CN115851484A

CN115851484A - Aerobic denitrification pseudomonas ZZRD2, biological denitrification microbial inoculum and application thereof

Info

Publication number: CN115851484A
Application number: CN202210926711.6A
Authority: CN
Inventors: 周金波; 朱诗君; 金树权; 汪峰; 王芳; 柴伟纲; 刘健
Original assignee: Ningbo Academy of Agricultural Sciences
Current assignee: Ningbo Academy of Agricultural Sciences
Priority date: 2022-08-03
Filing date: 2022-08-03
Publication date: 2023-03-28

Abstract

The invention belongs to the technical field of environmental microorganisms, and particularly relates to an aerobic denitrifying bacterium, a biological denitrification microbial inoculum and application thereof. The aerobic denitrifying bacteria are pseudomonas bacteria (Pseudomonas sp) with the strain number of ZZRD2, and the strain has high denitrification capability, can effectively remove nitrate in sewage, and provides microbial resources for biological denitrification and treatment of nitrogen-containing sewage.

Description

Aerobic denitrification pseudomonas ZZRD2, biological denitrification microbial inoculum and application thereof

Technical Field

The invention belongs to the technical field of environmental microorganisms, and particularly relates to an aerobic denitrifying pseudomonas ZZRD2, a biological denitrifying microbial inoculum and application thereof.

Background

Nitrogen is one of the material bases of the biogeochemical cycle, nitrogen fertilizer is artificially synthesized and widely used, and nitrogen compounds are greatly accumulated and released in the nature, so that the input amount of nitrogen in a flow domain is continuously increased, and more water bodies are polluted by nitrogen to different degrees. The pollution sources of nitrogen in the water body are more, and the pollution sources comprise domestic sewage, industrial wastewater, farmland drainage and other wastewater. Excessive input of nitrogen in the water environment can cause a series of water environment problems such as seasonal anoxia, eutrophication, large-area outbreak of harmful algae and the like in a large range of water bodies.

Biological denitrification refers to a process of converting organic nitrogen and ammonia nitrogen in sewage into nitrogen finally through ammoniation, nitration reaction and denitrification reaction under the combined action of microorganisms. The method has the characteristics of economy, effectiveness, no secondary pollution and the like, and becomes one of the research hotspots in recent years. The nitrogen in the water body oxidizes organic nitrogen and inorganic nitrogen into NO through the ammoniation, the ammoxidation and the nitrous acid oxidation of microorganisms ^3- N, then NO by denitrification ^3- Reduction of-N to NO, N ₂ O or N ₂ Thereby achieving the purpose of removing nitrogen in the water body.

There are reports of new results on technical studies in this respect, and some microorganisms for denitrification, such as denitrifying bacteria, etc., are disclosed, however, it has been found that some aerobic denitrifying bacteria have weak activity in the actual biological denitrification of sewage, and cannot meet the requirement of the field for biological denitrification at the present stage, so that screening of high-efficiency aerobic denitrifying bacteria is an important and continuous task.

Disclosure of Invention

The invention aims to provide an aerobic denitrifying pseudomonas ZZRD2, a biological denitrifying microbial inoculum and application thereof, wherein the pseudomonas ZZRD2 has high denitrifying capability and can effectively remove nitrate in sewage.

The invention provides an aerobic denitrifying Pseudomonas (Pseudomonas sp) ZZRD2; the preservation number of the pseudomonas ZZRD2 is CGMCC No.16778.

The invention provides a biological denitrifying bacterial agent, which comprises pseudomonas ZZRD2.

Preferably, the effective components of the biological denitrifying bacteria agent comprise one or more of pseudomonas ZZRD2 bacteria, pseudomonas ZZRD2 fermentation liquor and pseudomonas ZZRD2 metabolite.

Preferably, the concentration of pseudomonas ZZRD2 in the biological agent is 1 × 10 ⁹ CFU/mL。

The invention also provides application of the aerobic denitrifying bacteria or the biological denitrification microbial inoculum in the technical scheme in sewage denitrification treatment.

Preferably, the wastewater comprises nitrogen-containing wastewater.

Preferably, the nitrogen-containing sewage comprises one or more of domestic sewage, industrial wastewater, farmland drainage and aquaculture sewage.

The invention also provides a sewage denitrification treatment method, which treats sewage by using the pseudomonas ZZRD2 or the biological denitrification microbial inoculum in the technical scheme.

Preferably, the time for carrying out the sewage denitrification treatment is more than or equal to 30 hours, the temperature is 20-40 ℃, and the pH is 7-10.

Preferably, the carbon source for denitrification of sewage comprises sodium acetate and/or sodium oxalate, the C/N ratio is 15.0-37.7, and the rotation speed is 100-160 r/min.

Has the advantages that:

the invention provides an aerobic denitrifying Pseudomonas (Pseudomonas sp) ZZRD2, and biological preservation is completed. The pseudomonas ZZRD2 provided by the invention has high denitrification capability, can effectively remove nitrate in sewage, and provides a microbial resource for biological denitrification and treatment of nitrogen-containing sewage.

Biological preservation information

Pseudomonas (Pseudomonas) ZZRD2 is preserved in China general microbiological culture Collection center (CGMCC) at 26.11.2018, with the preservation address of No. 3 Siro-1 of Beijing, chaoyang, and the preservation number of CGMCC No.16778.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 shows the nitrate nitrogen degradation rate of 6 strains in example 1 for 24 hours;

FIG. 2 is a colony morphology feature of the D2 strain of example 1;

FIG. 3 is the 16S rRNA phylogenetic tree of strain D2 in example 1;

FIG. 4 is the effect of different treatment times on the growth of ZZRD2 strain and the concentration of nitrate nitrogen in example 2;

FIG. 5 is the effect of different temperatures on the ability of ZZRD2 strain to degrade nitrate nitrogen in example 2;

FIG. 6 is a graph showing the effect of different pH on the ability of ZZRD2 strain to degrade nitrate nitrogen;

FIG. 7 shows the effect of different carbon sources on the ability of ZZRD2 strain to degrade nitrate nitrogen;

FIG. 8 is a comparison of the nitrate nitrogen degradation rate of the ZZRD2 strain of example 3 for different dosages of the same strain, wherein the three columns for each treatment represent 24h, 48h and 72h in order from left to right.

Detailed Description

The invention provides a strain of aerobic denitrifying Pseudomonas (Pseudomonas sp) ZZRD2; the preservation number of the pseudomonas ZZRD2 is CGMCC No.16778.

The pseudomonas ZZRD2 is separated from sludge of a sewage treatment plant in the northeast of the Jiangtong of Ningbo city. In the invention, the 16S rDNA sequence of the pseudomonas ZZRD2 is preferably shown as SEQ ID No. 1: <xnotran> 5'-TCCATGCAAGTCGAGCGGATGAGTGGAGCTTGCTCCATGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGACGGTACA-3'. </xnotran> The similarity of the strain and Pseudomonas stutzeri ATCC 17588= LMG 11199 reaches 99.93%. The development tree was constructed after multiple sequence alignment analysis by MEGA5.0 software (FIG. 3), and it was confirmed that this strain belongs to Pseudomonas bacteria (Pseudomonas sp.).

The biological characteristics of the pseudomonas ZZRD2 are light yellow, round, fuzzy in edge and wrinkled.

The pseudomonas ZZRD2 has better degradation capability on nitrate nitrogen and can be used for denitrification treatment of sewage.

The invention provides a biological denitrifying bacterial agent, which comprises pseudomonas ZZRD2. In the invention, the effective component of the biological denitrogenation microbial inoculum comprises one or more of pseudomonas ZZRD2 thallus, pseudomonas ZZRD2 fermentation liquor and pseudomonas ZZRD2 metabolite, and is more preferably in the form of thallus or fermentation liquorAre present. The concentration of pseudomonas ZZRD2 in the biological agent is preferably 1 × 10 ⁹ CFU/mL。

When thalli is used as the effective component of the biological denitrification bacterial agent, the invention preferably comprises the following components: culturing the pseudomonads ZZRD2 in an LB liquid culture medium for 48 hours, washing and centrifuging the pseudomonads ZZRD2 strain obtained by culture by adopting sterile water, and mixing the obtained thallus with the sterile water to obtain the biological denitrifying bacteria agent.

The culture of the invention is preferably shaking culture, and the rotation speed of the shaking culture is preferably 100-160 rpm, and more preferably 160rpm. The shaking culture is preferably performed using a shaker in the present invention. The temperature for the culture according to the present invention is preferably 10 to 40 ℃, more preferably 30 ℃. The times of washing and centrifuging are preferably the same, and the times of centrifuging are preferably 1 to 3, and more preferably 3; the rotation speed of the centrifugation is preferably 2000-5000 rpm, and more preferably 3000rpm; the time for the centrifugation is preferably 5 to 15min, more preferably 10min. The mass-to-volume ratio of the bacteria to the sterile water is preferably 5 to 10:90 to 95, more preferably 10:90.

the invention also provides application of the aerobic denitrifying bacteria or the biological denitrification microbial inoculum in the technical scheme in sewage denitrification treatment. Compared with the existing denitrifying bacteria, the pseudomonas ZZRD2 provided by the invention has higher denitrification capability, and can effectively remove nitrate in sewage.

The sewage water of the present invention preferably includes nitrogen-containing sewage water, and more preferably includes one or more of domestic sewage water, industrial wastewater water, farmland drainage water and aquaculture sewage water. When the bacteria are used as the effective component of the biological denitrification bacterial agent, the use concentration of the aerobic denitrifying bacteria in the process of carrying out the sewage denitrification treatment is preferably 1 x 10 ⁹ CFU/mL. In the process of carrying out the sewage denitrification treatment, the time for the sewage denitrification treatment is preferably more than or equal to 30 hours, and more preferably 30 hours; the temperature is preferably 20 to 40 DEG CMore preferably 25 to 35 ℃, and still more preferably 30 ℃; the pH is preferably 7 to 10, more preferably 8 to 9, and still more preferably 9. The carbon source for the denitrification treatment of wastewater according to the present invention preferably includes sodium acetate and/or sodium oxalate, more preferably sodium acetate or sodium oxalate, and still more preferably sodium oxalate. The C/N ratio in the denitrification of the wastewater is preferably 15.0 to 37.7, more preferably 22.6 to 30.2, and even more preferably 22.6; the rotation speed is preferably 100 to 160r/min, more preferably 130 to 160r/min, and still more preferably 160r/min.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

The culture medium and the formula thereof involved in the examples are as follows:

enrichment culture medium: 9.63g of sodium citrate, 0.85g of sodium nitrate, 1.36g of monopotassium phosphate, 0.27g of ammonium sulfate, 1g of yeast extract, 0.19g of magnesium sulfate, 1ml of trace elements, 1L of distilled water with the pH value of 7.2-7.3, and sterilizing for 30min at 121 ℃.

Bromothymol blue plate (BTB): KNO ₃ 1.0g, trisodium citrate 1.0g ₂ PO ₄ 1.0g，FeSO ₄ ·7H ₂ O 0.05g，CaCl ₂ 0.2g，MgSO ₄ ·7H ₂ O1.0 g,1% bromothymol blue 1mL (1% in ethanol), agar 20g, water to 1L, pH7.3, 121 ℃, sterilized for 30min.

DM denitrification culture solution: KNO ₃ 0.5g.L ^-1 、KH ₂ PO ₄ 1g.L ^-1 、MgSO ₄ ·7H ₂ O 0.1g.L ^-1 Sodium succinate 2.8g.L ^-1 And sterilizing at 121 ℃ for 30min.

LB liquid medium: 5g of yeast powder, 10g of tryptone, 10g of NaCl, 1L of distilled water and pH7.2-7.3. Sterilizing at 121 deg.C for 15min.

Example 1

Separation, screening and identification of pseudomonas ZZRD 2:

1. adding a proper amount of sludge sample into an enrichment medium containing 100mL, and carrying out 160r.min at 30 DEG C ^-1 And (5) performing shake culture. After 3d, 5mL of the enriched medium was added to fresh enriched medium and the culture was continued (this process was repeated 3 times). The enriched bacterial suspension is treated according to the formula 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 And (3) performing gradient dilution, coating the solution on a bromothymol blue plate (BTB), culturing the solution at 30 ℃ for 2-3D, picking single colonies with blue halos, continuously separating and purifying (repeatedly carrying out partition streaking on the bromothymol blue plate (BTB) until the single colonies are obtained) until purified strains are obtained, and separating 6 strains which are respectively named as A, B, C, D2, E and B2.

2. And (3) detecting the denitrification capability of the strain:

respectively inoculating the 6 purified strains obtained in the step 1 (bacterial colonies are dipped by using a sterile toothpick) into 100mL of LB culture medium (each strain is arranged in 3 parallel), and placing the bacterial liquid into a sterile centrifuge tube for 5000r.min when the bacterial liquid is turbid by visual inspection ^-1 Centrifuging for 10min; the supernatant was discarded, and the cells were washed with sterile water and centrifuged again (this operation was repeated 3 times). Inoculating to DM denitrifying culture solution at 1% (V/V) at 30 deg.C for 160r.min ^-1 Under the condition, after 3 days of culture, the concentration of nitrate nitrogen in the DM denitrification culture solution is subjected to ultraviolet visible spectrophotometry, the DM denitrification culture solution without inoculated bacterial solution is used as blank treatment, and the nitrate nitrogen degradation rate is calculated, wherein the degradation rate is% = (blank treatment nitrate nitrogen content-microbial inoculum treatment nitrate nitrogen content)/blank treatment nitrate nitrogen content is multiplied by 100, and the details are not repeated. The screened strain D2 has better denitrification capability, as shown in figure 1.

As can be seen from fig. 1: the 6 strains all have certain effect of degrading nitrate nitrogen, wherein the D2 strain has the best degradation effect, and the degradation rate is 62.8%. Thus, further studies were conducted on the D2 strain.

3. The morphological characteristics of the bacterial colony are as follows: a single colony of the D2 strain is picked up, streaked on an LB plate in a partition mode, and cultured for 48 hours at the temperature of 30 ℃, so that the colony of the D2 strain is light yellow, round, fuzzy in edge and wrinkled and has the morphological characteristics shown in figure 2.

4. Sequencing and identification of the isolated strains:

the strain D2 is named ZZRD2 and subjected to sequencing analysis to obtain a 16S rRNA sequence, which is shown in SEQ ID No. 1. BLAST comparison with GenBank database sequences was performed, and 20 representative strain sequences with 99% similarity to the strain were selected, wherein the similarity with the closest relativity to Pseudomonas stutzeri ATCC 17588= LMG 11199 was 99.93%. Developmental trees were constructed after multiple sequence alignment analysis by MEGA5.0 software (FIG. 3). The results showed that this strain belongs to the genus Pseudomonas (Pseudomonas sp.).

Example 2

Pseudomonas ZZRD2 (hereinafter referred to as ZZRD 2) denitrification performance test

1. Using an inoculating loop to pick a ZZRD2 strain and inoculate the strain into an activation culture medium (LB liquid culture medium: 5g of yeast powder, 10g of tryptone, 10g of NaCl, 1000mL of distilled water, pH7.2-7.3, subpackaging in 250mL triangular flasks, 100mL of each flask, sterilizing at 121 ℃ for 15 min), culturing for 48h by using a shaking table, centrifuging and washing for 3 times by using sterile water, finally centrifuging 10 mL of bacterial liquid, removing supernatant, and adding the bacterial into 100mL of sterile water to prepare the bacterial liquid for later use.

2. Influence of treatment time on growth amount of ZZRD2 strain and nitrate nitrogen concentration

The inoculation amount is 10% (V/V) (viable count of bacterial liquid is 1X 10) ⁹ CFU/mL, the same description is omitted), inoculating the bacterial liquid obtained in step 1 into DM denitrification culture solution, culturing the inoculated culture solution at the pH value of 7.0 and the rotation speed of 160r/min at 30 ℃ for 48h, sampling 1 time every 2h after inoculation, and determining OD ₆₀₀ The values and nitrate nitrogen concentrations were calculated by treating the DM denitrification culture medium without inoculated bacterial solution as a blank, and the results are shown in FIG. 4:

from fig. 4, it can be derived that: the ZZRD2 strain grows rapidly in the culture medium and after 30 hours the growth tends to be flat. Meanwhile, in 0-30 h, nitrate nitrogen in the culture solution is rapidly degraded, the degradation rate tends to be flat, and the growth amount of the strain is in direct proportion to the degradation rate of the nitrate nitrogen.

3. Effect of temperature on the Denitrification Activity of ZZRD2 Strain

The degradation condition of the ZZRD2 strain on nitrate nitrogen under different temperature conditions is tested, and the following culture medium is designed according to an aerobic denitrification culture medium: potassium nitrate 0.2g/L, potassium dihydrogen phosphate 0.105g/L, trace elements 2mL/L, pH7, 121 deg.C sterilization for 30min. The thalli activated by the LB culture medium is centrifuged and then suspended in sterile water, the suspension is repeatedly added into an aerobic denitrification culture medium according to the inoculum size of 10 percent after 3 times, and the nitrate nitrogen degradation rate is measured after the thalli are respectively cultured for 48 hours under the conditions of 10 ℃, 20 ℃,30 ℃ and 40 ℃ with the rotating speed of 160r/min. Each experiment was performed in 3 parallel groups, and the culture medium without inoculation was used as a blank, and the results are shown in FIG. 5:

from fig. 5, it can be seen that: the temperature has certain influence on degrading nitrate nitrogen of the ZZRD2 strain. Along with the increase of the temperature, the degradation rate of the bacterial strain ZZRD2 to nitrate nitrogen is gradually increased, and when the temperature reaches 30 ℃, the degradation rate of the nitrate nitrogen of the bacterial strain is the highest and is 71.4%. Whereas above 30 ℃ the degradation rate decreases instead with increasing temperature, and when the temperature reaches 50 ℃ the strain loses its degradation capacity.

4. Effect of pH on the denitrifying Activity of ZZRD2 Strain

The degradation condition of the ZZRD2 strain on nitrate nitrogen under different pH conditions is tested, and the following culture medium is designed according to an aerobic denitrification culture medium: 0.2g/L of potassium nitrate, 0.105g/L of monopotassium phosphate, 2mL/L of trace elements, pH of 4, 5, 6, 7, 8, 9, 10 and 11 respectively, sterilizing at 121 ℃ for 30min, centrifuging the thalli activated by the LB culture medium, then resuspending in sterile water, repeating for 3 times, adding the thalli into an aerobic denitrification culture medium according to the inoculum size of 10% (V/V), rotating at 160r/min, culturing at 30 ℃ for 48h, and then measuring the concentration of nitrate nitrogen. Each experiment was performed in 3 parallel groups, and the culture medium without inoculation was used as a blank, and the results are shown in FIG. 6:

from fig. 6, it can be derived that: when the pH value is 4.0, the strain ZZRD2 basically has no denitrification activity, the degradation rate of nitrate nitrogen is rapidly increased along with the increase of the pH value, and the removal rate of the nitrate nitrogen is up to 78.6% at the pH value of 9.0, and then the degradation rate is reduced along with the increase of the pH value.

5. Effect of different carbon sources on the Mirabilite Activity of ZZRD2 Strain

Adopting DM denitrification culture solution as basic culture medium, respectively adding glucose, ethanol, sodium oxalate and sodium acetate,the initial concentration of nitrate nitrogen is 10mg.L ^-1 The inoculum size was 10%. And culturing the inoculated culture solution at the pH value of 7.0 and the rotating speed of 160r/min for 48h at the temperature of 30 ℃ and then determining the concentration of nitrate nitrogen. Each experiment was performed in 3 parallel groups, and the result is shown in FIG. 7, in which the blank was treated with a DM denitrification culture medium without inoculation:

from fig. 7, it can be seen that: different carbon sources have influence on the denitrification of the strain ZZRD2, the denitrification of the strain ZZRD2 is relatively stronger when sodium acetate and sodium oxalate are used as carbon sources, and particularly when sodium oxalate is used as a carbon source, the nitrate nitrogen removal rate is the highest and is 70.9%. Thus indicating that the sodium oxalate is the most suitable carbon source for the aerobic denitrification of the strain D2.

6. Effect of different C/N ratios on the denitrifying Activity of ZZRD2 Strain

The initial concentration of nitrate nitrogen is 10mg.L ^-1 Adjusting the C/N ratio of the DM-denitrification culture solution to be 7.5, 15, 22.6, 30.2 and 37.7 respectively under the condition that the inoculation amount is 10 percent (V/V), measuring the nitrate nitrogen concentration after the inoculated culture solution is cultured for 48 hours under the conditions of pH value of 7.0, rotating speed of 160r/min and 30 ℃, calculating the degradation rate by taking the DM-denitrification culture solution without inoculation as a blank treatment, and determining the influence of different C/N on the capability of the bacterial strain to degrade the nitrate nitrogen, wherein the results are shown in Table 1:

TABLE 1 nitrate nitrogen degradation rate of ZZRD2 strain compared with different C/N

C/N	The degradation rate of nitrate nitrogen is%
		7.5	54.56
15.0	59.56
		22.6	65.81
30.2	67.62
		37.7	69.74

From table 1 it can be derived: different C/N ratios have influence on the denitrification activity of the strain ZZRD2, the degradation rate of nitrate nitrogen is improved along with the increase of the carbon-nitrogen ratio, and when the carbon-nitrogen ratio reaches more than 22.6, the degradation rate of nitrate nitrogen is not changed greatly, which shows that the denitrification activity of the strain can be effectively improved by proper carbon-nitrogen ratio.

7. Effect of different rotational speeds on the denitrifying Activity of ZZRD2 Strain

Inoculating bacteria in DM (Dioscorea nipponica) denitrification culture solution, adjusting the shaking table speeds to be 100r/min,130r/min and 160r/min under the condition that the inoculation amount is 10% (V/V), measuring the nitrate nitrogen concentration after shaking table culture is carried out for 48 hours at the temperature of 30 ℃, taking the DM denitrification culture solution without inoculating bacteria as blank treatment, calculating the degradation rate, determining the influence of different rotating speeds on the capability of the bacterial strains for degrading the nitrate nitrogen, and showing the results in a table 2:

TABLE 2 Effect of different shaking speeds on the nitrate nitrogen degradation rate of ZZRD2 Strain

Rotational speed	The degradation rate of nitrate nitrogen is%
		100r/min	65.00
130r/min	65.29
		160r/min	68.19

As can be seen from table 2: dissolved oxygen is the major contributor in the denitrification process. The test sets 3 different sets of rotational speeds, namely 100r/min,130r/min and 160r/min. The rotating speed is between 100 and 160r/min, the rotating speed has certain influence on the degradation rate of the bacterial strain ZZRD2 on nitrate nitrogen, but the difference is not obvious.

Example 3

Application of pseudomonas ZZRD2 in simulated artificial wastewater

The test wastewater formula is as follows: potassium nitrate 0.2g/L, potassium dihydrogen phosphate 0.105g/L, and trace element solution 2mg/L (trace element solution: znSO) ₄ ·7H ₂ O 2.2g/L，Ca Cl ₂ ·2H ₂ O 5.5g/L，MnCl ₂ ·4H ₂ O 5.06g/L，FeSO ₄ ·7H ₂ O 5.0g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 1.1g/L，CuSO ₄ ·5H ₂ O 1.57g/L，CoCl ₂ ·6H ₂ O1.61 g/L, pH = 7.0), pH 7.0, sterilization at 121 ℃ for 30min.

The flask test was performed in a 500mL triangular flask, each containing 250mL of an artificial wastewater sample.

And (3) experimental design: 4 treatments were set, each 3 replicates.

The amounts of Pseudomonas ZZRD2 cells (concentration of Pseudomonas ZZRD2 in the cells was 1X 10) were measured as shown in Table 3 ⁹ CFU/mL) was placed in a 500mL triangular flask of simulated artificial wastewater in a shaker at 30 ℃ and 160rpm. 1 in Table 3: 10 ⁴ The volume ratio of the bacteria liquid to the sewage is defined, and so on.

Table 3 experimental design table in example 3

Note: the concentration of Pseudomonas ZZRD2 in the bacterial liquid in Table 3 is not less than 1X 10 ⁹ CFU/mL。

Sampling time: samples were taken 24h, 48h and 72h after dosing.

Measurement indexes are as follows: NO3 ^- -N. And (3) detection of nitrate nitrogen: centrifuging the bacterial liquid at 5000r.min ^-1 Centrifuging at speed for 10min, collecting supernatant, measuring by ultraviolet spectrophotometry, taking treatment 5 as blank treatment group, and calculating degradation rate, with the result as shown in FIG. 8:

from fig. 8, it can be derived that: when the volume ratio of the bacterial liquid to the wastewater is 1 ⁴ In the process, the bacterial strain grows slowly in the wastewater for 24 hours, 48 hours and 72 hours, and the degradation rates of the bacterial strain on the nitrate nitrogen in the wastewater are respectively only 12.9%, 13.7% and 12.0%; when the volume ratio of the bacterial liquid to the wastewater is 1 ³ In the process, the bacterial strain grows slowly in the wastewater for 24 hours, 48 hours and 72 hours, and the degradation rates of the bacterial strain on the nitrate nitrogen in the wastewater are respectively only 18.2%, 14.5% and 14.3%. The volume ratio of the bacterial liquid to the wastewater is 1 ² And 1, the strain grows rapidly, the degradation rate of nitrate nitrogen in the wastewater is higher, and the volume ratio of the bacterial liquid to the wastewater is 1 ² The degradation rates of the nitrate nitrogen in the wastewater for 24 hours, 48 hours and 72 hours are 47.0-58.5 percent and 60.9 percent in sequence, and the degradation rate is highest in 72 hours. When the volume ratio of the bacterial liquid to the wastewater is 1.

From the above embodiments, it follows that: the pseudomonas ZZRD2 provided by the invention has high denitrification capability, and can effectively remove nitrate in sewage.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. An aerobic denitrifying pseudomonad (Pseudomonas sp) ZZRD2 is characterized in that the preservation number of the pseudomonad ZZRD2 is CGMCC No.16778.

2. A biological denitrification bacterial agent comprising the Pseudomonas ZZRD2 of claim 1.

3. The biological denitrificaion agent as claimed in claim 2, wherein the effective component of the biological denitrificaion agent comprises one or more of pseudomonas ZZRD2 thallus, pseudomonas ZZRD2 fermentation liquor and pseudomonas ZZRD2 metabolite.

4. The biological denitrifying bacteria agent of claim 2 or 3, wherein the concentration of Pseudomonas ZZRD2 in the biological agent is 1X 10 ⁹ CFU/mL。

5. Use of the pseudomonad ZZRD2 according to claim 1 or of the biological denitrification bacterial agent according to any one of claims 2 to 4 for denitrification of wastewater.

6. The use of claim 5, wherein the contaminated water comprises nitrogen-containing contaminated water.

7. The use of claim 6, wherein the nitrogen-containing wastewater comprises one or more of domestic wastewater, industrial wastewater, farm drainage, and aquaculture wastewater.

8. A method for denitrification treatment of sewage, characterized in that the sewage is treated by the pseudomonas ZZRD2 of claim 1 or the biological denitrification microbial inoculum of any one of claims 2 to 4.

9. The method according to claim 8, wherein the denitrification treatment is carried out for 30 hours or more at a temperature of 20 to 40 ℃ and a pH of 7 to 10.

10. The method according to claim 8, wherein the carbon source for the denitrification treatment of the wastewater comprises sodium acetate and/or sodium oxalate, the C/N ratio for the denitrification treatment is 15.0 to 37.7, and the rotation speed is 100 to 160r/min.