CN109439568B

CN109439568B - Denitrifying bacterium, application thereof and microbial agent

Info

Publication number: CN109439568B
Application number: CN201811200369.1A
Authority: CN
Inventors: 杜莉莉; 包海花; 王明中; 李骚
Original assignee: Zhongyuan Environmental Protection Co ltd
Current assignee: Zhongyuan Environmental Protection Co ltd
Priority date: 2018-10-16
Filing date: 2018-10-16
Publication date: 2020-06-09
Anticipated expiration: 2038-10-16
Also published as: CN109439568A

Abstract

The invention relates to a denitrifying bacterium, namely Comamonas sp DB-1, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16354. The strain can perform denitrification nitrogen removal under various different carbon sources, and in a high-concentration nitrate system, the DB-1 strain has a quick denitrification process start and complete conversion; secondly, the C/N ratio is lower when the nitrate is converted, the used carbon source is less, and the cost of an external carbon source for sewage denitrification treatment is saved; the existence of ammonia nitrogen does not interfere the denitrification process of the DB-1 strain, so that the range of sewage which is suitable for being treated by the strain is wider; can resist high alkaline environment and has better applicability.

Description

Denitrifying bacterium, application thereof and microbial agent

Technical Field

The invention belongs to the technical field of environmental microorganisms, and relates to denitrifying bacteria and application thereof and a microbial agent.

Background

Nitrogen is one of indispensable vital elements and has important influence on human survival and development, but due to the fact that nitrogen is over-developed and utilized by human, a series of environmental pollution problems are caused, and the health and life of human are threatened. The nitrogen can be discharged in a large amount to cause the water body to be anoxic through the nitrification, so that the water body is black and smelly; can stimulate the excessive propagation of algae in the water body to cause eutrophication; the accumulation of all nitrogen cycle intermediates, such as nitrate and nitrite, in addition to molecular nitrogen in the nitrogen cycle can have serious human and environmental consequences.

The nitrogen content in the sewage body can be controlled by a physical and chemical method and a biological denitrification method at present. The physical chemical denitrification technology has wide application range and has corresponding effect on nitrogen-containing wastewater with various concentrations, but has the defects of high cost, complex operation, easy generation of secondary pollution and the like. The biological denitrification technology well overcomes the defects of a physical chemical method, has the advantages of simple process, low cost, easy popularization and the like, and is the most important in the denitrification treatment of the sewage at presentOne of the most efficient and economical processes. The core of the method is nitration reaction and denitrification reaction, because of different flora and sewage treatment process parameters, the nitration reaction and the denitrification reaction are carried out in two isolated reactors or in the same reactor which causes alternate anoxic and aerobic environments in time and space. The traditional biological denitrification process also has some disadvantages: firstly, the proliferation speed of common denitrification flora is slow, the high concentration is difficult to maintain for denitrification, and secondly, the pH value of the sewage is reduced due to the acidity generated in the nitrification process, so that the denitrification reaction is adversely affected; thirdly, when the process is used for treating sewage with low C/N ratio, a large amount of carbon-containing organic matters need to be added to ensure the denitrification effect, so that the cost is increased; fourthly, the traditional denitrification reaction is not thorough, and the generated intermediate products NO and N₂The long-term accumulation of gases such as O and the like aggravates the damage of greenhouse effect to the environment.

Disclosure of Invention

In view of the above, the present invention aims to provide a denitrifying bacterium, an application thereof, and a microbial agent prepared from the denitrifying bacterium.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a denitrifying bacterium is Comamonas sp DB-1, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16354.

The denitrifying bacteria DB-116 SrRNA gene sequence is shown in SEQ ID No. 1.

The denitrifying bacteria have round bacterial colonies, clear edges, white, moist and bright bacterial colonies, no pigment on the back of the bacterial colonies, and easy picking of the bacterial colonies on a flat plate; is a rod-shaped, gram-negative bacterium.

2. Provides the application of denitrifying bacteria DB-1 in sewage treatment.

Further, the application is to remove nitrate nitrogen in sewage by using the denitrifying bacteria DB-1 strain.

Further, the colony count of denitrifying bacteria in the treatment was 4.0X 10⁵-4.0×10⁷cfu/ml。

3. The microbial agent containing denitrifying bacteria DB-1 is prepared by the following steps: inoculating the seed liquid of the strain DB-1 into a culture solution according to 0.5-2% v/v for enrichment culture, introducing sterile air into a tank in the fermentation process, stirring at the speed of 180-250rpm, fermenting at the temperature of 28-35 ℃ for 35-45h, and collecting the culture solution, namely the microbial agent of the denitrifying bacteria DB-1.

Further, the preparation method of the microbial agent comprises the following steps: inoculating the seed liquid of the strain DB-1 into a culture solution according to 1% v/v for enrichment culture, introducing sterile air into a tank in the fermentation process, stirring at the speed of 200rpm, fermenting at the temperature of 30 ℃ for 40h, and collecting the culture solution, namely the microbial agent of the denitrifying bacteria DB-1.

The invention has the beneficial effects that: the invention obtains a novel denitrifying bacteria DB-1 strain through long-term anaerobic acclimation in a high-nitrogen environment, and the comparison result with an NCBI database proves that the strain is a new strain and has more practicability through 16s rDNA sequencing. A large number of experiments prove that the DB-1 strain has ideal total nitrogen degradation performance, which is shown in the following steps: 1) in a high-concentration nitrate system, the denitrification process of the DB-1 strain is started quickly, the transformation is thorough, and the transformation can be completed under the condition of low bacteria concentration, which means that the preparation cost of the microbial inoculum is low when the industrial application is carried out; 2) the C/N ratio is lower when the nitrate is converted, and the used carbon source is less, so that the cost of an external carbon source for sewage denitrification treatment is saved; 3) the existence of ammonia nitrogen does not interfere the denitrification process, so that the range of sewage suitable for being treated by the strain is wider; 4) DB-1 strain denitrification can tolerate the alkaline environment of pH8.0, and has better applicability; 5) the denitrification process of the DB-1 strain is complete without intermediate harmful gases such as NO and N₂O and the like are generated, and the environmental pollution is small.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the results of 16S rDNA sequence alignment analysis of strain DB-1;

FIG. 2 is a plate colony map of the selected strain DB-1.

Biological material preservation

The strain DB-1 is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No.3 of Xilu No.1 of Suo Xinyang district, Beijing) in 8.8.29 days in 2018, the preservation number is CGMCC No.16354, and the strain is classified and named as Comamonas sp.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Culture medium for isolation of DB strain:

1. liquid denitrification culture medium formula (g/L): anhydrous sodium acetate: 2g of the total weight of the mixture; dipotassium hydrogen phosphate: 0.4 g; magnesium sulfate: 0.6 g; iron chloride: 0.5 g; calcium chloride: 0.1 g; dipotassium hydrogen phosphate: 0.2 g; sodium nitrate: 1g of a compound; trace elements: 2 ml; the pH value is 7.0-7.2.

2. Solid denitrification identification medium (g/L): 1% BTB: 10g of a mixture; anhydrous sodium acetate: 2g of the total weight of the mixture; dipotassium hydrogen phosphate: 0.4 g; magnesium sulfate: 0.6 g; iron chloride: 0.5 g; calcium chloride: 0.1 g; dipotassium hydrogen phosphate: 0.2 g; sodium nitrate: 1g of a compound; trace elements: 2 ml; pH is 7.0-7.2; agar: 2 percent.

3. Formula (g/L) of the slant preservation culture medium: beef extract: 1g of a compound; peptone: 10g of a mixture; sodium chloride: 5g of the total weight of the mixture; pH 7.2-7.4; agar: 2 percent; and (3) sterilization conditions: 121 ℃ and 30 min.

Example 1 Strain screening

DB-1 strain isolation process: taking 1ml of mixed solution of pond bottom sludge of a certain sewage plant, inoculating to a liquid denitrification culture medium for long-term anaerobic acclimation in a high-nitrogen environment, inoculating to the culture medium, adding paraffin for sealing, isolating from air to maintain an anaerobic environment, performing an initial screening test for 6-8 days, performing a later characteristic test for acclimation culture in a seed culture solution for 3-4 days until the seed culture solution grows to OD₆₀₀About 1. After acclimatization, diluting with sterile water by 10 times gradient, and taking 10 times of the diluted solution^-4、10^-5、10^-6Coating on solid denitrification identificationOn the culture medium, 3 dilutions were performed until there were clear colonies on the plate, the size and color of the colonies were observed, and a single colony large in size and having a blue halo on the periphery was picked up as shown in FIG. 2, in which the encircled strain No.3, DB-1, of the present invention was obtained. Inoculating to slant culture medium, preserving at 4 deg.C in refrigerator, and screening 11 strains, numbered 1-11. And respectively inoculating the strains obtained by primary screening into a liquid denitrification culture medium, standing for 3 days, inoculating into 10ml of sterilized liquid culture solution by using an inoculation amount of 5%, growing for 6 days at room temperature, and measuring the nitrate nitrogen index of each culture tube. Comparison of the experimental results was performed as shown in table 1. It can be seen that among the 11 strains obtained by the preliminary screening, the 3 rd, 8 th and 11 th strains were completely transformed, and their denitrification effects were at a uniform level. The three strains are identified, wherein the strain No. 8 (named DB-3) is identified as pseudomonas, the sequence of the pseudomonas is shown as SEQ ID No.3, and the pseudomonas is a denitrifying bacterium in the traditional sense, and the strain is used as a reference.

TABLE 1 initial screening of 6-day nitrate nitrogen degradation index results for the strains

The selected No.3 strain is named DB-1, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No.1 Siro-1 of North Chen-Yang district, Beijing) in 29/8 of 2018, the preservation number is CGMCC No.16354, and the strain is classified and named as Comamonas sp.

The selected No. 11 strain is named DB-2, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No.1 Hospital No.3 of North West Lu of the Korean district, Beijing) in 2018, 8 and 29 days, the preservation number is CGMCC No.16355, and the strain is classified and named as Stenotrophomonas sp.

Example 2

Sequencing and classification identification of the strain DB-1:

extracting 16s rDNA for PCR amplification: 16s rDNA was amplified by PCR using primers 27F/1492R; and (3) PCR product purification: carrying out ExoSP-IT purification on the single-strip PCR product, and carrying out gel cutting purification on the PCR product with non-specific strips; sanger sequencing, bidirectional sequencing. The DB-1 bidirectional sequencing splicing sequence is shown as SEQ ID No.1, and the 16S rDNA sequence and the comparison analysis result of the strain DB-1 are shown as figure 1. As can be seen from FIG. 1, DB-1 has 99% homologous similarity to Commonas sp in database, and can only be identified as the same genus, and also has 1% difference, i.e., 13.58bp in 1358bp is different from the 16s rDNA sequence of the published strain, so that it is identified as a new strain and preserved and survived.

The strain DB-1 is subjected to morphological identification through biological characteristic observation, and the result is as follows:

DB-1 is streaked and inoculated on a specific solid culture medium, and is cultured for 1-2 days at room temperature, so that circular denitrifying bacteria colonies with clear edges appear, the colonies are white, wet and bright, the back surfaces of the colonies are free of pigments, and the colonies are easy to pick on a flat plate; DB-1 is rod-shaped, gram-negative bacteria.

Example 3

Time course of the conversion of nitrate by the DB-1 strain:

the DB-1 and DB-2 strains are inoculated to a liquid denitrification culture medium under the same condition of 5 percent of inoculum size as the reference strain DB-3, the content of nitrate in the culture solution is sampled and detected every 24 hours, and the result is shown in Table 2, the DB-1 strain has high denitrification reaction starting speed in a high-concentration nitrate nitrogen system, and the nitrate nitrogen is degraded by 50 percent in the first day compared with DB-3 in an adaptation period; in addition, the denitrification reaction of the DB-1 strain is fast, the strain is completely degraded in the third day, and the DB-3 strain is completely degraded in the fifth day.

TABLE 2 determination of total nitrogen degradation curves for three strains within 6 days

Example 4

And (3) determining the number of bacteria in a strain DB-1 transformation culture solution: the culture solution cultured for 3 days in example 3 was counted for viable bacteria on a plate, and the number of bacteria in the DB-1 culture was calculated to be 4.0X 10⁶cfu/ml。

Example 5

Research on Denitrification Properties of DB-1

① Effect of ammoniacal nitrogen on Strain DB-1 Denitrification

In the presence of ammoniacal Nitrogen (NH) equivalent to 1g/L of sodium nitrate₄)₂SO₄In the presence of the culture medium, the strains DB-1, DB-2 and DB-3 were subjected to aerobic shaking and anaerobic static culture, respectively, and the denitrification results are shown in Table 3. From the results, it was found that DB-1 is a facultative bacterium, and the presence of ammonia nitrogen does not affect the anaerobic denitrification process.

TABLE 3 influence of ammoniacal nitrogen on the denitrification index of the strains

② the strains DB-1 and DB-3 are tested in different carbon source environments, and the experimental data are shown in Table 4. the results show that the DB-1 strain can achieve ideal degradation effect by utilizing more carbon sources.

TABLE 4 degradation results of 6-day growth indexes of strains in different carbon source environments

③ the data of DB-1 and DB-3 degrading nitrate under different C/N ratio of the same carbon source are shown in Table 5. from the results, DB-1 can achieve the ideal denitrification effect under lower C/N.

TABLE 5 degradation results of 6-day growth indexes of strains under different C/N environments

④ the strains DB-1 and DB-3 have denitrogenation effect in high pH environment, the data are shown in Table 6, the DB-1 can reach ideal denitrogenation effect in high alkalinity environment.

TABLE 6 degradation results for 4 days of high pH environmental strain growth

Example 6

Preparing DB-1 microbial inoculum: inoculating 10ml of seed solution cultured for three days into 1L of culture solution for enrichment culture to serve as seed solution for fermentation, inoculating the seed solution into a sterilized fermentation tank for fermentation, controlling the fermentation temperature to be 30 ℃, and introducing sterile air into the tank in the fermentation process; stirring speed is 200rpm, culture time is about 40h, and culture solution, namely denitrifying bacteria DB-1 microbial inoculum, is collected.

Example 7

Inoculating DB-1 strain into the pilot plant at 1% inoculation amount for 2 days, and culturing at 0.8-1M flow rate³The water quality of inlet water and outlet water of the pilot plant is continuously monitored for 10 days from 9 and 4 days in 2018 to 9 and 13 days in 2018, and the data are shown in Table 7. From the data in Table 7, it is clear that the nitrate nitrogen is reduced to 0.5mg/L and the total nitrogen is below 2 mg/L.

TABLE 7 Denitrification project test detection data

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> original environmental protection products Ltd

<120> denitrifying bacterium, application thereof and microbial agent

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>1358

<212>DNA

<213>Comamonas sp.

<400>1

gcgcacttcg gatgctgacg agtggcgaac gggtgagtaa tacatcggaa cgtgcctagt 60

agtgggggat aactactcga aagagtggct aataccgcat gagatctacg gatgaaagca 120

ggggatcgca agaccttgtg ctactagagc ggccgatggc agattaggta gttggtggga 180

taaaagctta ccaagccgac gatctgtagc tggtctgaga ggacgatcag ccacactggg 240

actgagacac ggcccagact cctacgggag gcagcagtgg ggaattttgg acaatgggcg 300

caagcctgat ccagcaatgc cgcgtgcagg atgaaggcct tcgggttgta aactgctttt 360

gtacggaacg aaaagccctg ggttaatacc ctggggtcat gacggtaccg taagaataag 420

caccggctaa ctacgtgcca gcagccgcgg taatacgtag ggtgcgagcg ttaatcggaa 480

ttactgggcg taaagcgtgc gcaggcggtt ttgtaagaca gaggtgaaat ccccgggctc 540

aacctgggaa ctgcctttgt gactgcaagg ctagagtacg gcagaggggg atggaattcc 600

gcgtgtagca gtgaaatgcg tagatatgcg gaggaacacc gatggcgaag gcaatcccct 660

gggcctgtac tgacgctcat gcacgaaagc gtggggagca aacaggatta gataccctgg 720

tagtccacgc cctaaacgat gtcaactggt tgttgggtct taactgactc agtaacgaag 780

ctaacgcgtg aagttgaccg cctggggagt acggccgcaa ggttgaaact caaaggaatt 840

gacggggacc cgcacaagcg gtggatgatg tggtttaatt cgatgcaacg cgaaaaacct 900

tacccacctt tgacatgtac ggaatccttt agagatagag gagtgctcga aagagagccg 960

taacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020

caacgagcgc aacccttgcc attagttgct acgaaagggc actctaatgg gactgccggt 1080

gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttata ggtggggcta 1140

cacacgtcat acaatggctg gtacaaaggg ttgccaaccc gcgaggggga gctaatccca 1200

taaagccagt cgtagtccgg atcgcagtct gcaactcgac tgcgtgaagt cggaatcgct 1260

agtaatcgtg gatcagaatg tcacggtgaa tacgttcccg ggtcttgtac accccgcccg 1320

tcacaccatg ggaacgggtc tcgccagaag taggtaga 1358

<210>2

<211>1393

<212>DNA

<213>Stenotrophomonas sp.

<400>2

tgcagtcgac ggtagcacag aggagcttgc tccttgggtg acgagtggcg gacgggtgag 60

gaatgcatcg gaatctactc tttcgtgggg gataacgtag ggaaacttac gctaataccg 120

catacgacct acgggtgaaa gcaggggacc ttctaggcct tgcgcgattg aatgagccga 180

tgtccgatta gctagttggc ggggtaatgg cccaccaagg cgacgatcgg tagctggtct 240

gagaggatga tcagccacac tggaactgag acacggtcca gactcctacg ggaggcagca 300

gtggggaata ttggacaatg ggcgcaagcc tgatccagcc ataccgcgtg ggtgaagaag 360

gccttcgggt tgtaaagccc ttttgttggg aaagaaaagc agccggctaa tacccggttg 420

ttctgacggt acccaaagaa taagcaccgg ctaacttcgt gccagcagcc gcggtaatac 480

gaagggtgca agcgttactc ggaattactg ggcgtaaagc gtgcgtaggt ggttgtttaa 540

gtctgtcgtg aaagccctgg gctcaacctg ggaactgcga tggaaactgg gcgactagag 600

tgtggtagag ggtagcggaa ttcctggtgt agcagtgaaa tgcgtagata tcaggaggaa 660

catccatggc gaaggcagct acctgggcca acactgacac tgaggcacga aagcgtgggg 720

agcaaacagg attagatacc ctggtagtcc acgccctaaa cgatgcgaac tggatgttgg 780

gtgcactttg gcacgcagta tcgaagctaa cgcgttaagt tcgccgcctg gggagtacgg 840

tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agtatgtggt 900

ttaattcgat gcaacgcgaa gaaccttacc tggccttgac atgctgagaa ctttccagag 960

atggattggt gccttcggga actcagacac aggtgctgca tggctgtcgt cagctcgtgt 1020

cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgtccttagt tgccagcacg 1080

taatggtggg aactctaagg agaccgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140

caagtcatca tggcccttac ggccagggct acacacgtac tacaatggtg gggacagagg 1200

gctgcaaact cgcgagagta agccaatccc agaaacccca tctcagtccg gattggagtc 1260

tgcaactcga ctccatgaag tcggaatcgc tagtaatcgc agatcagcac tgctgcggtg 1320

aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga 1380

agcaggtagc tta 1393

<210>3

<211>1391

<212>DNA

<213>Pseudomonas sp.

<400>3

ccatgcagtc gagcggatga ggggtgcttg cactctgatt cagcggcgga cgggtgagta 60

atgcctaaga atctgcccga tagtggggga caacgtttcg aaaggaacgc taataccgca 120

tacgtcctac gggagaaagt gggggatctt cggacctcac gctatcggat gagcctaggt 180

cggattagct agttggtgag gtaatggctc accaaggcga cgatccgtaa ctggtctgag 240

aggatgatca gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300

gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360

ttcggattgt aaagcacttt aagttgggag gaagggctgt cggctaatac cctgcagttt 420

tgacgttacc aacagaataa gcaccggcta acttcgtgcc agcagccgcg gtaatacgaa 480

gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt tcagcaagtt 540

ggatgtgaaa gccccgggct caacctggga actgcatcca aaactactga gctagagtac 600

ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtatatatag gaaggaacac 660

cagtggcgaa ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720

aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ctgttgggtt 780

ccttgagaac ttagtagcga agctaacgcg ataagttgac cgcctgggga gtacggccgc 840

aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900

ttcgaagcaa cgcgaagaac cttacctggc cttgacatgc tgagaacttt ccagagatgg 960

attggtgcct tcgggaactc agacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020

agatgttggg ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgttat 1080

ggtgggcact ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 1140

tcatcatggc ccttacggcc agggctacac acgtgctaca atggtcggta cagagggttg 1200

ccaagccgcg aggtggagct aatctcacaa aaccgatcgt agtccggatc gcagtctgca 1260

actcgactgc gtgaagtcgg aatcgctagt aatcgtgaat cagaatgtca cggtgaatac 1320

gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgct ccagaagtag 1380

ctagtctaac c 1391

Claims

1. A denitrifying bacterium is characterized in that the strain is Comamonas sp DB-1, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 16354.

2. The denitrifying bacterium of claim 1, wherein the bacterial 16SrRNA gene sequence is shown as SEQ ID No. 1.

3. The denitrifying bacterium of claim 1, wherein the colony of the strain DB-1 is white, rod-shaped, gram-negative.

4. Use of the denitrifying bacteria of claim 1 in sewage treatment.

5. Use according to claim 4, characterized in that it is the use of said strain for the removal of nitrate nitrogen from sewage.

6. Use according to claim 4, wherein the number of colonies of denitrifying bacteria in the treatment is 4.0 x 10⁵-4.0×10⁷cfu/ml。

7. The microbial agent containing the denitrifying bacteria of claim 1, which is prepared by a method comprising: inoculating the seed liquid of the strain DB-1 into a culture solution according to 0.5-2% v/v for enrichment culture, introducing sterile air into a tank in the fermentation process, stirring at the speed of 180-250rpm, fermenting at the temperature of 28-35 ℃ for 35-45h, and collecting the culture solution, namely the microbial agent of the denitrifying bacteria DB-1.

8. The microbial agent according to claim 7, wherein the preparation method comprises: inoculating the seed liquid of the strain DB-1 into a culture solution according to 1% v/v for enrichment culture, introducing sterile air into a tank in the fermentation process, stirring at the speed of 200rpm, fermenting at the temperature of 30 ℃ for 40h, and collecting the culture solution, namely the microbial agent of the denitrifying bacteria DB-1.