019-05-31 | 广东博沃特生物科技有限公司 | One plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its application |
Non-Patent Citations (2)
| Title |
|---|
| 一株异养硝化细菌的分离鉴定及其脱氮效果;尹丽等;《环境科学与技术》;20141215(第12期);摘要 * |
| 一株异养硝化细菌的筛选鉴定及硝化性能研究;陈佗;《西部皮革》;20190425(第08期);摘要 * |
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Inventor after: Zhang Xiaoping Inventor after: Huang Zhiyuan Inventor after: Bian Fangyuan Inventor after: Li Qiaoling Inventor after: Zhong Hao Inventor after: Xiu Chengming Inventor after: Zhong Zheke Inventor before: Zhang Xiaoping |
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