CN107686820B - Aerobic denitrifying bacteria and application thereof in water denitrification - Google Patents
Aerobic denitrifying bacteria and application thereof in water denitrification Download PDFInfo
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- CN107686820B CN107686820B CN201710812319.8A CN201710812319A CN107686820B CN 107686820 B CN107686820 B CN 107686820B CN 201710812319 A CN201710812319 A CN 201710812319A CN 107686820 B CN107686820 B CN 107686820B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- 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
- C12R2001/38—Pseudomonas
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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
- 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
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
Abstract
The invention discloses aerobic denitrifying bacteria and application thereof in water body denitrification. The aerobic denitrifying bacteria of the invention belong to Pseudomonas sp DN13-01, are preserved in Guangdong province microorganism strain preservation center, and have the preservation numbers as follows: GDMCC No.60228, with preservation address of No. 59 building 5 of No. 100 Dazhou school of Xieli Zhonglu, Guangzhou, Guangdong province, and preservation date of 2017, 9 and 4 days. The Pseudomonas (Pseudomonas sp.) DN13-01 has the advantages that when the initial concentration of nitrite nitrogen is 14mg/L, the removal rate of nitrite nitrogen after 12 hours of culture reaches 100%, nitrate nitrogen and ammonia nitrogen are not accumulated while nitrite nitrogen in a water body is removed, and secondary pollution cannot be caused, so that the Pseudomonas (Pseudomonas sp.) DN13-01 and the denitrification function thereof have good application prospects in water body denitrification.
Description
Technical Field
The invention belongs to the technical field of microorganism and environmental science, and particularly relates to a high-efficiency aerobic denitrifying bacterium, namely Pseudomonas (Pseudomonas sp) DN13-01 and application thereof in water body denitrification.
Background
With the rapid development of industry and agriculture and the aggravation of human activities, nitrogen becomes an important pollutant in various water bodies, and seriously threatens ecological safety, environmental safety and human health. Therefore, how to solve the problem of higher nitrite in the water body has important practical significance.
Aerobic denitrifying bacteria are bacteria that utilize nitrogen oxides as electron acceptors in the presence of oxygen to perform the denitrification process. Many aerobic denitrifying bacteria have unique advantages in removing nitrite, can finally reduce the nitrite into gas or assimilate the nitrite to separate from the aquaculture water body, have no residue, do not cause secondary pollution, can improve and maintain the ecological balance of aquaculture, reduce diseases, and have important function in biological nitrogen removal.
The aerobic denitrifying bacteria have diversity in physiology, biochemistry and phylogeny. Numerous studies have shown that aerobic denitrifying bacteria are mainly distributed in the genera Pseudomonas (Pseudomonas), Alcaligenes (Alcaligenes), Paracoccus (Paracoccus), Bacillus (Bacillus), and the like. Patent 201611257678.3 (application No. 2016.12.30) discloses a strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa) with a nitrate removal rate of 40-100% after culturing for 70h at a pH of above 4.5, but without nitrite nitrogen NO removal2 --N. Patent 201510124201.7 (application 2015.03.20) discloses a strain of Pseudomonas brassiceranum, which has a nitrate degradation rate of 100% after 8 days of culture, but also accumulates nitrite. Patent 201310568985.3 (application date 2013.11.15) discloses a strain of Pseudomonas aureofaciens (Pseudomonas xanthomarina) which has a 90% denitrification rate after 72h incubation. Patent 201010506931.0 (application date 2010.10.14) discloses that a pseudomonas CCTCC M2010209 has a nitrite removal rate of 17.35% after 24 hours, wherein the nitrite removal rate is 100mg/L at the initial concentration. Patent 201410229739.X (application date 2014.05.28) discloses a pseudomonas CGMCCC No.9089 can reduce nitrate, meanwhile, nitrite is not accumulated basically, and after 48 hours of culture, the total nitrogen removal rate is 98.94%. Patent 201511015597.8 (application date 2015.12.31) discloses a strain of pseudomonad (pseudomonas aeruginosa) which has 70-80% removal rate of nitrite nitrogen after culturing for 36 h.
Disclosure of Invention
The invention aims to: according to the lack of novel high-efficiency aerobic denitrifying bacteria, a high-efficiency aerobic denitrifying bacterium separated from natural boundary, namely Pseudomonas sp DN13-01, and application thereof in water body denitrification are provided.
In order to achieve the aim, the invention provides a method for performing secondary domestication on deep sea sediments
1.9095THas the highest similarity of 16S rRNA genes (99.29%), but Pseudomonas bauzanensis cannot reduce nitrite (doi:10.1099/ijs.0.026104-0, Int J Syst Evol Microbiol). And Pseudomonas (Pseudomonas sp.) DN13-01 has 96.27% of 16S rRNA gene similarity with Pseudomonas CCTCC M2010209 in patent 201010506931.0; and patent
201410229739.X Pseudomonas CGMCC No.9089 has 93.75% of 16S rRNA gene similarity; all are less than the bacteria classification threshold (97%), which indicates that the Pseudomonas (Pseudomonas sp.) DN13-01 is a novel strain.
The biological characteristics of the Pseudomonas (Pseudomonas sp.) DN13-01 are as follows: gram-negative, capable of staining in NaNO2The bacterial colony grows on a solid culture medium which is the only nitrogen source, and during culture, the bacterial colony is milky white, semitransparent and round, the surface of the bacterial colony is moist and smooth, the cell is rod-shaped, and the bacterial colony can grow by using nitrite as the only nitrogen source.
The nucleotide sequence of 16S rRNA of the Pseudomonas sp 13-01 is shown as SEQ ID NO: 1. The sequence is subjected to homology comparison analysis on NCBI and EzBioCloud websites, and the result shows that the sequence has higher similarity with the 16S rDNA sequence of Pseudomonas bauzanensis, and the similarity is 99.2%.
The invention relates to the field of Pseudomonas(Pseudomonas sp.) DN13-01 in the presence of NO2 -NO after 12h of cultivation in a Medium with-N (14mg/L) as sole Nitrogen Source2 -The removal rate of-N reaches 100%.
Compared with the prior art, the invention has the following advantages:
the invention discovers a novel high-efficiency aerobic denitrifying bacterium, namely Pseudomonas DN13-01, wherein the removal rate of nitrite nitrogen after 12 hours of culture reaches 100% when the initial concentration of nitrite nitrogen in the Pseudomonas DN13-01 is 14mg/L, and nitrate nitrogen and ammonia nitrogen are not accumulated while nitrite nitrogen in a water body is removed, so that secondary pollution cannot be generated, and therefore, the Pseudomonas DN13-01 and the denitrification function thereof have good application prospects in water body denitrification.
Preservation information: pseudomonas sp (Pseudomonas sp.) DN13-01, deposited in the microbial strain collection center of Guangdong province with the collection number: GDMCC No.60228, with preservation address of No. 59 building 5 of No. 100 institute of Tokyo, Guangzhou, Guangdong province, and preservation date of 2017, 9 months and 4 days.
Drawings
The invention and the advantageous effects are explained in detail below with reference to the drawings and the detailed description.
FIG. 1 shows the colony morphology of Pseudomonas sp DN13-01 on a solid plate.
FIG. 2 is a photograph under a light microscope (100X) of the genus Pseudomonas sp DN13-01 of the present invention.
FIG. 3 is a diagram showing the growth tendency of Pseudomonas (Pseudomonas sp.) DN13-01 and the ability of removing nitrite nitrogen in the present invention, wherein the square represents the nitrite nitrogen concentration, and the circle represents the growth tendency of the strain.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results. Unless otherwise indicated, the methods described in the examples are conventional, and the reagents used are conventional or formulated in a conventional manner.
Example 1 isolation of Pseudomonas (Pseudomonas sp.) DN13-01
1. Enrichment culture:
preparing a culture medium for enrichment, wherein the formula is as follows: sodium succinate 2.5g, sodium citrate dihydrate 2.5g, NaNO21g，K2HPO41g，KH2PO41g，MgSO4·7H20.2g of O, 1g of L-asparagine and pH 7.4, metering the volume to 1000mL, and subpackaging into 250mL triangular bottles with each bottle being 100 mL. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. 1 percent of composite carbon source, 0.2 percent of composite vitamin and 0.2 percent of composite microelement after sterile filtration are added.
Wherein, the composite carbon source: 6.9g of D-glucose, 6.9g of D-fructose, 6.9g of D-lactose, 9.5 g of sodium acetate, 6.4mL of 90% lactic acid, 7g of mannitol, 7mL of absolute ethyl alcohol, 6.3mL of glycerol, 4.8 g of sodium benzoate and 4.6g of salicylic acid, wherein the D-glucose, the mannitol, the absolute ethyl alcohol, the glycerol, the sodium benzoate and the salicylic acid are dissolved in 500mL of water, the pH value is 7.4, and the filtration sterilization is performed through a 0.22 mu m filter membrane.
Vitamin complex: biotin/Vh 10mg, folic acid/Vb 9100 mg, pyridoxine/Vb 6100 mg, thiamine/Vb 1200mg, nicotinic acid 200mg, calcium pantothenate 100mg, Vb 122 mg, riboflavin/Vb 220 mg, lipoic acid 20mg, EDTA-Na400mg, pH adjustment to 7.5, and filtration sterilization with 0.22 μm filter membrane.
Compounding trace elements: ZnSO4·7H2O 0.1g，MnCl2·4H2O 0.4g，H3BO31.24g， CoCl2·2H2O0.5g，CuCl2·2H2O 0.5g，NiSO4·6H2O 0.02g，NaMoO4·2H2O 0.4g， KI 0.2g，CaCl25g，FeSO4·7H2O1.1 g, EDTA-Na 10g, dissolved in 1000mL of H2O, pH 7.4, 0.22 μm filter.
2g of marine sediment sample is taken and put into a conical flask filled with the culture medium, and the culture is carried out for 7d at 28 ℃ and 180rpm for enrichment of denitrifying bacteria. After one week of incubation, the culture was transferred to a new flask containing the medium at 28 ℃ and 180rpm for 7 days for secondary enrichment.
2. Separating and purifying
For the secondary enrichment culture, a solid plate is coated by adopting a dilution coating method (the solid culture medium is added with 1.7 percent of agar based on the formula of the enrichment culture medium); the colony morphology was visually observed (FIG. 1) and streaked for purification, and the purified colonies were picked up in 5mL Duchen tube-containing liquid medium (enriched medium, but NaNO)2The concentration is 10mM, namely the concentration of N is 140mg/L), the aerogenesis condition is observed by incubating at 28 ℃, and the bacterial strain is found to be capable of converting nitrite into a gas product. For the purified strain, glycerol tubes and freeze-dried tubes were grown up and seeded, and the photograph thereof under an optical microscope (100 ×) is shown in FIG. 2.
3.16S rDNA identification
Extracting genome DNA of the strain, amplifying by using a bacterial 16S rRNA gene amplification universal primer 27F/1492R to obtain a PCR product, sending the PCR product to the Mergiz biomedical science and technology Co., Ltd (Guangzhou division) in the Shanghai for sequencing, and performing homology comparison analysis on a sequencing result and a16S rDNA sequence in an EzBioCloud website database, wherein the result analysis shows that the similarity of the sequencing result and the 16S rRNA gene sequence of the strain Pseudomonas bauzanensis is highest (99.2%). Based on the above results analysis, the strain of the present invention was preliminarily identified as Pseudomonas sp, taxonomically named: pseudomonas (Pseudomonas) DN 13-01.
4. Quantitative determination of denitrification performance of Pseudomonas sp (Pseudomonas sp.) DN13-01
Inoculating Pseudomonas sp DN13-01 into liquid medium (enriched medium, but in which NaNO is present)2At a concentration of 10mM, i.e. NO2 -N concentration of 14mg/L), shaking at 28 ℃ and 180rpm, and sampling after 0, 4, 8, 12, 16, 24, 32 and 40 hours of cultivation, wherein one part of the sample is used for detecting the OD 600 value, and the other part of the culture solution is centrifuged to take the supernatant, and the N concentration is quantitatively detected (Grignard reagent method). As shown in FIG. 3, Pseudomonas sp DN13-01 is used for water bodyIn NO2 -the-N has good degradation effect, and can be used for degrading NO with initial concentration of 14mg/L in water body when the bacteria grow to OD 600 of about 0.4 (about 12h)2 -Complete degradation of N; meanwhile, a diphenylamine color development method is used for detecting nitrate in the water body, a nano reagent is used for detecting ammonium root in the water body, and the result shows that the Pseudomonas (Pseudomonas sp) DN13-01 does not accumulate nitrate nitrogen and ammonia nitrogen in the process of degrading nitrite and has no secondary pollution.
Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the protection scope of the claims of the present invention. In addition, although specific terms are used herein, they are used for convenience of description only and do not limit the present invention in any way.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
Guangdong Bo Wo Tech Biotech Ltd
<120> aerobic denitrifying bacteria and application thereof in water denitrification
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1402
<212>DNA/RNA
<213> Pseudomonas sp.DN 13-01 16S rRNA16S rRNA)
<400>1
tgcagtcgag cggaagaagg gagcttgctc ccggatttag cggcggacgg gtgagtaaca 60
cctgggaatc tgcctgatag tgggggataa cgtccggaaa cgggcgctaa taccgcgtac 120
gtcctacggg agaaagcagg ggatcttcgg accttgcgct atcagatgag cccaggtcgg 180
attagcttgt tggtgaggta atggctcacc aaggcgacga tccgtaactg gtctgagagg 240
atgatcagtc acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 300
aatattggac aatgggggca accctgatcc agccatgccg cgtgtgtgaa gaaggtcttc 360
ggattgtaaa gcactttaag ttgggaggaa gggctttagg ctaataccct gaagttttga 420
cgttaccgac agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg 480
tgcaagcgtt aatcggaatt actgggcgta aagcgcgcgt aggtggttca gtaagatggg 540
tgtgaaatcc ccgggctcaa cctgggaact gcatccataa ctgctgggct agagtacggt 600
agagggtagt ggaatttcct gtgtagcggt gaaatgcgta gatataggaa ggaacaccag 660
tggcgaaggc gactacctgg actgatactg acactgaggt gcgaaagcgt ggggagcaaa 720
caggattaga taccctggta gtccacgccg taaacgatgt caactagccg ttgggaacct 780
tgagttctta gtggcgcagc taacgcacta agttgaccgc ctggggagta cggtcgcaag 840
attaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900
gaagcaacgc gaagaacctt acctggcctt gacatgcaga gaactttcca gagatggatt 960
ggtgccttcg ggaactctga cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgtaa cgagcgcaac ccttgtcctt agttaccagc acgttatggt 1080
gggcactcta aggagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca 1140
tcatggccct tacggccagg gctacacacg tgctacaatg ggggatacaa agggttgcca 1200
agccgcgagg tggagctaat cccataaagt ctctcgtagt ccggattgga gtctgcaact 1260
cgactccatg aagtcggaat cgctagtaat cgtggatcag aatgccacgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcacca gaagtagcta 1380
gtctaacctt cggaggacgg ta 1402
Claims (3)
1. An aerobic denitrifying bacterium, characterized in that it is classified and named as Pseudomonas (A)Pseudomonassp.) DN13-01, which is preserved in Guangdong province microorganism culture collection center with the preservation number as follows: GDMCC No.60228, with preservation address of No. 59 building 5 of No. 100 Dazhou school of Xieli Zhonglu, Guangzhou, Guangdong province, and preservation date of 2017, 9 and 4 days.
2. The use of the aerobic denitrifying bacteria of claim 1 in denitrification of water bodies.
3. The use of aerobic denitrifying bacteria according to claim 2, wherein said Pseudomonas (A), (B) or (C) is usedPseudomonassp.) DN13-01 is cultured in a liquid culture medium taking nitrite nitrogen as a unique nitrogen source for 12 hours, and the removal rate of the nitrite nitrogen reaches 100 percent.
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| CN108587947B (en) * | 2018-04-10 | 2020-05-08 | 暨南大学 | Phosphate solubilizing bacteria, composite microbial inoculum of phosphate solubilizing bacteria and DEHP degrading bacteria and application of phosphate solubilizing bacteria and DEHP degrading bacteria in soil improvement |
| CN112251387B (en) * | 2020-11-05 | 2022-05-10 | 广东省微生物研究所（广东省微生物分析检测中心） | Denitrifying bacteria and application thereof |
| CN112625942B (en) * | 2020-12-01 | 2022-06-14 | 华南理工大学 | Aerobic denitrifying bacterium and application thereof |
| CN113151077B (en) * | 2021-04-14 | 2023-03-21 | 广西大学 | Pseudomonas aeruginosa and screening method and application thereof |
| CN113293111B (en) * | 2021-06-07 | 2022-02-11 | 姚雨欣 | Bacillus marinus with denitrification function and application thereof |
| CN113604379B (en) * | 2021-07-06 | 2023-01-20 | 广州大学 | Pseudomonas holothurians with heterotrophic nitrification-aerobic denitrification function and application thereof |
| CN114381402B (en) * | 2022-01-20 | 2022-12-16 | 广州大学 | Acid-resistant and alkali-resistant aerobic denitrifying bacterium and microbial inoculum for rapid denitrification and application thereof |
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| CN102199561A (en) * | 2011-03-21 | 2011-09-28 | 哈尔滨工业大学 | Aerobic denitrifying bacterium treating nitrite as nitrogen source and screening method thereof |
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Address after: 510070 No. 58, courtyard, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Patentee after: GUANGDONG BOWOTE BIOTECHNOLOGY Co.,Ltd. Address before: No. 56, courtyard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong 510070 Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) Patentee before: GUANGDONG BOWOTE BIOTECHNOLOGY Co.,Ltd. |
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| AV01 | Patent right actively abandoned | ||
| AV01 | Patent right actively abandoned | ||
| AV01 | Patent right actively abandoned |
Granted publication date: 20200403 Effective date of abandoning: 20220916 |
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| AV01 | Patent right actively abandoned |
Granted publication date: 20200403 Effective date of abandoning: 20220916 |