CN110283739B

CN110283739B - A salt-tolerant denitrifying bacteria and its application

Info

Publication number: CN110283739B
Application number: CN201910441591.9A
Authority: CN
Inventors: 祝惠; 王鑫壹; 阎百兴; 于翔霏; 陈欣
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2019-05-24
Filing date: 2019-05-24
Publication date: 2021-04-23
Anticipated expiration: 2039-05-24
Also published as: CN110283739A

Abstract

The invention discloses a salt-tolerant denitrifying bacterium and application thereof, and relates to the technical field of environmental microorganisms. The invention aims to solve the technical problem that nitrite nitrogen in saline aquaculture wastewater cannot be removed because the nitrogen removal capability of the existing common denitrifying bacteria is inhibited by high salinity. The strain is Shewanella allowachii sp.F2, is preserved in the China general microbiological culture Collection center in 2019, 3 months and 25 days, and has the preservation number of CGMCC No: 17433. the strain can denitrify under the condition of salt content and carry out denitrification. The strain is used for removing nitrite nitrogen pollutants in water.

Description

Salt-tolerant denitrifying bacterium and application thereof

Technical Field

The invention relates to the technical field of environmental microorganisms.

Background

With the rapid development of seawater culture, most of the seawater culture enters a semi-intensive or intensive culture mode, and nitrite nitrogen in culture water rises year by year due to various factors such as high stocking density, excessive feeding residual baits and the like. Nitrite nitrogen is one of eutrophication factors of water bodies and is an intermediate product of nitrification and denitrification, so that nitrite nitrogen widely exists in natural water bodies and is easy to cause great harm to the health of aquatic animals and human beings. Nitrite in water has high toxicity, and people can cause mental retardation and even death after drinking high-concentration nitrite water for a long time; aquatic animals are more sensitive to nitrite toxicity due to accumulation of nitrite, and various physiological flocculation phenomena such as insufficient oxygen content, tachypnea, fast heartbeat and the like in aquatic animal tissues are caused. Therefore, the reduction or removal of nitrite in water is very important for the safety of water.

The method for removing nitrite nitrogen in water comprises a physical method, a chemical method and a biological method, and the biological method is widely researched because the biological method can effectively remove various nitrogen-containing compounds and does not bring secondary pollution, so that the method is one of the most effective and green and environment-friendly methods. The removal of nitrite nitrogen in water by a microbial method is mainly realized by nitrification and denitrification of microorganisms, wherein the nitrification is a process of oxidizing nitrite nitrogen into nitrate nitrogen under the action of nitrifying bacteria (nitrite oxidizing bacteria) as an electron donor; the denitrification takes nitrite nitrogen as an electron acceptor, and the nitrite nitrogen is reduced into nitrogen oxide or nitrogen under the action of denitrifying bacteria, so that the process of thoroughly removing the nitrite nitrogen in the water body is realized. The denitrification can fundamentally solve the problem that nitrogen compounds in the water body exceed the standard, and is considered as the most effective nitrogen removal mode in biological nitrogen removal. However, the wastewater generated in the mariculture process contains a large amount of soluble salts, so that the osmotic pressure is increased, and the cells of general microorganisms are easily dehydrated seriously, and protoplasm is separated, so that the growth and metabolism are inhibited, and even the microorganisms die. The nitrogen removal capability of common denitrifying bacteria is inhibited by high salinity, and nitrite nitrogen in the saline culture wastewater cannot be removed. Therefore, the treatment of nitrite nitrogen in saline aquaculture wastewater by using microbial denitrification becomes a research hotspot and difficulty in the field.

Disclosure of Invention

The invention provides a salt-tolerant denitrifying bacterium and application thereof, aiming at solving the technical problem that the nitrogen removal capability of the existing common denitrifying bacterium is inhibited by high salinity and nitrite nitrogen in salt-containing aquaculture wastewater cannot be removed.

A salt-tolerant denitrifying bacterium is Shewanella allowacea sp.F2, is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No. 1 of Beijing republic of Shangyang, the preservation date is 2019, 3 and 25 days, the number is CGMCC No: 17433.

f2 of the Shewanella allowaensis was analyzed by 16S rDNA sequence alignment, and the homology with the 16S rDNA sequence of the Shewanella allowaensis was 99.81%. The strain F2 belongs to the genus Shewanella Alishhewanella sp.

The salt-tolerant denitrifying bacteria disclosed by the invention are applied to denitrification under the salt-containing condition.

Further, the salt-containing condition is that the NaCl concentration is less than or equal to 30 g/L.

Further, the salt-containing condition is that the pH is 9-10, and NaNO is₂The initial concentration is 0.4-0.8 g/L.

Further, the inoculation amount of the salt-tolerant denitrifying bacteria is 5-7% (v/v).

The invention has the beneficial effects that:

according to the invention, an abnormal Shewanella sp is screened out, and found to have the denitrification capability of efficiently removing nitrite nitrogen; it is further found that the bacteria have certain salt tolerance and effectively exert the denitrification capability in salt-containing conditions. By utilizing the physiological and biochemical characteristics and the metabolic mechanism of the bacteria with salt-tolerant denitrification capability, the problem existing in the treatment of the saline culture wastewater by common denitrifying bacteria can be better solved.

F2 is a denitrifying bacterium, when the inoculation amount is 5% (v/v), the denitrifying performance is very obvious in the 36 th hour of culture, and NO is treated in the 48 th hour₂ ^--N、NO₃ ^-the-N removal rates are respectively 98.97 percentAnd 94.81% NO at 120h₂ ^--N、NO₃ ^-the-N removal rate is up to more than 96%. F2 has high denitrification performance. In addition, the results of salt tolerance measurement show that the Alischewanella sp.F2 has certain salt tolerance, but the growth rate is slow due to too high salinity. The Alischewanella sp.F2 has high-efficiency NO under the condition of NaCl salinity of 0-30 g/L₂ ^--N、NO₃ ^-The N removal capacity and the removal rate are all about 99 percent.

Therefore, the strain Alischewanella sp.F2 has good practical value and application prospect in treating high-concentration nitrite wastewater under certain salt-containing conditions.

the strain is used for removing nitrite nitrogen pollutants in water.

Drawings

FIG. 1 is a photograph showing the gas generation phenomenon during the culture of F2 in Shewanella Alischer sp.;

FIG. 2 is an electropherogram of PCR amplification products of genomic DNA of Shewanella sp.F2, an alternative form of Shewanella; f2 is the strain of the invention, Alishewanella sp.F2;

FIG. 3 is a phylogenetic tree diagram of 16S rDNA gene sequences of Shewanella allowaensis sp.F2 and similar strains according to the present invention; wherein F2 is the strain of the invention, Alishewanella sp.F2;

FIG. 4 shows the example of the specific Shewanella Alischewanella sp.F2 vs. NO₂ ^--a removal effect of N, wherein

Represents NO₂ ^-Concentration of-N, ■ representsDividing rate;

FIG. 5 shows the example of the specific Shewanella Alischewanella sp.F2 vs. NO₃ ^--a removal effect of N, wherein

Represents NO₃ ^-N concentration, ■ for removal rate;

FIG. 6 shows the NO in the denitrification medium at different salinity₂ ^--N end concentration profile, wherein

Represents NO₂ ^-N concentration, ■ for removal rate;

FIG. 7 shows the NO in the denitrification medium at different salinity₃ ^--N end concentration profile, wherein

Represents NO₃ ^-N concentration, ■ represents removal rate.

Detailed Description

The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments.

The first embodiment is as follows: the salt-tolerant denitrifying bacterium is Shewanella allowacea sp.F2, is preserved in the China general microbiological culture Collection center, is preserved in No. 3 West Lu No. 1 Hospital, the republic of Beijing, the sunward area, the West Cheng, the West Lu, No. 3, is preserved in the China general microbiological culture Collection center, the preservation date is 2019, 3 months and 25 days, and the serial number is CGMCC No: 17433.

f2 bacterial colonies of the Shewanella Alishewanella sp.in the embodiment are white and circular, the diameter is 1-2 mm, the edges are neat, the surface is smooth, viscous and glossy. The thallus is rod-shaped, the length is 1-2 μm, and the width is 0.3-0.5 μm.

The physiological and biochemical characteristics of F2 in this embodiment of Shewanella Alishewanella sp.are shown in Table 1:

TABLE 1 physiological and biochemical characteristics of Alischewanella sp.F2

+: growth or positive; -: no growth or negative.

The second embodiment is as follows: the method for screening the Shewanella allowachii sp.F2 is realized according to the following steps:

the method comprises the following steps: sucking 2mL of muddy water sample collected from a seawall in Dalian city, Liaoning province (northern latitude 39 degrees 38 '31' east longitude 122 degrees 58 '19'), placing the muddy water sample into a 250mL conical flask, and adding 200mL of strain denitrification culture medium; sealing the bottle mouth by using a sealing film to manufacture an anoxic environment; standing and culturing at the constant temperature of 30 ℃ for 5-7 days to obtain an enrichment culture solution;

step two: performing gradient dilution on the enriched culture solution obtained in the step one by adopting a multiple dilution method, respectively sucking 0.5mL of each dilution suspension into a strain screening plate culture medium (which is placed overnight and does not grow with mixed bacteria), uniformly coating the enriched culture solution by using a glass triangular coating rod, and pouring the enriched culture solution into a second layer of sterilized screening plate culture medium with the temperature not higher than 40 ℃ to manufacture an anoxic environment for culturing denitrifying bacteria; sealing the periphery of the flat plate by using a sealing film; inverting the plate after the second layer of culture medium is solidified, putting the plate into a constant temperature incubator at the temperature of 30 ℃, and culturing until obvious bacterial colonies grow out; selecting the separated strain, streaking and purifying the strain for multiple times on a double-layer strain screening plate culture medium until no mixed bacteria is observed under a microscope, then inoculating the strain into 200mL of denitrification culture medium, and standing and expanding the culture at constant temperature under the anoxic condition of 30 ℃;

step three: inoculating the bacterial liquid obtained by the enlarged culture in the step two into 200mL of fresh bacterial strain denitrification culture medium according to the inoculation amount of 5% (v/v), culturing for 5d under the anoxic condition of the temperature of 30 ℃, and observing the turbidity condition of the culture medium and whether gas is generated in the culture process; as shown in FIG. 1, the cultures after 5d all became turbid and developed different degreesThe bubbles of (4); by NO in the denitrifying culture medium of the strain₂ ^--N、NO₃ ^-Screening by using the removal rate of-N as an index, and selecting NO₂ ^--N、NO₃ ^-And (4) separating and purifying the bacterial liquid with the N removal efficiency of more than 96% to obtain a bacterial strain F2 with the efficient denitrification function, namely completing the screening of the denitrifying bacteria.

Wherein the denitrification medium (/ L) of the strain consists of 5g of CH₃COONa, 1g of K₂HPO₄0.8g of NaNO₂0.03g of CaCl₂1g of Na₂CO₃0.06g of FeSO₄·7H₂O, 0.2g MgSO₄·7H₂O and 1000mL of deionized water, wherein the pH value is 10, and the mixture is sterilized for 30min at the temperature of 121 ℃ under high pressure;

the strain screening plate culture medium (/ L) is prepared by adding agar 20g per liter based on the strain denitrification culture medium, sterilizing at 121 deg.C under high pressure for 30min at pH 10.

F2 is identified by Shewanella allowachii Alishewanella sp:

16S rDNA sequencing and evolutionary tree making:

extraction of total genomic DNA of strain F2: culturing strain, collecting thallus, cracking thallus, extracting DNA, depositing, washing, pumping out (drying) to obtain total genome DNA, taking 5 microliter, and detecting with 1% agarose gel electrophoresis.

PCR amplification of strain F2 genomic DNA: the total DNA is taken as a template, and the amplification primers are a pair of universal primers.

The forward primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively;

the reverse primer is 1492R: 5'-GGTTACCTTGTTA CGACTT-3' are provided.

PCR amplification was performed using the genomic DNA as a template. The PCR reaction system is as follows:

the PCR reaction was carried out in a 25. mu.L system. The reaction system comprises the following components: template DNA 0.5. mu. L, dNTP (mix) 1. mu.L, Taq Buffer (with MgCl. RTM. MgCl)₂)2.5 μ L, Taq enzyme 0.2 μ L, primer F0.5 μ L, primer R0.5 μ L, adding double distilled water to 2%5μL。

PCR amplification requires pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles in total, repair extension at 72 ℃ for 10min, termination of the reaction at 4 ℃, and the obtained fragments are used for sequencing.

The 16S rDNA of the Shewanella allowaensis sp.F2 is shown as SEQ ID NO: 1 is shown.

mu.L of PCR amplification product was detected by 1% agarose gel electrophoresis.

The electrophoretogram of the PCR amplification product is shown in FIG. 2.

After the verification, the gel strip was cut off, and the PCR product was purified using a DNA gel recovery kit (Shanghai Bioengineering Co., Ltd.).

The evolutionary position of the phylogenetic tree of the strain in the similar strain is analyzed by the N-J algorithm, the result is shown in figure 3, and the homology of the strain F2 with the 16S rDNA sequence of Shewanella sp, Shewanella allowaensis is 99.81 percent as can be seen from figure 3.

According to the manual of common bacteria system identification and the manual of Bergey' S system bacteriology, the strain F2 is identified as Shewanella allowachii (Alischewanella sp.) by combining physiological and biochemical characteristics and 16S rDNA sequencing, and the homology of the 16S rDNA sequence is 99.81 percent. The named strain F2 was: f2, the preservation date is 3 months and 25 days in 2019, the preservation address is China general microbiological culture Collection center, and the preservation number is CGMCC No: 17433.

the third concrete implementation mode: the salt tolerance of F2 in the Shewanella allowachii of this embodiment is determined as follows:

preparing screening separation culture media with different salinity gradients, namely NaCl concentrations of 0g/L, 30g/L, 50g/L, 70g/L and 100g/L respectively; inoculating the strain F2 to a culture medium plate with various salinity by streaking, putting the strain into a 30 ℃ constant temperature incubator for culture for 5d, and taking whether the strain grows as the basis of whether the strain resists the corresponding salinity; the culture medium without denitrifying bacteria was used as a blank, and 3 cells were used in parallel.

After 1d of culture, no bacterial colony appears in each salinity culture medium; after 2d culture, obvious colony growth is realized on a culture medium containing 0g/L and 30g/L NaCl, and trace colony growth is realized on a culture medium containing 50g/L, 70g/L and 100g/L NaCl; after 5 days of culture, obvious colony growth is observed on a culture medium containing 0-70 g/L NaCl, and trace colony growth can be observed on a culture medium containing 100g/L NaCl. The strain F2 is proved to have certain salt tolerance, but the growth speed is slow due to overhigh salinity.

The fourth concrete implementation mode: f2 denitrifying Properties of the Shewanella Alishawanella sp.F2

Inoculating to sterilized denitrifying culture medium with different salinity at an inoculation amount of 5% (v/v), sealing the bottle mouth, performing static culture at constant temperature of 30 ℃ for 5 days, and measuring NO in the culture solution with each salinity₂ ^--N、NO₃ ^-N concentration, the denitrification performance of the strain is tested.

After 5d culture, the strain F2 has certain NO under the condition of 0-70 g/L NaCl salinity₂ ^--N、NO₃ ^-N-removing ability, but too high salinity inhibits the growth of strain F2, thereby reducing its NO tolerance₂ ^--N、NO₃ ^--N removal capacity. Under the condition of NaCl salinity of 0-30 g/L, the strain F2 has high-efficiency NO₂ ^--N、NO₃ ^-The N removal capacity and the removal rate are all about 99 percent. Under the condition of NaCl salinity of 30-100 g/L, over-high salinity inhibits growth of the strain F2, so that the strain F2 can inhibit NO₂ ^--N、NO₃ ^-the-N removal capacity is significantly reduced.

The following examples were used to demonstrate the beneficial effects of the present invention:

example one

Separating and purifying the strains:

(1) the culture medium used

A. Strain denitrification medium (/ L): from 5g of CH₃COONa, 1g of K₂HPO₄0.8g of NaNO₂0.03g of CaCl₂1g of Na₂CO₃0.06g of FeSO₄·7H₂O, 0.2g MgSO₄·7H₂O and 1000mL of deionized water, and the pH value is 10;

B. strain screening plate medium (/ L): adding 20g of agar per liter on the basis of the denitrification culture medium of the strain, wherein the pH value is 10;

C. strain preservation inclined tube medium (/ L): the same medium as the plate medium for screening the above strains.

The above culture medium is sterilized at 121 deg.C under high pressure for 30min before use.

(2) Separation and purification of strain F2

The method comprises the following steps: sucking 2mL of muddy water sample collected from a seawall in Dalian city, Liaoning province (northern latitude 39 degrees 38 '31' east longitude 122 degrees 58 '19'), placing the muddy water sample into a 250mL conical flask, adding 200mL of strain denitrification culture medium, sealing the flask opening by using a sealing film, and manufacturing an anoxic environment; standing and culturing at the constant temperature of 30 ℃ for 5-7 days to obtain an enrichment culture solution;

step two: and (3) performing gradient dilution on the enriched culture solution obtained in the step one by adopting a multiple dilution method, respectively sucking 0.5mL of each dilution suspension into a strain screening plate culture medium (which is placed overnight and grows without mixed bacteria), uniformly coating the enriched culture solution by using a glass coating rod, pouring the enriched culture solution into a second layer of sterilized screening plate culture medium with the temperature not higher than 40 ℃, manufacturing an anoxic environment for culturing denitrifying bacteria, and finally sealing the periphery of a plate by using a sealing film. Inverting the flat plate after the second layer of culture medium is solidified, putting the flat plate in a constant temperature incubator at the temperature of 30 ℃, and culturing until obvious bacterial colonies grow out;

step three: selecting the strain separated in the second step, and streaking and purifying the strain on a double-layer strain screening plate culture medium for multiple times until no mixed bacteria are observed under a microscope, wherein the strain F2 is considered to be purified completely; and selecting the purified bacterial colony, streaking on a strain preservation inclined tube culture medium, and storing in a refrigerator at 4 ℃ for later use after an obvious bacterial colony grows out.

Determination of Denitrification Performance of the Strain

Carrying out enrichment culture on the strain F2, sealing a bottle opening, carrying out static culture at a constant temperature of 30 ℃, and taking the strain F2 as an enrichment seed bacterial liquid after a culture medium is turbid and generates gas; inoculating with 200mL of sterilized denitrification medium at an inoculation amount of 5% (v/v) and charging with 250mPlacing in an L-shaped conical flask, sealing at constant temperature of 30 deg.C, and standing for 5 d; taking a fixed amount of culture solution at regular time, and measuring NO in the culture solution₂ ^--N and NO₃ ^-N, detecting the denitrification performance of the strain. The culture medium without denitrifying bacteria was used as a blank, and 3 cells were used in parallel.

Determination of NO by Strain F2₂ ^-The effect of-N removal is shown in FIG. 4, where

Represents NO₂ ^-N concentration, ■ for removal rate; strain F2 for NO₃ ^-The effect of-N removal is shown in FIG. 5, where

Represents NO₃ ^-N concentration, ■ for removal rate; as can be seen from the figure, NO in the denitrifying medium after 36h of culture₃ ^--N and NO₃ ^-the-N concentration is obviously reduced, and the strain F2 is used for NO₂ ^--N、NO₃ ^-The removal rates of-N were 74.31% and 74.56%, respectively; after 48h of culture, strain F2 was protected against NO₂ ^-The removal rate of-N reached a maximum of 98.97% of NO at that time₃ ^-The removal rate of-N was 94.81%; after 96h of culture, strain F2 was paired with NO₃ ^-The removal rate of-N reached a maximum of 99.05% and NO was present at this time₂ ^-The removal rate of-N was 98.44%.

Determination of salt tolerance of the Strain

The growth of the strain was observed by varying the salinity of the screening isolation medium.

Preparing screening separation culture media with different salinity gradients, namely NaCl concentrations of 0g/L, 30g/L, 50g/L, 70g/L and 100g/L respectively; and (3) streaking and inoculating the strain F2 to a culture medium plate with various salinity, and culturing for 5d in a 30-DEG C constant-temperature incubator by taking whether the strain grows as the basis of whether the strain resists the corresponding salinity. The culture medium without denitrifying bacteria was used as a blank, and 3 cells were used in parallel.

As shown in Table 2, after 2 days of culture, significant colony growth was observed on the medium containing 0g/L and 30g/L NaCl, and trace colony growth was observed on the medium containing 50g/L, 70g/L and 100g/L NaCl; after 5 days of culture, obvious colony growth is observed on the culture medium containing 0g/L, 30g/L, 50g/L and 70g/L NaCl, and trace colony growth can be observed on the culture medium containing 100g/L NaCl, which shows that the strain F2 has certain salt tolerance, but the growth speed is slow due to overhigh salinity.

TABLE 2 growth of Strain F2 in different salinity gradient media

++: obvious colonies appear on the surface of the culture medium, which indicates that the strain F2 grows normally;

+: micro-colonies appear on the surface of the culture medium, which indicates that the strain F2 has a growth phenomenon but has a slow growth speed;

-: no colonies appeared on the surface of the medium, indicating that strain F2 did not grow.

Influence of salinity on the Denitrification Performance of the strains according to the invention

Denitrifying culture mediums with different salinity gradients are prepared, namely NaCl concentrations of 0g/L, 30g/L, 50g/L, 70g/L and 100g/L respectively. The strain F2 is transferred into sterilized culture media with various salinity by the inoculation amount of 5% (v/v), the bottle mouth is sealed, and the strain is subjected to static culture for 5-7 d at the constant temperature of 30 ℃. Determination of NO in culture broth₂ ^--N、NO₃ ^-N concentration, the denitrification performance of the strain F2 was tested. The culture medium without denitrifying bacteria was used as a blank, and 3 cells were used in parallel.

Determination of NO by Strain F2₂ ^-The effect of-N removal is shown in FIG. 6, where

Represents NO₂ ^-N concentration, ■ for removal rate; strain F2 for NO₃ ^-The effect of-N removal is shown in FIG. 7, where

Represents NO₃ ^-N concentration, ■ representing the removal rate, it can be seen from the figure that strain F2 is responsible for NO₂ ^--N、NO₃ ^-The removal ability of-N is significantly affected by the salt content. After 5 days of culture, in the denitrification culture solution containing 0g/L and 30g/L NaCl, the strain F2 is used for NO₂ ^--N、NO₃ ^-Significant removal of-N, NO₂ ^--N、NO₃ ^-The removal rate of-N is about 99 percent, which indicates that the strain F2 normally grows under the condition of containing 30g/L NaCl and NO₂ ^--N、NO₃ ^-The N-removing ability is not affected by salt. Strain F2 for NO in a broth containing 50g/L and 70g/L NaCl₂ ^--N、NO₃ ^-The removing capability of-N is poor, the removing rate is in the range of 24-31%, which indicates that the strain F2 grows slowly under the condition of containing 50-70 g/L NaCl, and NO is₂ ^--N、NO₃ ^-the-N removing ability is significantly reduced by the influence of salt. Strain F2 vs NO in a broth containing 100g/L NaCl₂ ^-The removal rate of-N is kept to be about 26 percent, and the removal rate of NO is kept₃ ^-The absence of N removal indicates that the growth of the strain F2 was inhibited in the presence of 100g/L NaCl, NO₂ ^--N、NO₃ ^-the-N removal capability is severely affected.

Optimum salt-tolerant denitrification condition of strain

200mL of sterilized denitrification medium containing 30g/L NaCl was placed in a 250mL conical flask, and the strain F2 of the present invention was cultured by standing in a sealed state at 30 ℃ to examine NaNO at different pH values (3.0, 5.0, 7.0, 9.0, 10.0, 11.0)₂Initial concentrations (0.4g/L, 0.8g/L, 1.6g/L, 2.4g/L, 3.2g/L) and inoculum sizes (1% (v/v), 3% (v/v), 5% (v/v), 7% (v/v), 10% (v/v)) of strain F2 for NO₂ ^--N、NO₃ ^--N removal capacity.

The result shows that the strain F2 can adapt to a slightly alkaline environment under the condition of NaCl salinity of 30g/L, and the strain F2 can be used for NO treatment within the pH range of 9-10₂ ^--N、NO₃ ^-The removal rate of N reaches more than 99% and 96% respectively; optimum NaNO for Strain F2₂The initial concentration is 0.4-0.8 g/L, and the strain F2 is NO₂ ^--N、NO₃ ^-The removal rate of N reaches more than 98 percent and 94 percent respectively; the optimal inoculation amount of the strain F2 is 5-7% (v/v), and the strain F2 is used for NO₂ ^--N、NO₃ ^-The removal rate of-N reaches more than 99% and 96% respectively.

Sequence listing

<110> institute of geography and agroecology of northeast China academy of sciences

<120> one strain of salt-tolerant denitrifying bacteria and application thereof

<160> 3

<210> 1

<211> 1033

<212> DNA

<213> Shewanella allowae (Alischewanella sp.)

<220>

<223> Shewanella allowae (Alischewanella sp.) F2

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tagcggcgga cgggtgagta atgcgtagga agctgcccga tagaggggga taccagttgg 60

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gctatcggat gcgcctacgt gggattagct agttggtggg gtaatggctc accaaggcga 180

cgatccctag ctggtttgag aggatgatca gccacactgg aactgagaca cggtccagac 240

tcctacggga ggcagcagtg gggaatattg gacaatgggc gcaagcctga tccagccatg 300

ccgcgtgtgt gaagaaggcc ttcgggttgt aaagcacttt cagtggggag gaagggtgtt 360

gtgttaatag tacagcactt tgacgttacc cacagaagaa gcaccggcta actccgtgcc 420

agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc gtaaagcgca 480

cgcaggcggc tttttaagtc ggatgtgaaa gccccgggct caacctggga attgcatctg 540

atactgggaa gctagagtat gtgagagggg ggtagaattc caagtgtagc ggtgaaatgc 600

gtagagattt ggaggaatac cagtggcgaa ggcggccccc tggcacaata ctgacgctca 660

ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 720

tgtctactag ctgttcgcgg ccttgtgttg tgagtagcgc agctaacgca ttaagtagac 780

cgcctgggga gtacggtcgc aagattaaaa ctcaaatgaa ttgacggggg cccgcacaag 840

cggtggagca tgtggtttaa ttcgacgcaa cgcgaagaac cttacctact cttgacatct 900

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tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc aacccttatc 1020

cttagttgcc agc 1033

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<213> Artificial sequence

<220>

<223> Forward primer 27F

<400>2

agagtttgatcctggctcag 20

<210>3

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<213> Artificial sequence

<220>

<223> reverse primer 1492R

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ggttaccttgttacgactt 19

Claims

1. the denitrifying bacteria of a strain of salt tolerance, is characterized in that: this bacterial strain is other kind Shewanella (Alishewanella sp.) F2, is preserved in China Microorganism Culture Collection General Microorganism Center, and the preservation address is Beijing Chaoyang No. 3, No. 1 Courtyard, Beichen West Road, District, the preservation date is March 25, 2019 in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC No: 17433.

2. The application of the salt-tolerant denitrifying bacteria of claim 1 for denitrification and denitrification under saline conditions.

3. The application according to claim 2, wherein the salt-containing condition is that NaCl concentration≤30g/L.

4 . The application according to claim 2 , wherein the pH of the reaction conditions for denitrification is 9-10, and the initial concentration of NaNO ₂ is 0.4-0.8 g/L. 5 .

5 . The application according to claim 2 , wherein the inoculum of the salt-tolerant denitrifying bacteria is 5-7% (v/v). 6 .