CN112195126B - Denitrification strain, microbial agent and application - Google Patents
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Abstract
The invention discloses a denitrification bacterial strain, a microbial agent and application, wherein the denitrification bacterial strain is bacillus subtilis ZL-1 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 20555; the preservation time is 26 days at 08 months in 2020; the denitrification strain has the capabilities of both heterotrophic nitrification and aerobic denitrification, can efficiently degrade nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in nitrogen-containing sewage, can tolerate the concentrations of the nitrate nitrogen and the nitrite nitrogen as high as 2500mg/L, and is suitable for biological treatment of high-concentration nitrogen-containing wastewater.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, and particularly relates to a denitrification strain, a microbial agent and application.
Background
The traditional biological denitrification process is a nitrification-denitrification biological denitrification process, wherein the nitrification refers to that ammonia nitrogen is firstly converted into nitrite nitrogen and then converted into nitrate nitrogen under the action of nitrifying bacteria, and the denitrification process refers to that the nitrate nitrogen or the nitrite nitrogen is converted into gaseous substances such as nitrogen and the like to be separated from a water body under the action of denitrifying bacteria, so that the aim of denitrification of the water body is fulfilled. Because nitrification needs to be carried out under aerobic conditions and denitrification needs to be carried out under anoxic conditions, the traditional denitrification process usually sets nitrification and denitrification in different structures for reaction, and therefore, the traditional biological denitrification process has the defects of high capital cost, long treatment process flow and the like. Therefore, the discovery and application of a plurality of novel and efficient denitrifying microorganism strains can promote the improvement of the efficiency of the traditional biological denitrification process and the research and application of the novel biological denitrification process.
In addition, under the condition of high resistance to high-concentration nitrate nitrogen and nitrite nitrogen, the normal biological treatment method is used, and the biological denitrification reaction is completed in a longer period, so that the obtained high-concentration nitrate nitrogen resistant degradation strain has important significance for treating actual wastewater.
Disclosure of Invention
The invention aims to provide a denitrification strain, a microbial agent and application, wherein the denitrification strain has the capabilities of heterotrophic nitrification and aerobic denitrification, can efficiently degrade nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in nitrogen-containing sewage, can tolerate the nitrate nitrogen concentration of 2500mg/L, and is suitable for biological treatment of high-concentration nitrogen-containing wastewater.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a denitrification strain, which is Bacillus subtilis ZL-1 and is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, wherein the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC NO. 20555; the preservation time is 26 days at 2020 and 08 months.
Preferably, the 16S rRNA gene sequence of the denitrified strain is shown as SEQ ID NO: 1 is shown.
The invention provides a microbial agent containing the denitrification strain in a second aspect.
Preferably, the microbial agent is obtained by inoculating the denitrified strain into a culture medium and culturing.
Preferably, the carbon source in the culture medium is sodium acetate, and the nitrogen source is ammonium chloride, potassium nitrate or sodium nitrite.
The third aspect of the invention provides an application of the denitrification strain or the microbial agent in denitrification treatment of nitrogen-containing wastewater.
The invention provides a nitrogen removal treatment method of nitrogen-containing wastewater, which comprises the following steps:
and inoculating the denitrification strain or the microbial agent into nitrogen-containing wastewater for biological denitrification.
Preferably, the temperature of the wastewater is controlled to be 25-35 ℃ in the biological denitrification process.
Preferably, the pH value of the wastewater is controlled to be 6-9 in the biological denitrification process.
Preferably, the total concentration of the nitrite nitrogen or the nitrate nitrogen in the nitrogen-containing wastewater is not higher than 2500 mg/L. .
Compared with the prior art, the invention has the beneficial effects that at least:
the denitrification strain has the capabilities of both heterotrophic nitrification and aerobic denitrification, can efficiently degrade nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in nitrogen-containing sewage, can tolerate the concentrations of the nitrate nitrogen and the nitrite nitrogen as high as 2500mg/L, and is suitable for biological treatment of high-concentration nitrogen-containing wastewater.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 shows the effect of Bacillus subtilis ZL-1 on the degradation rate of nitrate nitrogen and the concentration of strains under different nitrate nitrogen concentrations in example 2 of the present invention;
FIG. 2 is a degradation curve of Bacillus subtilis ZL-1 under the condition of nitrate nitrogen of 2000mg/L in example 2 of the invention;
FIG. 3 is a graph showing the effect of Bacillus subtilis ZL-1 on the degradation rate of nitrite nitrogen and the concentration of strains under different nitrite nitrogen concentrations in example 3 of the present invention;
FIG. 4 is a degradation curve of Bacillus subtilis KK-1 in nitrite nitrogen at 2000mg/L in example 3 of the invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
This example is the isolation and characterization of a strain of Bacillus subtilis ZL-1
(A) The culture medium is separated from an anoxic denitrification tank of a certain municipal sewage treatment plant in Beijing by adopting an enrichment culture method, and comprises the following specific steps:
(1) preparing a culture medium: a250 mL triangular flask is filled with 100mL nitrate nitrogen inorganic salt culture medium mixed system which comprises culture medium solution, inorganic salt trace element solution and potassium nitrate. Adding 1.2mL of trace element solution into 1L of culture medium, and sterilizing at 120 deg.C for 30 min.
The culture medium solution is (g/L): 5g CH3COONa、1g KH2PO4、1g K2HPO4·3H2O、 0.2g MgSO4·7H2O; the trace element solution is (g/L): 1g of FeSO4·7H2O，1g MnSO4·H2O， 0.25g Na2MoO4·2H2O，0.1g H3BO4，0.25g CuCl2·2H2O，0.25g ZnCl2，0.1g NH4·VO3，0.25g Co(NO3)2·6H2O，0.1g NiSO4·6H2O, dissolved in 900mL of distilled water, 5mL of concentrated H2SO4Distilled water was added to 1000 mL.
(2) Domestication and culture: 5mL of anoxic denitrification tank activated sludge collected from a certain municipal sewage treatment plant in Beijing is inoculated into the culture medium, the culture is carried out for 2 to 3 days in a shaking table with the rotation speed of 200r/min at the temperature of 30 ℃, 5mL of bacterial liquid is inoculated into 100mL of new culture medium, and the transfer is repeated for 6 times. The concentration of nitrite nitrogen in the new culture medium is gradually increased. The initial concentration is 500mg/L, and the concentration of nitrite nitrogen in each transfer is increased by 500mg/L until 3500mg/L of nitrite nitrogen in the 6 th transfer.
(3) Separation and purification: acclimatized and cultured bacterial solution according to the formula 10-3、10-4、10-5、10-6And 10-7Diluting, respectively coating 0.1mL of each concentration of diluent on a culture medium plate containing 2000mg/L of nitrite nitrogen, carrying out inverted culture at 35 ℃ for 3-5 days, picking out a single colony, and carrying out streaking separation and purification for 3 times to obtain a purified strain ZL-1.
(B) The method comprises the following steps of classifying and identifying ZL-1 strains by a 16S rRNA gene sequencing method:
(1) preparation of total bacterial DNA: genomic DNA of strain ZL-1 was extracted using a Tiangen genome extraction kit and used as a template for PCR.
(2) RCR amplification of the 16S rRNA gene:
the following amplification primers were used
27F:5’-AgAgTTTgATCMTggCTCAg-3’[M＝C,A]
1492R:5’-CggYTACCTTgTTACgACTT-3’[Y＝T,C]
Intermediate primers: 533F 5' -gTgCCAgCMgCCgCgGgTAA-3
The PCR reaction system is shown in the following table:
the PCR reaction conditions are as follows: (94 ℃ C. for 3min) → (94 ℃ C. for 1min → 55 ℃ C. for 0.5min → 72 ℃ C. for 1min) × 30 cycles → (72 ℃ C. for 1 min).
(4) Purifying, cloning, sequencing and analyzing PCR products;
the PCR product is purified by agarose gel electrophoresis and then connected with a pMD18-T vector, transformed into escherichia coli DH5 alpha, then recombinant plasmids are extracted, and 16S rRNA gene sequences are determined, wherein the specific gene sequences are shown as SEQ ID NO: 1 is shown. The gene sequence is logged in a national information center website (http:// www.ncbi.nlm.nih.gov) of the biotechnology, nucleotide sequence Blast comparison is carried out, a plurality of nucleotide sequences which are homologous with the 16S rRNA gene sequence of related strains are obtained, and the result shows that the homology of the ZL-1 strain and the 16S rRNA gene sequence of Bacillus subtilis is more than 99 percent, so that the separated strain is identified as Bacillus subtilis.
(C) The morphological identification characteristics of the bacillus subtilis ZL-1 strain are as follows:
gram-positive bacteria, white and opaque colony, ridge and smooth and neat edge, the bacterial strain can tolerate nitrate nitrogen or nitrite nitrogen of 2500mg/L in the growth process, and the optimal growth conditions are as follows: pH7.0-8.0, and temperature 30 ℃.
Example 2
Degradation rate of bacillus subtilis ZL-1 on nitrate nitrogen with different concentrations and thallus concentration (OD600 value)
The cultures of the Bacillus subtilis ZL-1 strain were inoculated into 100mL of the above-mentioned medium containing potassium nitrate as a sole nitrogen source at pH7.0, a temperature of 30 ℃ and initial concentrations of nitrate nitrogen of 500mg/L, 1000mg/L, 1500mg/L, 2000mg/L, 2500mg/L, 3000mg/L and 3500mg/L, respectively, and sampled after shaking culture at 200r/min for 48 hours.
And (3) measuring the content of the residual nitrate nitrogen in the sample by adopting an ultraviolet spectrophotometry, and calculating the degradation rate. The initial concentration of nitrate nitrogen was plotted on the abscissa and the degradation rate of nitrate nitrogen and the OD600 value (cell concentration) were plotted on the ordinate, and the results are shown in FIG. 1.
As can be seen from the figure 1, the bacillus subtilis ZL-1 has good degradation effect on nitrate nitrogen when the concentration of the nitrate nitrogen is lower than 2000mg/L, and the degradation rate can reach more than 99%; when the concentration of the nitrate nitrogen is as high as 2500mg/L, the degradation rate can still reach more than 90 percent; when the initial concentration of the nitrate nitrogen is higher than 3000mg/L, the degradation rate of the bacillus subtilis ZL-1 to the nitrate nitrogen is gradually reduced, but the bacillus subtilis ZL-1 still has certain degradation capability.
The content of nitrate nitrogen remaining in the sample was measured by ultraviolet spectrophotometry, and a degradation curve was plotted with time as abscissa and the concentration of nitrate nitrogen remaining as ordinate, with the results shown in fig. 2.
As can be seen from FIG. 2, when the initial nitrate nitrogen concentration of the culture medium is 2000mg/L, the Bacillus subtilis ZL-1 of the invention can degrade more than 99% of the nitrate nitrogen in the culture medium within 48h, and the final nitrate nitrogen concentration in the culture solution is lower than 1 mg/L.
Example 3
Degradation rate of bacillus subtilis ZL-1 on nitrite nitrogen with different concentrations and thallus concentration (OD600 value)
The initial concentration of nitrite nitrogen is respectively 500mg/L, 1000mg/L, 1500mg/L, 2000mg/L, 2500mg/L, 3000mg/L and 3500mg/L at the pH of 7.0 and the temperature of 30 ℃, the culture of the strain ZL-1 of the bacillus subtilis is inoculated into 100mL of the culture medium which takes sodium nitrite as the only nitrogen source, and the sample is taken after shaking culture is carried out for 48h at 200 r/min.
And (3) measuring the content of the residual nitrite nitrogen in the sample by adopting an ultraviolet spectrophotometry, and calculating the degradation rate. The initial concentration of nitrite nitrogen was plotted on the abscissa, and the degradation rate of nitrite nitrogen and the OD600 value (cell concentration) were plotted on the ordinate, and the results are shown in FIG. 3.
As can be seen from the figure 3, the bacillus subtilis ZL-1 has good degradation effect on nitrite nitrogen when the concentration of the nitrite nitrogen is lower than 2000mg/L, and the degradation rate can reach more than 99%; when the nitrite nitrogen concentration is 2500mg/L, the degradation rate can still reach more than 90%; when the initial concentration of the nitrite nitrogen is higher than 3000mg/L, the degradation rate of the bacillus subtilis ZL-1 on the nitrite nitrogen is gradually reduced, but a certain degradation capability is still achieved.
And (3) measuring the content of the residual nitrite nitrogen in the sample by adopting an ultraviolet spectrophotometry, and drawing a degradation curve by taking time as a horizontal coordinate and the residual concentration of the nitrite nitrogen as a vertical coordinate, wherein the result is shown in figure 4.
As can be seen from FIG. 4, when the initial nitrite nitrogen concentration of the culture medium is 2000mg/L, the Bacillus subtilis ZL-1 of the present invention can degrade more than 99% of the nitrite nitrogen in the culture medium within 48h, and the final nitrite nitrogen concentration in the culture solution is lower than 1 mg/L.
Example 4
Degradation condition of bacillus subtilis ZL-1 on ammonia nitrogen
Inoculating the culture of the strain ZL-1 of the bacillus subtilis into 100mL of the culture medium which takes ammonium chloride as a unique nitrogen source at the pH of 7.0 and the temperature of 30 ℃ and the initial concentration of ammonia nitrogen of 1000mg/L, carrying out shaking culture at 200r/min for 48h,
measuring the content of residual ammonia nitrogen in the sample by adopting an ultraviolet spectrophotometry, and calculating the degradation rate; the calculation result shows that the ammonia nitrogen residual quantity is 1mg/L, and the ammonia nitrogen removal rate reaches 99%.
The bacillus subtilis ZL-1 can be used for treating nitrite nitrogen, nitrate nitrogen solution and ammonia nitrogen solution to degrade the nitrite nitrogen, the nitrate nitrogen and the ammonia nitrogen in the nitrite nitrogen, nitrate nitrogen and ammonia nitrogen, and can be used for biologically treating industrial wastewater containing the nitrite nitrogen, nitrate nitrogen and ammonia nitrogen. The bacillus subtilis ZL-1 can efficiently degrade nitrite nitrogen, nitrate nitrogen and ammonia nitrogen, and after the bacillus subtilis ZL-1 is inoculated in a culture liquid culture medium which takes the nitrite nitrogen and the nitrate nitrogen as nitrogen sources (for example, the concentration of sodium nitrite or potassium nitrate is 5g/L) and sodium acetate as carbon sources (for example, the concentration of sodium nitrite or potassium nitrate is 10g/L), the nitrite nitrogen and the nitrate nitrogen can be efficiently degraded within the range of 30 ℃ and pH 6-10, and the degradation rate of the nitrite nitrogen and the nitrate nitrogen within 48h can reach more than 99%.
The specific application method comprises the following steps: in a liquid culture medium which takes sodium acetate as a carbon source and sodium nitrite or potassium nitrate as a nitrogen source, bacillus subtilis ZL-1 is subjected to shake culture at 25-35 ℃ for 36-60 h, then a bacterial solution is inoculated into wastewater with the total content of nitrite nitrogen and nitrate nitrogen lower than 2500mg/L (for example, the bacterial solution can be inoculated according to the inoculation amount of 5-10%), and after the shake culture at 25-35 ℃ for 48h, more than 90% of nitrate nitrogen and nitrite nitrogen can be removed.
The following three application examples (examples 5 to 7) further illustrate the application of the bacillus subtilis ZL-1 of the present invention in the treatment of nitrite-containing nitrogen, nitrate-nitrogen solution, and ammonia nitrogen solution (in these examples, nitrite-containing nitrogen, nitrate-nitrogen, and ammonia nitrogen wastewater).
Example 5
Application of bacillus subtilis ZL-1 strain in treatment of industrial wastewater containing nitrate nitrogen
The bacillus subtilis ZL-1 is subjected to shake culture for 48h at 30 ℃ in an inorganic salt culture liquid culture medium which takes sodium acetate as a carbon source (10g/L) and potassium nitrate as a nitrogen source (5g/L), then the bacterial liquid is inoculated into wastewater of a certain chemical plant with the nitrate nitrogen content of 1100mg/L according to the inoculum concentration of 5%, the shake culture is performed for 48h at 30 ℃, the residual amount of the nitrate nitrogen is 1mg/L, and the removal rate of nitrate nitrogen reaches 99%.
The residual amount of nitrate nitrogen in a control group without adding the bacillus subtilis ZL-1 is 870mg/L, and the removal rate of nitrate nitrogen is only 21%.
Example 6
Application of bacillus subtilis ZL-1 strain in treatment of industrial wastewater containing nitrite nitrogen
The bacillus subtilis ZL-1 is subjected to shake culture for 48h at 30 ℃ in an inorganic salt culture liquid culture medium which takes sodium acetate as a carbon source (10g/L) and potassium nitrite as a nitrogen source (5g/L), then the bacterial liquid is inoculated into certain fermentation workshop production wastewater with the nitrite nitrogen content of 2500mg/L according to the inoculum concentration of 10%, the shake culture is performed for 48h at 30 ℃, the nitrite nitrogen residual quantity is 240mg/L, and the nitrite nitrogen removal rate reaches 90%.
The residual quantity of nitrite nitrogen in a control group without adding the bacillus subtilis ZL-1 is 1800mg/L, and the removal rate of nitrite nitrogen is only 28%.
Example 7:
application of bacillus subtilis ZL-1 strain in treatment of nitrogen-containing industrial wastewater
The bacillus subtilis ZL-1 is subjected to shake culture for 48 hours at 30 ℃ in an inorganic salt culture liquid culture medium which takes sodium acetate as a carbon source (10g/L) and potassium nitrate as a nitrogen source (5g/L), then the bacillus subtilis ZL-1 is inoculated into certain fine chemical production wastewater with the ammonia nitrogen content of 291mg/L, the nitrite nitrogen content of 356mg/L, the nitrate nitrogen content of 460mg/L and the total nitrogen content of 1400mg/L according to the inoculation amount of 5%, the shake culture is carried out for 48 hours at 30 ℃, the total nitrogen residual amount is 5mg/L, and the total nitrogen removal rate reaches 99%.
The total nitrogen residual quantity of the control group without adding the bacillus subtilis ZL-1 is 360mg/L, and the total nitrogen removal rate is only 26%.
In conclusion, the bacillus subtilis ZL-1 strain and the microbial agent containing the same, which are claimed by the invention, can efficiently degrade nitrite nitrogen, nitrate nitrogen and ammonia nitrogen, and the bacillus subtilis ZL-1 strain and the microbial agent containing the same are applied to the treatment of wastewater containing high-concentration nitrite nitrogen, nitrate nitrogen or ammonia nitrogen, so that the problem of low degradation rate of the nitrate nitrogen and the nitrate nitrogen in the prior art when the actual wastewater containing the high-concentration nitrite nitrogen and the nitrate nitrogen is biologically treated can be solved; in addition, the bacillus subtilis ZL-1 strain can tolerate nitrite nitrogen and nitrate nitrogen as high as 2500mg/L, can efficiently degrade the nitrite nitrogen and the nitrate nitrogen in a 2000mg/L nitrate nitrogen and nitrate nitrogen sub-solution, and can degrade ammonia nitrogen at the same time, so that the bacillus subtilis ZL-1 strain has important practical significance for biologically treating nitrogen-containing wastewater.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
SEQUENCE LISTING
<110> Shenmei science and technology Co., Ltd
<120> a denitrogenation strain, microbial inoculum and application thereof
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1376
<212> DNA
<213> Artificial
<400> 1
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agtcgagttg cagactgcga tccgaactga gaacagattt gtgggattgg cttaacctcg 180
cggtttcgct gccctttgtt ctgtccattg tagcacgtgt gtagcccagg tcataagggg 240
catgatgatt tgacgtcatc cccaccttcc tccggtttgt caccggcagt caccttagag 300
tgcccaactg aatgctggca actaagatca agggttgcgc tcgttgcggg acttaaccca 360
acatctcacg acacgagctg acgacaacca tgcaccacct gtcactctgc ccccgaaggg 420
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cacatcagac ttaagaaacc gcctgcgagc cctttacgcc caataattcc ggacaacgct 900
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Claims (9)
1. A denitrification strain is characterized in that the denitrification strain is Bacillus subtilis ZL-1 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 20555; the preservation time is 26 days after 08 months in 2020.
2. A microbial agent comprising the denitrification strain of claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent is obtained by inoculating the denitrified strain in a culture medium and culturing.
4. The microbial inoculant according to claim 3, wherein the carbon source in the culture medium is sodium acetate and the nitrogen source is ammonium chloride, potassium nitrate or sodium nitrite.
5. Use of the denitrification strain of claim 1 or the microbial agent of any one of claims 3 to 4 in denitrification treatment of nitrogen-containing wastewater.
6. A denitrification treatment method for nitrogen-containing wastewater is characterized by comprising the following steps:
the denitrification strain of claim 1 or the microbial agent of any one of claims 3 to 4 is inoculated into nitrogen-containing wastewater for biological denitrification.
7. The denitrification treatment method according to claim 6, wherein the temperature of the wastewater in the biological denitrification process is controlled to be 25-35 ℃.
8. The denitrification treatment method according to claim 6, wherein the pH of the wastewater in the biological denitrification process is controlled to 6-9.
9. The denitrification treatment method according to claim 6, wherein the total concentration of nitrite nitrogen or nitrate nitrogen in the nitrogen-containing wastewater is not higher than 2500 mg/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011119157.8A CN112195126B (en) | 2020-10-19 | 2020-10-19 | Denitrification strain, microbial agent and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011119157.8A CN112195126B (en) | 2020-10-19 | 2020-10-19 | Denitrification strain, microbial agent and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112195126A CN112195126A (en) | 2021-01-08 |
| CN112195126B true CN112195126B (en) | 2022-05-03 |
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| Isolation and denitrification characteristics of two heterotrophic nitrification-aerobic denitrifying bacteri;huang mingming等;《Environmental Science & Technology (China)》;20191231;第42卷(第12期);第123-130页 * |
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