CN112870620A - Application of chaetomium cupreum in denitration of nitrocellulose - Google Patents
Application of chaetomium cupreum in denitration of nitrocellulose Download PDFInfo
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- 229920001220 nitrocellulos Polymers 0.000 title claims abstract description 100
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 50
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 25
- 238000000034 method Methods 0.000 abstract description 19
- 230000008569 process Effects 0.000 abstract description 11
- 229910002651 NO3 Inorganic materials 0.000 abstract description 8
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 abstract description 8
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 abstract description 6
- 230000007246 mechanism Effects 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
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- 239000001913 cellulose Substances 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 description 11
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- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 4
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 4
- 238000009629 microbiological culture Methods 0.000 description 4
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 3
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- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241000219784 Sophora Species 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/06—Explosives, propellants or pyrotechnics, e.g. rocket fuel or napalm
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/26—Organic substances containing nitrogen or phosphorus
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Abstract
The invention discloses an application of chaetomium cupreum in nitrocellulose denitration. The chaetomium cupreum strain converts nitrocellulose, denitration is completed in the conversion process, nitrate and nitrite can be converted into self nutrient substances in the process, better growth of the strain is realized, and pollution is reduced; the components of the culture medium are simple, the raw materials are easy to obtain, and the method is suitable for industrial production; the single microbial strain is used for carrying out denitration treatment on the nitrocellulose, the denitrification degree can be controlled more conveniently, and good bedding is carried out for the later mechanism research of reducing the nitrogen of the nitrocellulose. The method does not need alkaline hydrolysis pretreatment before denitration conversion, and does not damage the main chain of cellulose; the high and large hair shells do not need an anaerobic environment in the denitration process of the nitrocellulose, and the conversion environment is relatively simple; after the mixed culture is cultured with the nitrocellulose for 14 days, the nitrogen content of the nitrocellulose with the original nitrogen content of 13.03 percent can be reduced to 10.43 percent.
Description
Technical Field
The invention belongs to the fields of environmental engineering and biology, and relates to an application of chaetomium cupreum in denitration of nitrocellulose.
Background
The nitrocellulose is a high molecular polymer material formed by esterifying cellulose and concentrated nitric acid, has wide application field, and can be applied to paint, celluloid, propellant and the like. Nitrocellulose monomer C6H7O2(ONO2)3The nitrogen content was 14.1%. Nitrocellulose with too high a nitrogen content is unstable in performance and extremely explosive, so it is essential to find a method for reducing the nitrogen content of nitrocellulose.
Chemically, the nitrogen reduction treatment mode of the nitrocellulose is alkaline hydrolysis, nitrate and nitrite generated by the alkaline hydrolysis cause great pollution to soil and water, and in addition, the reaction degree is difficult to control by a chemical method, so that excessive nitrogen reduction is easy to occur, and the performance of the nitrocellulose as a material is influenced. The biological method has few reports on the nitrogen reduction treatment of the nitrocellulose. In the existing reports of nitrogen reduction experiments of nitrocellulose by a biological method, the degradation of nitrocellulose by microorganisms is influenced by the steric hindrance of nitro groups, so that the degradation can not directly act on the main chain of nitrocellulose, and the performance of nitrocellulose can not be influenced. Most of the biological methods reported in the prior art use mixed bacteria microorganisms to act on the microorganisms, for example, nitrocellulose is treated by the microorganisms in sludge, and the effect of reducing nitrogen is found to be certain. However, the method is complicated, the mechanism of the microorganism for reducing the nitrogen content of the nitrocellulose is not clear, and the requirement of industrial treatment is difficult to achieve. Moreover, microorganisms in the sludge mostly perform anaerobic respiration and facultative anaerobic respiration, which is inconvenient for researching the specific nitrogen reduction mechanism. Therefore, the denitration and nitrogen reduction of the nitrocellulose by using the single microbial strain not only makes up for the defect of environmental pollution by a chemical method, but also can research the denitration mechanism of the single microorganism.
Disclosure of Invention
Aiming at the defects of the prior art, the application of chaetomium cupreum in the denitration of nitrocellulose can convert nitrate and nitrite into self nutrient substances in the denitration process of the nitrocellulose, and the culture medium has simple components, is a simple potato culture medium, has easily obtained raw materials and simple components, and is suitable for industrial production; and the chaetomium cupreum strain is a fungus, has strong growth capacity, is not easy to contaminate and has the advantages of the strain. The single bacterial strain is used for carrying out nitrogen reduction research on the nitrocellulose, so that the method is more convenient, and the mechanism of nitrogen removal and nitrogen reduction is easier to research in the later period.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
an application of chaetomium cupreum in the denitration of nitrocellulose is characterized in that the chaetomium cupreum (Chaetomium elatum) The preservation number of (2) is CGMCC NO. 3.5617.
The characteristics of this strain are as follows:
(1) colony characteristics: the colony is in a loose or tight villiform shape on the culture medium, the surface of the colony is white, and spores are easy to scatter; the hyphae are thick and long, spread without limitation and can extend to the whole culture dish;
(2) the nitrate culture experiment of the strain is positive, can perform denitrification, and can utilize nitrate nitrogen and nitrite nitrogen for growth.
The application comprises the following steps:
step 1, culturing the strain and preparing spore suspension
Activating a chaetomium cupreum strain on a potato agar culture medium, taking a ring of spores by using an inoculating ring, inoculating the ring of spores into 1 mL of sterile water to prepare a spore suspension, and adding 200 mu L of the spore suspension into 100mL of liquid potato culture medium for culture;
step 2, conversion of substrate
When the bacterial strain is cultured until a large number of mycelium spheres appear and the color of the culture solution is red, adding nitrocotton to enable the final concentration to be 4g/L, and carrying out culture conversion;
step 3, re-extraction of the substrate
After the culture conversion is finished, the culture solution is filtered, and the organic solution is used for re-extracting the nitrocotton.
The suction filtration replaces the centrifugation to reduce the loss rate, because the nitrocotton is too light, the heavy extraction process is generally centrifuged at 8000rpm for 15min, but because the nitrocotton is too light, the rotating speed is difficult to be completely centrifuged and precipitated, the rotating speed is increased, the operation risk is increased, so that the loss rate of the heavy extraction of the nitrocotton is higher, and the suction filtration has important significance for the heavy extraction.
As a modification, the ingredient of the potato culture medium in the step 1 is 20.0 of glucose in g/L; 1000 mL of potato leachate.
In a further improvement, in the potato leachate obtained in the step 1, 200 g of potatoes are added with 1000 mL of distilled water for boiling, and the filtrate is obtained by filtration and is supplemented with the distilled water to 1000 mL.
As a refinement, the transformation of step 2 is specifically as follows: weighing 0.2g of nitrocotton, and adding the nitrocotton into 10mL of sterile deionized water; sterilizing the soaked nitrocotton with high-temperature high-pressure steam at 115 ℃ for 20min to obtain sterile nitrocotton solution, and drying the nitrocotton under the sterile condition to obtain sterile 0.2g of nitrocotton because the nitrocotton is insoluble in water; and thirdly, adding sterile 0.2g of nitrocotton into the culture solution which has a large amount of mycelial spheres and turns red, so that the final concentration of the nitrocotton is 4g/L for culture and transformation.
The improvement is that after the suction filtration in the step 3 is finished, the sediment on the filter paper is taken; washing the precipitate with ultrapure water and 1M NacL solution in sequence, and repeating the washing for three times; after washing, carrying out suction filtration to obtain a precipitate; adding 40ml of acetone into 1g of the precipitate, adding an acetone solution into the precipitate, dissolving for 2 hours at 40 ℃, and centrifuging to obtain a supernatant; gradually dripping a solution mixed by water and ethanol according to the volume ratio of 2:1 into the supernatant until the nitrocellulose is not separated out any more; drying the separated nitrocellulose, weighing, calculating the loss rate, and calculating according to the weight ratio of the dried nitrocellulose/0.2 extracted from each 100mL of culture solution; the precipitated nitrocellulose was dried and analyzed for N content using an elemental analyzer having an instrument model of Elemantar: Vario EL Cube.
As a modification, the temperature of the culture transformation in the step 2 is 28 ℃, and the pH value in the system is 7.0-7.5.
Has the advantages that:
the invention utilizes chaetomium cupreum (Chaetomium elatum) Preliminary experiments on the denitration and denitrification of the nitrocellulose show that at the temperature of 28 ℃ and the pH value of 7.0-7.5, the bacterial strain can effectively reduce the nitrogen content of the nitrocellulose, and nitrate nitrogen and nitrite nitrogen generated by the nitro-group removal of the nitrocellulose are converted into nitrogen sources for utilization, so that the bacterial strain grows better, better denitration and denitrification effects are generated, the bacterial strain is circulated and reciprocated, no harmful nitrate or nitrite is generated, and green and clean are realized. Therefore, the single microbial strain is used for carrying out denitrification and denitration treatment on the nitrocellulose, the denitrification degree of the nitrocellulose can be more conveniently controlled, a green and clean treatment mode is realized in the process, and a good bedding is provided for the later research on the mechanism of reducing the nitrogen of the nitrocellulose.
Compared with the prior art, the invention has the following advantages:
(1) the chaetomium cupreum (provided by the invention)Chaetomium elatum) Before denitration, alkaline hydrolysis pretreatment is not needed, and the main chain of cellulose is not damaged;
(2) the chaetomium cupreum (provided by the invention)Chaetomium elatum) The process of denitrification and denitration treatment of the nitrocellulose can be carried out by oxygen, and an anaerobic environment is not needed; the nitrate nitrogen and nitrite nitrogen after denitrification in the culture medium can be used as nitrogen sources to realize the growth of the strains so as to better realize denitrification and denitration;
(3) the chaetomium cupreum (provided by the invention)Chaetomium elatum) The denitrification treatment and the denitrification treatment of the nitrocellulose do not produce harmful nitrate and nitrite and do not pollute water quality and soil.
Drawings
FIG. 1 shows chaetomium officinarum (Chaetomium elatum) A comparison graph of denitrification results of nitrocellulose under different culture days;
FIG. 2 shows Chaetomium fortunei (Chaetomium elatum) In thatFTIR comparison plots of denitrogenation of nitrocellulose at different culture days;
FIG. 3 shows Chaetomium fortunei (Chaetomium elatum) The method comprises the steps of denitrification process diagram of nitrocellulose, A-substrate co-culture, B-acetone dissolution and extraction, C-organic solvent separation, D-suction filtration, E-weighing and F-element analysis.
Detailed description of the preferred embodiments
Sulfate reducing bacteria Chaetomium cupreum used in the inventionChaetomium elatum) Purchased from China general microbiological culture Collection center (CGMCC). The processing steps not mentioned in the following examples are all conventional in the art.
Example 1: preparation of spore suspension and culture of strain
The research uses a chaetomium cupreum (which is from China general microbiological culture Collection center and has the preservation number of CGMCC NO: 3.5617)Chaetomium elatum) The culture medium is inoculated from the slant of the test tube, and the inoculation mode adopts spore suspension for inoculation.
Preparation of spore suspension: a chaetomium cupreum (with the preservation number of CGMCC NO: 3.5617) from the China general microbiological culture collection centerChaetomium elatum) One loopspore was collected from the tube slant with a loop and inoculated into 1 mL of sterile water to prepare a spore suspension.
Liquid culture of the strain: 200 μ L of spore suspension was added to 100mL of liquid potato medium for cultivation.
The potato culture medium comprises the following components: glucose 20.0, unit g/L; potato leachate 1000 mL (200 g potato, 1000 mL distilled water added boiling, filtering to get filtrate, supplementing 1000 mL with distilled water.)
The culture temperature is 28 ℃, and the culture pH is 7.0-7.5; after 2 days of culture, cells were visually observed.
The above-cultured strains are described:
(3) colony characteristics: the colony is in a loose or tight villiform shape on the culture medium, the surface of the colony is white, and spores are easy to scatter; the hyphae are thick and long, spread without limitation and can extend to the whole culture dish;
(4) the nitrate culture experiment of the strain is positive, can perform denitrification, and can utilize nitrate nitrogen and nitrite nitrogen for growth.
Example 2: chaetomium fortunei on different days (Chaetomium elatum) Comparison of Nitrogen reduction results on Nitrocellulose
A strain of chaetomium cupreum (CGMCC NO: 3.5617) purchased from China general microbiological culture collection center with the collection number of CGMCC NOChaetomium elatum) Taking a circumsporozoite from the inclined plane of the test tube by using an inoculating loop, inoculating the circumsporozoite into 1 mL of sterile water to prepare a spore suspension, and then adding 200 mu L of the spore suspension into 100mL of liquid potato culture medium for culture. Culturing at 28 deg.C in shaking table at 200rpm for 2 days until macroscopic thallus is generated, adding 4g/L nitrocellulose as substrate, and performing substrate conversion reaction with high chaetomium cupreum strain.
After the conversion reaction is carried out for 7 days, the cultured substrate nitrocellulose is re-extracted by using a suction filtration device, so that the loss rate of the nitrocellulose is reduced. After the suction filtration is finished, taking a precipitate on the filter paper; washing the precipitate with ultrapure water and 1M NacL solution in sequence, and repeating the washing for three times; after washing, carrying out suction filtration to obtain a precipitate; adding acetone solution (40 mL acetone is added into 1g of precipitate), dissolving for 2 h at 40 ℃, and centrifuging to obtain supernatant; gradually dripping a solution mixed by water and ethanol according to the volume ratio of 2:1 into the supernatant until the nitrocellulose is not separated out any more; drying the separated nitrocellulose, weighing and calculating the loss rate, and calculating according to the weight extraction of the dried nitrocellulose in each 100mL of culture solution)/0.2; the precipitated nitrocellulose was dried and analyzed for N content using an elemental analyzer having an instrument model of Elemantar: Vario EL Cube. The original medicine nitrocellulose is detected by using an element analyzer in chaetomium cupreum ()Chaetomium elatum) Since a decrease in the nitrogen content was observed in the nitrocellulose after 7 days of treatment, the experiment was repeated and the culture time was extended to see whether the nitrogen reduction effect was further increased with the lapse of time in order to verify whether the nitrocellulose had the nitrogen reduction effect.
After the culture for 2-8 days is finished, the re-extracted nitrocellulose is subjected to element analysis,discovery of Chaetomium Sophora Gagnep: (Chaetomium elatum) It does have a nitrogen-reducing effect on nitrocellulose, and the nitrogen-reducing effect is more pronounced with increasing time, as shown in detail in figure 1.
Example 3: chaetomium fortunei on different days (Chaetomium elatum) FTIR results of treated nitrocellulose were compared.
And (3) comparing the nitrocellulose treated by the sulfate reducing bacteria on different days with the original nitrocellulose by Fourier infrared spectra. 1635 cm-1Is formed by-ONO2Asymmetric stretching vibration, 1274 cm-1Is formed by-ONO2Symmetric telescopic vibration, 1061 cm-1Is in C-O-C telescopic vibration at 996 cm-1Is subjected to C-O-C telescopic vibration of 824 cm-1C-O-C stretching vibration is adopted. It was found that the major groups of nitrocellulose did not disappear and no new groups were produced, and the absorption peak of nitro groups was found in chaetomium cupreum ((C.))Chaetomium elatum) There was a significant decrease after treatment and the decrease was more pronounced the longer the time, see figure 2 for details.
The application of the chaetomium cupreum strain in denitrification and denitration of nitrocellulose realizes the co-transformation of the strain and a substrate, so that the denitration process of nitrocotton is completed in the growth and culture process of the strain, and the transformation process of the substrate is simplified; the chaetomium officinarum strains can take the toxic substances to the environment, such as nitrate and nitrite generated by the denitrification of the nitrocellulose, as the nutrient sources of the strains, so that the whole process is green and clean; the invention replaces the centrifugation in the re-extraction process with the suction filtration device, thereby not only improving the re-extraction efficiency of the nitrocotton, but also being more convenient for suction filtration than centrifugation and more beneficial to industrialization.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (7)
1. Application of chaetomium cupreum in nitrocellulose denitration and application thereofCharacterized in that the chaetomium officinarum (Chaetomium elatum) The preservation number of (2) is CGMCC NO. 3.5617.
2. The application of chaetomium cupreum in nitrocellulose denitration according to claim 1, is characterized by comprising the following steps:
step 1, culturing the strain and preparing spore suspension
Activating a chaetomium cupreum strain on a potato agar culture medium, taking a ring of spores by using an inoculating ring, inoculating the ring of spores into 1 mL of sterile water to prepare a spore suspension, and then adding 200 mu L of the spore suspension into 100mL of liquid potato culture medium for culture;
step 2, conversion of substrate
When the bacterial strain is cultured until a large number of mycelium spheres appear and the color of the culture solution is red, adding nitrocotton to enable the final concentration to be 4g/L, and carrying out culture conversion;
step 3, re-extraction of the substrate
After the culture conversion is finished, the culture solution is filtered, and the organic solution is used for re-extracting the nitrocotton.
3. The use of chaetomium aurantiacum in the denitrification of nitrocellulose according to claim 2, wherein the potato medium in step 1 comprises glucose 20.0 in g/L; 1000 mL of potato leachate.
4. The use of chaetomium gaurea for denitration of nitrocellulose as claimed in claim 3, wherein the potato leachate is prepared by boiling 200 g of potatoes in 1000 mL of distilled water, filtering to obtain a filtrate, and supplementing the filtrate with distilled water to 1000 mL.
5. The application of chaetomium cupreum in denitration of nitrocellulose as claimed in claim 2, wherein the transformation in step 2 is specifically as follows: weighing 0.2g of nitrocotton, and adding the nitrocotton into 10mL of sterile deionized water; sterilizing the soaked nitrocotton with high-temperature high-pressure steam at 115 ℃ for 20min to obtain sterile nitrocotton solution, and drying the nitrocotton under the sterile condition to obtain sterile 0.2g of nitrocotton because the nitrocotton is insoluble in water; and thirdly, adding sterile 0.2g of nitrocotton into the culture solution which has a large amount of mycelial spheres and turns red, so that the final concentration of the nitrocotton is 4g/L for culture and transformation.
6. The application of chaetomium cupreum in denitration of nitrocellulose as claimed in claim 2, wherein after the suction filtration in step 3 is finished, a precipitate on filter paper is taken; washing the precipitate with ultrapure water and 1M NacL solution in sequence, and repeating the washing for three times; after washing, carrying out suction filtration to obtain a precipitate; adding 40ml of acetone into 1g of the precipitate, adding an acetone solution into the precipitate, dissolving for 2 hours at 40 ℃, and centrifuging to obtain a supernatant; gradually dripping a solution mixed by water and ethanol according to the volume ratio of 2:1 into the supernatant until the nitrocellulose is not separated out any more; drying the separated nitrocellulose, weighing, calculating the loss rate, and calculating according to the weight ratio of the dried nitrocellulose/0.2 extracted from each 100mL of culture solution; the precipitated nitrocellulose was dried and analyzed for N content using an elemental analyzer having an instrument model of Elemantar: Vario EL Cube.
7. The use of chaetomium aurantiacum in the denitrification of nitrocellulose according to claim 2, wherein the temperature for culturing and transforming in step 2 is 28 ℃, and the pH value in the system is 7.0-7.5.
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| CN102041291A (en) * | 2009-10-21 | 2011-05-04 | 中国石油化工股份有限公司 | Method for screening denitrifying bacterium |
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| CN108330081A (en) * | 2017-11-27 | 2018-07-27 | 天津工业大学 | One plant of heterotrophic nitrification-aerobic denitrification bacterium and its application |
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| CN102041291A (en) * | 2009-10-21 | 2011-05-04 | 中国石油化工股份有限公司 | Method for screening denitrifying bacterium |
| CN104250625A (en) * | 2014-05-28 | 2014-12-31 | 北京市环境保护科学研究院 | Aerobic denitrifying bacterium strain and application thereof to water denitrification |
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