CN110540946A - Denitrifying bacterium with complete denitrification capability and rapid denitrification capability and application thereof - Google Patents
Denitrifying bacterium with complete denitrification capability and rapid denitrification capability and application thereof Download PDFInfo
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Classifications
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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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- 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
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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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- 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
- C02F2101/163—Nitrates
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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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Abstract
The invention discloses a denitrifying bacterium Pseudomonas songnensis L103 with complete denitrification capability and rapid denitrification capability and application thereof, wherein the strain is preserved in China center for type culture Collection 3/20 in 2019 with the preservation number of CCTCC M2019184. The strain has genes for coding nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, the genes can code a complete denitrification enzyme system, catalyze NO 3-to be completely reduced to generate N2, and the denitrification rate can be as high as 1.62-2.36 g NO 3-/day/L. Therefore, the strain has important application value in the aspects of preparing the denitrification microbial agent for reducing nitrate leaching loss and underground water nitrate pollution in soil below a root layer, purifying nitrogen-containing sewage and the like.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a denitrifying bacterium with complete denitrification capability and rapid denitrification capability and application thereof.
Background
In agricultural production, nitrogen fertilizer application rates far exceed crop nitrogen requirements, leading to soil nitrate accumulation. The accumulated nitrate can be transported with water after encountering strong rainfall, so that the nitrate pollution of underground water is caused, and the human health is seriously threatened. In addition, excessive nitrogen enters lakes and wetlands, which causes a series of environmental problems such as water eutrophication and water quality deterioration.
The denitrification is a process (see the process represented by the formula below) in which microorganisms gradually reduce NO 3-and NO 2-to gaseous nitrogen oxides NO, N2O and N2 by using nitrate reductase (NAR), nitrite reductase (NIR), Nitric Oxide Reductase (NOR), nitrous oxide reductase (N2OR) and the like, and plays an important ecological role in nitrogen circulation. Based on the fact whether the method has the two aspects of complete denitrification capability and rapid denitrification capability, the efficient denitrification microorganisms are separated and screened, and powerful support is provided for reducing nitrate leaching loss in soil below a root layer by utilizing a denitrification technology and further reducing underground water nitrate pollution.
Researchers have now isolated a number of denitrifying bacteria from various environments such as water, soil and sediments, including Bacillus, Alcaligenes, Aquasispirillum and Pseudomonas. The strains have different living environments and nutritional conditions and different denitrification capabilities. Wherein, most strains only have the denitrification key enzyme NAR, NIR or NOR for converting NO3 < - >, NO2 < - > into NO and N2O, and lack the denitrification key enzyme N2OR for further completely converting NO2 into pollution-free N2, so that the complete denitrification process cannot be carried out, the content of greenhouse gas N2O in the atmosphere is increased to a certain extent, and the ecological environment is damaged.
Research shows that Pseudomonas bacteria play a crucial role in soil denitrification, and currently, the Pseudomonas denitrifying bacteria from soil environment are mainly Pseudomonas stutzeri (Pseudomonas stutzeri), but reports on Pseudomonas pini (Pseudomonas solanacearum) are very limited. Zhang et al (2014) obtained a strain NEAU-ST5-5T from saline-alkali soil of Pinyin plain in China, and the analysis indicates that the strain is a novel species of Pseudomonas and the strain is named as Pinyin Pseudomonas for the first time; the conventional nitrate reduction test shows that the strain is positive, but the characteristics of whether complete denitrification process can be carried out, the denitrification rate and the like are not clear.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide denitrifying bacteria L103 obtained by separating and purifying 100m aquifer soil of a farmland, the bacterial strain has complete denitrifying capability and rapid denitrification capability, can completely reduce NO 3-to generate N2, and has the denitrification rate as high as 1.62-2.36 g NO 3-/day/L.
The invention also aims to provide a denitrifying microorganism bacterial agent prepared by denitrifying bacteria L103, which is used for reducing nitrate leaching loss in soil below a root layer and further reducing nitrate pollution of underground water.
Another purpose of the invention is to provide an application of denitrifying bacteria L103 in the treatment process of nitrogen-containing sewage.
The invention is realized by the following technical scheme:
The invention provides a denitrifying bacterium (Pseudomonas sonnensis) L103 separated from 100m aquifer soil of agricultural ecosystem test station of Koelreuteria paniculata of institute of genetics and developmental biology, China academy of sciences, agricultural resource research center, 600kg of high nitrogen fertilizer application amount (N), hm-2. ya-1 long-term location test farmland, the strain is preserved and the preservation unit: china center for type culture Collection, collection address: china, wuhan university, date of preservation: 20/3/2019, deposit number: CCTCC M2019184.
The denitrifying bacteria L103 are facultative anaerobic gram-negative bacteria, can grow at a pH value of 6.6-7.8, are rod-shaped, are about 1.5 microns long, have a diameter of about 0.5 microns, have no flagella, and have motility.
The 16S rDNA sequence of the denitrifying bacteria L103 is shown in SEQ ID NO.1, and is a Pseudomonas sonnenensis strain from 100m aquifer soil of a farmland.
The denitrifying bacteria L103 have complete denitrification capability and rapid denitrification capability, so that the denitrifying bacteria L103 can be used for preparing a denitrifying microbial agent which can be used for reducing nitrate leaching loss in soil below a root layer, and further reducing nitrate pollution of underground water; the denitrifying bacteria can also be used for purifying and treating nitrogen-containing wastewater.
The invention has the beneficial effects that:
The invention separates 600kg (N) hm-2 ya-1 high nitrogen fertilizer application amount of agriculture ecological system test station of goldenrain agriculture ecological system test center of China academy of sciences genetics and developmental biology research center from 100m aquifer soil of farmland in long-term location test to obtain a denitrifying bacterium (Pseudomonas sonnensis) L103, wherein the denitrifying bacterium L103 has genes for coding nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, the genes can code a complete denitrification enzyme system, catalyze NO 3-to be completely reduced to generate N2, have complete denitrification capability and rapid denitrification capability, the denitrification rate can reach 1.6-2.3 g NO 3-/day/L, the denitrifying microbial agent prepared by the bacteria is used for reducing the nitrate leaching loss in soil below the root layer, thereby reducing the nitrate pollution of underground water and having good application prospect in the aspect of purifying and treating nitrogen-containing sewage.
Drawings
FIG. 1 is a scanning electron microscope observation image of denitrifying bacterium (Pseudomonas sonnensis) L103 of the present invention;
FIG. 2 is a phylogenetic tree of the 16S rDNA sequence of the denitrifying bacterium (Pseudomonas sonnensis) L103 of the present invention;
FIG. 3 is a denitrification rate map of denitrifying bacteria (Pseudomonas sonnensis) L103 of the present invention under an acidic environment (pH 6.6);
FIG. 4 is a denitrification rate map of denitrifying bacteria (Pseudomonas sonnensis) L103 of the present invention under alkaline conditions (pH 7.4).
For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.
Detailed Description
In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.
The first embodiment is as follows: isolation and purification of denitrifying bacteria (Pseudomonas sonnensis) L103
Sample source: the high nitrogen fertilizer application amount of an agricultural ecosystem test station of Koelreuteria paniculata of the institute of genetics and developmental biology of Chinese academy of sciences is 600kg (N) -hm-2-ya-1 for long-term positioning test of 100m aquifer soil of a farmland.
TSA medium (solid): tryptone 15g/L, soybean peptone 5g/L, sodium chloride 5g/L, agar 15g/L, adjusting pH to 7.2-7.4, sterilizing at 121 deg.C for 30min with high pressure steam.
TSB medium (liquid): tryptone 15g/L, soybean peptone 5g/L, sodium chloride 5g/L, adjusting pH to 7.2-7.4, sterilizing with steam at 121 deg.C for 30 min.
LB medium (liquid): 10g/L tryptone, 5g/L yeast extract and 5g/L sodium chloride, adjusting the pH value to 7.2-7.4, and performing high-pressure steam sterilization at 121 ℃ for 30 min.
Separation and purification steps: 1) taking 5g of fresh soil and 20mL of sterile water to be put into a 50mL sterile centrifuge tube, and putting the tube into a constant temperature shaking table at 28 ℃ and culturing for 1h at 180 r/min; 2) standing, taking a proper amount of supernatant, sequentially diluting with sterile water to obtain 10-1, 10-2 and 10-3 soil suspensions, respectively coating the soil suspensions on a solid TSA culture medium containing KNO3 with the final concentration of 2mM, sealing in an anaerobic culture bag (Haibo organism, Qingdao), and culturing at 28 ℃ until bacterial colonies appear; 3) observing the texture and the edge concave-convex character of the bacterial colony, the shape and the color of the hyphae and the aerial hyphae in the substrate, and whether the soluble pigment and the color thereof are generated, respectively selecting 5 different single bacterial colonies, inoculating the single bacterial colonies to a liquid LB culture medium, and placing the single bacterial colonies on a constant temperature shaking table at 28 ℃ and 180r/min for culturing for 12 hours; 4) respectively carrying out plate streaking culture on the bacterial liquid on a solid TSA culture medium until a single colony appears; 5) the single colony is picked repeatedly and streaked until a pure single colony is observed and the cell morphology is consistent under microscope observation.
example two: identification of denitrifying bacterium (Pseudomonas sonnensis) L103
1) Characterization of biological Properties
The colony of denitrifying bacteria L103 is in a white protuberant shape, and the edge is smooth; the cells were rod-shaped, about 1.5 μm long, about 0.5um in diameter, without flagella, but with motility.
Motility assay (semi-solid puncture method): semi-solid TSA culture medium (tryptone 15g/L, soybean peptone 5g/L, sodium chloride 5g/L, agar 7.5g/L) was prepared in a sterile 50mL centrifuge tube, and appropriate amount of objective strain was dipped with a sterile toothpick and then punctured and inoculated into the semi-solid TSA culture medium, which was placed in a constant temperature incubator, incubated overnight at 28 ℃ and visually observed with transmitted light. As a result, it was observed that denitrifying bacteria L103 grew as a cloud-like spread from the puncture line all around, but did not appear to grow on the puncture line at all, indicating the motility of denitrifying bacteria L103.
observation by a scanning electron microscope: 1) preparing: placing sterile glass slides with different sizes in a sterile glass culture dish in advance, adding a liquid TSB culture medium, inoculating a target strain, sealing by using a sealing film, and placing in a constant-temperature incubator at 28 ℃ for culturing until the logarithmic phase of growth (note: operation is gentle and no spill is caused); 2) cleaning: after the liquid TSB medium was slowly aspirated by a pipette and 10mL of a PBS solution (NaCl 8g/L, KCl 0.2.2 g/L, Na2HPO41.44g/L, KH2PO40.24g/L, pH7.4) was added thereto for washing, the PBS solution was slowly aspirated, leaving the coverslip and the cells attached to the coverslip; 3) fixing: adding 15mL of 2.5% glutaraldehyde solution, and fixing at room temperature in a dark place overnight; 4) and (3) dehydrating: slowly sucking off the glutaraldehyde solution by using a pipette, adding 15mL of 30% ethanol, standing for 15min, sucking off the ethanol, and repeating the dehydration operation by sequentially using 50%, 75%, 95% and 100% ethanol, wherein the dehydration is performed twice by using 100% ethanol; 5) and (3) drying: drying for 1.5h by using a critical point drying instrument (come card EMCPD300, Germany) (note: the most critical step, keeping in mind water residue or excessive drying); 6) and (4) observation: gently crushing the cover glass with thallus adhered thereon, uniformly distributing on a copper bench adhered with double-sided adhesive tape, gilding with high vacuum plating instrument (come card EMSCD050, Germany), observing, photographing and storing (see figure 1).
2) Systematic classification and identification
The invention adopts a Kit TIANAmp Bacteria DNA Kit (Tiangen, Beijing) and extracts the genomic DNA of denitrifying Bacteria L103 according to the operation steps. DNA was submitted to Shanghai Mergiz biomedical science and technology Limited and whole genome sequencing was performed using Illumina Hiseq sequencing platform.
Sequence splicing is carried out on the sequencing data by using SPAdes3.1.1 to obtain a 16S rDNA sequence (SEQ ID NO.1) of the denitrifying bacteria L103. Homology alignment of the 16S rDNA sequence of the denitrifying bacteria L103 was performed by NCBI (national Center for Biotechnology information) database website (http:// www.ncbi.nlm.nih.gov /), and a sequence having relatively high homology thereto was downloaded and a phylogenetic tree was created (see FIG. 2), and it was revealed that the 16S rDNA sequence of the denitrifying bacteria L103 had a similarity of up to 99.59% to the sequence of the known strain Pseudomonas sonnensis NEAU-ST5-5T and constituted a stable evolutionary branch with the sequence (boottrap value of 99). In addition, the Average Nucleotide Identity (ANI) value of the spliced denitrifying bacteria L103 whole genome sequence and Pseudomonas sonnensis NEAU-ST5-5T is up to 98.3%. Therefore, L103 obtained in the present invention was determined to be Pseudomonas, and it was named as Pseudomonas sonnenensis L103.
3) Identification of denitrifying functional gene
Sequence splicing is carried out on sequencing data by using SPAdes3.1.1, and denitrification functional gene annotation is carried out on the spliced sequence by using an RAST server. The results of the annotation showed that denitrifying bacteria L103 have 4 genes encoding nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, which are capable of encoding the complete denitrifying enzyme system, and thus the denitrifying bacteria L103 of the present invention have full denitrifying capability and are capable of completely reducing NO3 to produce N2.
EXAMPLES evaluation of denitrifying ability of denitrifying bacterium (Pseudomonas sonnensis) L103
The denitrifying bacterium Pseudomonas sonnenensis L103 is facultative anaerobic bacterium,
The denitrification capability of the strain under anaerobic and micro-anaerobic environments and acidic and alkaline environments is evaluated by using a GC-Robot automatic sampling and measuring system, and the result shows that denitrifying bacteria L103 can completely reduce NO 3-to generate N2 and have complete denitrification capability; in addition, the denitrification capability is realized within the pH value range of 6.6-7.8, the denitrification capability is increased along with the increase of the pH value, and the maximum denitrification rate can be as high as 1.62-2.36 g NO 3-/day/L; finally, the anaerobic and microaerobic environments have little influence on the denitrification capability of the denitrifying bacteria L103 in the alkaline environment, and the denitrification process is rapid.
1) Denitrification ability in anaerobic environment (gradient treatment of different pH values)
50mL 1/10 liquid TSB medium (KNO3 final concentration of 2mM) was placed in a 120mL serum bottle, a rotor (30X 8mM) was placed, the rubber stopper and aluminum cap were used to seal, and the air in the serum bottle was replaced with helium using an evacuation/aeration cleaning system. An absorbance value (i.e., an OD600 value) of the objective strain at a wavelength of 600nm was measured by an ultraviolet spectrophotometer, an inoculum size was calculated as OD600 value × inoculum size (mL) of 0.5, the objective strain was inoculated into a serum bottle by a syringe, and the concentrations of O2, NO, N2O and N2 in the serum bottle at 700rpm and 28 ℃ were continuously measured by a GC-Robot automatic sampling and measuring system (time interval 2 h).
The denitrification capacity of denitrifying bacteria L103 at different pH gradients was determined using a GC-Robot automatic sampling and determination system, as per the above procedure, with 3 replicates per treatment setup. The measurement results showed that denitrifying bacteria L103 were hardly able to grow at pH 5.8 and 6.2, and reduced all of KNO3 to N2 at pH 6.6, 7.0, 7.4 and 7.8 at 16h, 10h, 6h and 6h of culture, respectively, during which the maximum denitrification rates were about 0.77g of NO 3-/day/L, 1.62g of NO 3-/day/L, 2.02g of NO 3-/day/L and 2.36g of NO 3-/day/L, respectively, indicating that denitrifying bacteria L103 could grow at a pH in the range of 6.6 to 7.8, and the denitrification rate thereof increased with increasing pH within this range.
2) Denitrification ability in micro-anaerobic environment (acid environment and alkaline environment treatment)
After the air in the serum bottle was replaced with helium using the vacuum/gas-filled purge system, a 1% volume (0.7mL) of O2 was replenished using a syringe. The other operations are the same as those of the anaerobic environment. With reference to the denitrification rate measurement results of the denitrifying bacteria L103 under different pH value gradients, the pH value of 6.6 and the pH value of 7.4 are selected to represent the treatment in the acidic environment and the alkaline environment respectively, and each treatment is set for 3 times of repetition. The measurement results show that the denitrification capability of the denitrifying bacteria L103 in an alkaline environment (pH value of 7.4) is significantly higher than that in an acidic environment (pH value of 6.6). Wherein, denitrifying bacteria L103 reduces all KNO3, N2O and NO to generate N2 when cultured for about 56h in an acidic environment, and the denitrifying process is slow (figure 3); when the culture is carried out for about 12 hours in an alkaline environment, all KNO3, N2O and NO are reduced to produce N2, and the denitrification process is rapid (figure 4).
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
SEQUENCE LISTING
<110> research center of agricultural resources of institute of genetics and developmental biology of Chinese academy of sciences
<120> denitrifying bacterium with complete denitrification capability and rapid denitrification capability and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1459
<212> DNA
<213> Pseudomonas songnenensis
<400> 1
attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgaagag agcttgctct 60
ctgattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120
gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180
ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa aggctcacca 240
aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300
ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360
gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420
ggcagtaagc taataccttg ctgttttgac gttaccgaca gaataagcac cggctaactt 480
cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta atcggaatta ctgggcgtaa 540
agcgcgcgta ggtggttcgt taagttggat gtgaaagccc cgggctcaac ctgggaactg 600
catccaaaac tggcgagcta gagtatggca gagggtggtg gaatttcctg tgtagcggtg 660
aaatgcgtag atataggaag gaacaccagt ggcgaaggcg accacctggg ctaatactga 720
cactgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780
aaacgatgtc gactagccgt tgggatcctt gagatcttag tggcgcagct aacgcattaa 840
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 900
cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggccttg 960
acatgcagag aactttccag agatggattg gtgccttcgg gagctctgac acaggtgctg 1020
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc 1080
cttgtcctta gttaccagca cgttaaggtg ggcactctaa ggagactgcc ggtgacaaac 1140
cggaggaagg tggggatgac gtcaagtcat catggccctt acggcctggg ctacacacgt 1200
gctacaatgg tcggtacaaa gggttgccaa gccgcgaggt ggagctaatc ccataaaacc 1260
gatcgtagtc cggatcgcag tctgcaactc gactgcgtga agtcggaatc gctagtaatc 1320
gtgaatcaga atgtcacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380
atgggagtgg gttgctccag aagtagctag tctaaccttc ggggggacgg ttaccacgga 1440
gtgattcatg actggggtg 1459
Claims (6)
1. A denitrifying bacterium (Pseudomonas sonnensis) L103 with complete denitrification capability and rapid denitrification capability has a deposit number: CCTCC M2019184.
2. The denitrifying bacterium according to claim 1, wherein: the denitrification rate of the strain is 1.62-2.36 g NO 3-/day/L.
3. The denitrifying bacterium according to claim 1, wherein: the denitrifying bacteria are from 100m aquifer soil in farmland.
4. A denitrifying microbial inoculant, which is characterized in that: the denitrifying microorganism bacterium agent is prepared by the denitrifying bacterium of claim 1.
5. A method for reducing nitrate leaching loss in soil below a root layer is characterized in that: the use of the denitrifying microbial agent of claim 4.
6. Use of the denitrifying bacteria of claim 1 in a process for treating nitrogen-containing wastewater.
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| CN111763633A (en) * | 2020-05-28 | 2020-10-13 | 厦门市政环境科技股份有限公司 | Salt-tolerant denitrifying bacterium and application thereof |
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| CN107488611A (en) * | 2017-08-07 | 2017-12-19 | 厦门水务环境科技股份有限公司 | A kind of efficient denitrification denitrifying bacterium and its application |
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| CN107488611A (en) * | 2017-08-07 | 2017-12-19 | 厦门水务环境科技股份有限公司 | A kind of efficient denitrification denitrifying bacterium and its application |
Non-Patent Citations (1)
| Title |
|---|
| LEI ZHANG等: "Pseudomonas songnenensis sp. nov., isolated from saline and alkaline soils in Songnen Plain, China", 《ANTONIE VAN LEEUWENHOEK: JOURNAL OF MICROBIOLOGY AND SEROLOGY》 * |
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| CN111763633A (en) * | 2020-05-28 | 2020-10-13 | 厦门市政环境科技股份有限公司 | Salt-tolerant denitrifying bacterium and application thereof |
| CN111763633B (en) * | 2020-05-28 | 2022-03-25 | 厦门市政环境科技股份有限公司 | Salt-tolerant denitrifying bacterium and application thereof |
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