CN111454935A - Immobilized enzyme for sewage denitrification and preparation method and application thereof - Google Patents
Immobilized enzyme for sewage denitrification and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immobilized enzyme for sewage denitrification and a preparation method and application thereof, relating to the technical field of sewage treatment, wherein the preparation method of the immobilized enzyme comprises the following steps: s1 preparing nitrifying bacteria and denitrifying bacteria for later use; s2, carrying out ultrasonic crushing on the thalli, and then centrifuging to obtain a crude enzyme extracting solution; s3 adding active carbon into the crude enzyme solution for mixing and adsorption; immobilization of the S4 enzyme: adding the enriched enzyme solution into a carrier solution containing polyvinyl alcohol and sodium alginate, and then dropwise adding the mixed solution into 2% anhydrous CaCl2In a boric acid saturated solution, standing to obtainAnd washing the immobilized pellets by using deionized water to prepare the immobilized denitrase. The immobilized enzyme can be used for removing ammonia nitrogen in sewage, and has the advantages of simple preparation method, reusability, low production cost and the like.
Description
Technical Field
The invention relates to the technical field of sewage treatment, in particular to immobilized enzyme for sewage denitrification and a preparation method and application thereof.
Background
At present, the main factor of surface water pollution is that the total nitrogen content in water is too high (the total nitrogen comprises ammonia nitrogen and nitrate nitrogen), and the result is that the water is eutrophicated, the growth amount of green algae in the water is increased, the dissolved oxygen in the water is less, and the growth of aquatic animals and plants is not facilitated. Second, ammoniacal Nitrogen (NH) in the water body3) In the prior art, one of the methods for denitrification of sewage is that the high level of ammonia in the organism is not only prevented from being discharged to the outside, but also can permeate into the organism from water, so that the metabolism of aquatic organisms is reduced or stopped, some important organs including gills are damaged, the growth and development of the organs are inhibited, and even death is caused: the ammonia nitrogen and the nitrate nitrogen in the sewage are removed by adopting a mode of combining digestive bacteria and denitrifying bacteria.
The invention patent with application number 2019103687780 discloses a device and a method for treating domestic sewage by a short-cut denitrification-sludge fermentation coupling anaerobic ammonia oxidation system, wherein the method combines the action of nitrifying bacteria and denitrifying bacteria to remove total nitrogen in the domestic sewage. The invention patent of publication No. 105084682 discloses a method for improving the efficiency of ammonia nitrogen treatment in wastewater, in which nitrifying bacteria are also used for the relevant wastewater treatment. In the process of wastewater treatment by adopting the method, although the removal efficiency of nitrate nitrogen or ammonia nitrogen is higher in the wastewater treatment process, the problem of longer water treatment time exists, and the qualified time of wastewater treatment is more than 3 days generally. This results in a less efficient wastewater treatment.
Disclosure of Invention
In view of the disadvantages of the prior art, the first object of the present invention is to provide a method for preparing an immobilized enzyme for denitrification of wastewater, which comprises the steps of culturing bacterial cells, breaking cells, extracting intracellular enzyme and immobilizing the enzyme to obtain the immobilized enzyme with high denitrification efficiency.
A second object of the present invention is to provide an immobilized enzyme for denitrification of wastewater, which has an advantage of efficiently performing denitrification of wastewater in a short time.
The third purpose of the present invention is to provide an application of immobilized enzyme for denitrification of sewage, which has the advantages of simple application to denitrification of sewage, high denitrification efficiency and short cycle.
In order to achieve the first object, the invention provides the following technical scheme: a method for preparing an immobilized enzyme for denitrification of wastewater, the method comprising the steps of:
s1 preparing nitrifying bacteria and denitrifying bacteria for later use;
obtaining a crude enzyme extracting solution of S2: breaking cells by using ultrasonic in the thallus heavy suspension in the step S1, and centrifuging to obtain supernate, namely the enzyme crude extract;
enrichment of the S3 enzyme: adding activated carbon into the enzyme crude extract, and mixing and adsorbing to obtain a mixed solution A;
immobilization of the S4 enzyme: adding the mixed solution A into a carrier solution, mixing to obtain a mixed solution B, dripping the mixed solution B into a cross-linking agent, carrying out immobilized cross-linking at 4 ℃ for 12-36 h, and then washing with deionized water to obtain immobilized denitrification enzyme; the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 5% -15%, and the mass fraction of the sodium alginate is 0-2%.
By adopting the technical scheme, the nitrifying enzyme and the denitrifying enzyme in the cells are obtained by carrying out ultrasonic disruption on the obtained nitrifying bacteria thallus and denitrifying bacteria thallus, and then the enzyme is immobilized, so that the immobilized enzyme with better denitrification effect is obtained. Firstly, crude enzyme extract containing nitrification enzyme or denitrification enzyme in cells is obtained by adopting an ultrasonic disruption mode, the ultrasonic disruption mode can be implemented industrially, and the structural damage to the enzyme is small by adopting the ultrasonic disruption mode, so that active substances in the cells still have good activity after the cells are disrupted by adopting an ultrasonic method. And then, adsorbing the crude enzyme extracting solution by using activated carbon to obtain an enzyme extracting solution with higher purity, and purifying the enzyme by adopting a milder and effective mode. And finally, further immobilizing the target nitrification enzyme and denitrification enzyme to obtain higher and more stable enzyme activity, thereby finally obtaining better sewage denitrification effect, and the sewage denitrification efficiency and ammonia nitrogen removal rate are high.
Further, the conditions of the ultrasonication in the step S2 are: the machine is intermittently processed for 30-99 times, each time lasts for 3-5 s, the interval between every two adjacent machines lasts for 3-5 s, and the crushing power is 175-225W.
By adopting the technical scheme, the denitrification effect of the obtained immobilized enzyme is better under the cell disruption mode. The structural damage of the enzyme mixture with the denitrification effect is small by the operation parameters, so that the enzyme mixture with the denitrification effect has high biological activity, and further has a good denitrification effect when the enzyme mixture with the denitrification effect is used for sewage denitrification.
Further, the centrifugation of step S2 is high-speed freezing centrifugation, and the centrifugation conditions are as follows: centrifuging at 35000-45000 Xg for 25-35 min.
By adopting the technical scheme, the enzyme mixture with the denitrification effect obtained by concentration under the centrifugal condition has higher content, so that the enzyme mixture with the denitrification effect has better sewage denitrification effect finally.
Furthermore, in the step S3, the adding amount of the activated carbon is 2-6% of the mass of the crude enzyme extracting solution, and the adsorption time is 7.5-12.5 min.
By adopting the technical scheme, under the using amount of the active carbon and the adsorption time, impurity substances in the crude enzyme extracting solution are further removed, and the mass percentage of the enzyme mixture with the denitrification effect in the rest substances is higher, so that the content of the biological active substances with the actual denitrification effect in the purified enzyme mixture with the denitrification effect is higher, and the final denitrification effect of the immobilized enzyme is better.
Further, the crosslinking agent in the step S4 is a saturated boric acid solution, and anhydrous CaCl with a mass fraction of 2% is added into the saturated boric acid solution2And the pH value of the saturated boric acid solution is adjusted to 6.5-7.0 in advance.
By adopting the technical scheme, the gel particles prepared by using the cross-linking agent have high mechanical strength, long service life and good elasticity.
Further, in the step S4, the mixing mass ratio of the mixed solution a to the carrier solution is 1: 1-5.
By adopting the technical scheme, enough target enzymes can be fixed on the unit carrier, and an excellent mass transfer performance state can be guaranteed.
Further, the obtaining of the nitrifying and denitrifying bacteria comprises the steps of:
s1-1, respectively inoculating sludge from sewage plant treatment to nitrobacteria and denitrifying bacteria culture medium for strain culture; s1-2, respectively carrying out centrifugal separation on the culture solution, and removing supernatant to obtain concentrated thallus of nitrobacteria and denitrifying bacteria; s1-3, merging the obtained thalli of the nitrifying bacteria and the denitrifying bacteria, adding a buffer solution to re-suspend the concentrated thalli, and washing for multiple times to obtain a thalli re-suspension solution for later use.
By adopting the technical scheme, the enriched nitrifying bacteria liquid and denitrifying bacteria liquid with higher concentration can be obtained.
Further, the sample inoculated to the nitrifying bacteria culture medium is cultured for 25-35 days under the condition that the pH value is 7.0-8.0, and the sample inoculated to the denitrifying bacteria culture medium is cultured for 7-15 days under the conditions that the dissolved oxygen is lower than 0.5 mg/L, the pH value is 6.5-7.5 and the culture temperature is 25-35 ℃.
In order to achieve the second object, the invention provides the following technical scheme: an immobilized enzyme for sewage denitrification, which is prepared by the method.
By adopting the technical scheme, the prepared immobilized enzyme has high mechanical strength and long service life.
In order to achieve the third object, the invention provides the following technical solutions: the application of the immobilized enzyme for sewage denitrification is disclosed.
By adopting the technical scheme, the application method is simple, and the deamination nitrogen efficiency is higher.
In conclusion, the invention has the following beneficial effects:
firstly, because the invention adopts the operations of removing impurities of intracellular extracts and immobilizing enzyme after cell disruption, the immobilized enzyme with high ammonia nitrogen removal efficiency is obtained.
Secondly, in the invention, after the nitrifying bacteria and the denitrifying bacteria are crushed preferably by adopting an ultrasonic crushing mode, a better cell crushing result is obtained, so that intracellular extracts are released, a basis is further provided for obtaining more active substances with a denitrification effect subsequently, and the denitrification efficiency of the finally obtained immobilized enzyme is higher.
Detailed Description
The present invention will be described in further detail with reference to examples.
The raw materials of the invention are all sold in the market, wherein the component of the nitrification culture medium is (NH)4)2SO4500~1000mg/L,NaHCO3800-1200 mg/L, 0.2 mg/L of calcium, iron, magnesium and other trace elements, a phosphate buffer system and KNO as the component of a denitrification medium3Is 300 to 1000 mg/L of the total weight of the composition,CH3the chemical reagent is a phosphate buffer system, wherein OH is 500-1500 mg/L, the trace elements such as calcium, iron and magnesium are 0.2 mg/L, the chemical reagents are all domestic analytical purifiers and can be purchased from Beijing chemical factories, the trace element mixed liquid can be purchased from Jiangsu Punuo Biotechnology GmbH, and the active carbon product is an analytical purifier (AR) and can be purchased from Guangzhou chemical reagent factories.
Examples
Example 1
A method for preparing an immobilized enzyme for denitrification of wastewater, the method comprising the steps of:
s1 preparing nitrifying bacteria and denitrifying bacteria for later use, which specifically comprises the following steps:
s1-1, filtering the return sludge in the secondary sedimentation tank of the sewage plant through a 30-mesh nylon net to remove larger particles and other impurities, washing and centrifuging for 2-3 times by using normal saline, and respectively inoculating the concentrated activated sludge into nitrobacteria and denitrifying bacteria culture media to carry out strain culture;
culturing the sample inoculated to the culture medium of the nitrobacteria at the temperature of 30 ℃ and under the condition that the pH value is 7.5 for 30 days, and culturing the sample inoculated to the culture medium of the denitrifying bacteria at the temperature of 30 ℃, the dissolved oxygen of 0.3 mg/L and the pH value of 7.0 for 10 days;
s1-2, respectively centrifuging the culture solution under the following conditions: centrifuging at 4 deg.C and 6000r/min for 5min, and removing supernatant to obtain concentrated thallus of nitrifying bacteria and denitrifying bacteria;
s1-3, respectively adding buffer solution into the obtained thallus of the nitrifying bacteria and the denitrifying bacteria, combining the nitrifying bacteria buffer solution and the denitrifying bacteria buffer solution containing the thallus, centrifuging for 5min at the temperature of 4 ℃ and at 6000r/min, then washing the thallus for 2 times by using the buffer solution, and then re-suspending by using the buffer solution to obtain thallus re-suspension for later use.
Obtaining a crude enzyme extracting solution of S2: and (3) breaking the cells of the thallus heavy suspension in the step (S1) in an ultrasonic breaking mode, wherein the ultrasonic breaking conditions are as follows: intermittently treating for 99 times, each time for 4s, with the middle interval of 4s and the crushing power of 200W; followed by centrifugation under the following conditions: centrifuging for 30min under the condition of 40000Xg, and obtaining supernatant fluid which is the enzyme crude extract.
Enrichment of the S3 enzyme: adding activated carbon into the crude enzyme extract to make the adding amount of the activated carbon be 4% of the mass of the crude enzyme extract, mixing and adsorbing for 10min to obtain a mixed solution A, wherein the mixed solution A contains nitrification enzyme, denitrification enzyme and part of intracellular extract.
Immobilization of the S4 enzyme: adding the mixed solution A into a carrier solution, and mixing to obtain a mixed solution B, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 10%, the mass fraction of the sodium alginate is 1%, and the mass ratio of the mixed solution A to the carrier solution is 1: 2; then the mixed solution B is dripped into CaCl containing 2 percent of anhydrous2And then curing and crosslinking the solution at 4 ℃ for 24 hours, and washing the solution for 3 to 4 times by using deionized water to prepare the immobilized denitrase, wherein the immobilized enzyme is granular.
The immobilized enzyme for sewage denitrification prepared by the method is used for sewage denitrification treatment.
Example 2
A method for preparing an immobilized enzyme for denitrification of wastewater, the method comprising the steps of:
s1 preparing nitrifying bacteria and denitrifying bacteria for later use, which specifically comprises the following steps:
s1-1, filtering the return sludge in the secondary sedimentation tank of the sewage plant through a 30-mesh nylon net to remove larger particles and other impurities, washing and centrifuging for 2-3 times by using normal saline, and respectively inoculating the concentrated activated sludge into nitrobacteria and denitrifying bacteria culture media to carry out strain culture;
culturing the sample inoculated to the culture medium of the nitrobacteria for 25 days at the temperature of 25 ℃ and under the condition that the pH value is 7.0, and culturing the sample inoculated to the culture medium of the denitrifying bacteria for 7 days at the temperature of 25 ℃, the dissolved oxygen of 0.3 mg/L and the pH value of 6.5;
s1-2, respectively centrifuging the culture solution under the following conditions: centrifuging at 4 deg.C and 5500r/min for 8min, and discarding supernatant to obtain concentrated thallus of nitrifying bacteria and denitrifying bacteria;
s1-3, respectively adding buffer solutions into the obtained thallus of the nitrifying bacteria and the denitrifying bacteria, combining the nitrifying bacteria buffer solution and the denitrifying bacteria buffer solution containing the thallus, centrifuging for 8min at the temperature of 4 ℃ and at the speed of 5500r/min, then washing the thallus for 2 times by using the buffer solution, and then re-suspending by using the buffer solution to obtain a thallus re-suspension for later use.
Obtaining a crude enzyme extracting solution of S2: and (3) breaking the cells of the thallus heavy suspension in the step (S1) in an ultrasonic breaking mode, wherein the ultrasonic breaking conditions are as follows: intermittently treating for 99 times, each time for 3s, with the interval of 3s and the crushing power of 175W; then, the mixture is centrifuged for 35min under the condition of 35000Xg, and the supernatant is the enzyme crude extract.
Enrichment of the S3 enzyme: adding activated carbon into the crude enzyme extractive solution to make the addition amount of activated carbon 2% of the crude enzyme extractive solution, mixing, adsorbing, and stirring for 7.5min to obtain mixed solution A containing nitrifying enzyme, denitrifying enzyme and part of intracellular extract.
Immobilization of the S4 enzyme: adding the mixed solution A into a carrier solution, and mixing to obtain a mixed solution B, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 5%, the mass fraction of the sodium alginate is 0.1%, and the mass ratio of the mixed solution A to the carrier solution is 1: 1; then the mixed solution B is dripped into CaCl containing 2 percent of anhydrous2And then curing and crosslinking the solution at 4 ℃ for 12h, and washing the solution for 3 to 4 times by using deionized water to prepare the immobilized denitrase, wherein the immobilized enzyme is granular.
The immobilized enzyme for sewage denitrification prepared by the method is used for sewage denitrification treatment.
Example 3
A method for preparing an immobilized enzyme for denitrification of wastewater, the method comprising the steps of:
s1 preparing nitrifying bacteria and denitrifying bacteria for later use, which specifically comprises the following steps:
s1-1, filtering the return sludge in the secondary sedimentation tank of the sewage plant through a 30-mesh nylon net to remove larger particles and other impurities, washing and centrifuging for 2-3 times by using normal saline, and respectively inoculating the concentrated activated sludge into nitrobacteria and denitrifying bacteria culture media to carry out strain culture;
culturing the sample inoculated to the culture medium of the nitrobacteria for 35 days at the temperature of 35 ℃ and under the condition that the pH value is 8.0, and culturing the sample inoculated to the culture medium of the denitrifying bacteria for 15 days at the temperature of 35 ℃, the dissolved oxygen is 0.48 mg/L and the pH value is 7.5;
s1-2, respectively centrifuging the culture solution under the following conditions: centrifuging at 4 deg.C and 6500r/min for 4min, and discarding supernatant to obtain concentrated thallus of nitrifying bacteria and denitrifying bacteria;
s1-3, respectively adding buffer solution into the obtained thallus of the nitrifying bacteria and the denitrifying bacteria, combining the nitrifying bacteria buffer solution and the denitrifying bacteria buffer solution containing the thallus, centrifuging for 4min at the temperature of 4 ℃ and 6500r/min, then washing the thallus for 2 times by using the buffer solution, and then re-suspending by using the buffer solution to obtain thallus re-suspension for later use.
Obtaining a crude enzyme extracting solution of S2: and (3) breaking the cells of the thallus heavy suspension in the step (S1) in an ultrasonic breaking mode, wherein the ultrasonic breaking conditions are as follows: intermittently treating for 30 times, each time for 5s, the middle interval for 5s, and the crushing power for 225W; then, the mixture is centrifuged for 25min under the condition of 45000Xg, and the supernatant is the enzyme crude extract.
Enrichment of the S3 enzyme: adding activated carbon into the crude enzyme extractive solution to make the addition amount of activated carbon 6 wt% of the crude enzyme extractive solution, mixing, adsorbing, and stirring for 12.5min to obtain mixed solution A containing nitrifying enzyme, denitrifying enzyme and part of intracellular extract.
Immobilization of the S4 enzyme: adding the mixed solution A into a carrier solution, and mixing to obtain a mixed solution B, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 15%, the mass fraction of the sodium alginate is 2%, and the mass ratio of the mixed solution A to the carrier solution is 1: 4; then the mixed solution B is dripped into CaCl containing 2 percent of anhydrous2Then curing and crosslinking the solution at 4 ℃ for 36h, and washing the solution for 3 to 4 times by using deionized water to prepare the immobilized productDenitrogenating enzyme, immobilized enzyme is granular.
The immobilized enzyme for sewage denitrification prepared by the method is used for sewage denitrification treatment.
Examples 4 to 16
Examples 4-16 differ from example 1 in certain process parameters during the preparation of the immobilized enzyme, as specified in table 1.
Table 1 preparation of immobilized enzymes of examples 1-16
Comparative examples 1 to 7
Comparative examples 1 to 7 differ from example 9 in that certain process parameters differ during the preparation of the immobilized enzyme, as shown in table 2.
Table 2 preparation of immobilized enzymes of comparative examples 1 to 7
The immobilized enzyme is used for removing ammonia nitrogen from wastewater, 4% of immobilized denitrogenating enzyme is added according to the mass of the wastewater, and the ammonia nitrogen removal rate reaches 75% after 48 hours.
The immobilized enzymes prepared in examples 1 to 15 and comparative examples 1 to 3 were used in simulated organic nitrogen wastewater, which had the following composition: NH (NH)4Cl is 30 mg/L335 mg/L, and 40 mg/L of glucose3COONa·3H2O is 40 mg/L4·7H2O is 1.5 mg/L4·7H2O is 1.5 mg/L, and trace elements (MnSO)4、CuSO40.5 mg/L) when the ammonia nitrogen content is 9-10 mg/L is 50-60 mg/L, the addition amount of the immobilized enzyme in the simulated organic nitrogen wastewater is 4 percent (wt percent), and the wastewater is treated by the immobilized enzymeAfter 48h of treatment, the ammonia nitrogen content of the wastewater is detected, and the result of simulating the ammonia nitrogen removal rate of the wastewater is shown in Table 3. Wherein, the detection of ammonia nitrogen adopts a Nashin reagent colorimetric method.
Table 3 removal rate of ammonia nitrogen in wastewater by immobilized enzymes of examples 1 to 16 and comparative examples 1 to 10
The data in table 3 show that example 9 is the best embodiment. In comparative example 6, when the mass ratio of the solution a to the carrier liquid was 1:0.5, the gel concentration was low, the dropped gel floated on the surface of the crosslinking agent, and became oil drops, and no beading was observed, and a stable immobilized enzyme could not be formed in the latter stage, so this scheme was not employed.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (10)
1. A preparation method of immobilized enzyme for sewage denitrification is characterized by comprising the following steps:
s1 preparing nitrifying bacteria and denitrifying bacteria for later use;
obtaining a crude enzyme extracting solution of S2: taking the thallus heavy suspension in the step S1, breaking cells by adopting ultrasonic, and centrifuging to obtain supernate, namely the enzyme crude extract;
enrichment of the S3 enzyme: adding activated carbon into the enzyme crude extract, and mixing and adsorbing to obtain a mixed solution A;
immobilization of the S4 enzyme: adding the mixed solution A into a carrier solution, mixing to obtain a mixed solution B, dripping the mixed solution B into a cross-linking agent solution, carrying out immobilized cross-linking at 4 ℃ for 12-36 h, and then washing with deionized water to obtain immobilized denitrification enzyme; the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 5% -15%, and the mass fraction of the sodium alginate is 0% -2%.
2. The method of claim 1, wherein the ultrasonication in step S2 is carried out under the following conditions: the machine is intermittently processed for 30-99 times, each time lasts for 3-5 s, the interval between every two adjacent machines lasts for 3-5 s, and the crushing power is 175-225W.
3. The method of claim 1, wherein the centrifugation of step S2 is high-speed refrigerated centrifugation, and the centrifugation conditions are as follows: centrifuging at 35000-45000 Xg for 25-35 min.
4. The method for preparing the immobilized enzyme for denitrification of sewage according to claim 1, wherein the adding amount of the activated carbon in the step S3 is 2-6% of the mass of the crude enzyme extract, and the adsorption time is 7.5-12.5 min.
5. The method of claim 1, wherein the cross-linking agent in step S4 is a saturated boric acid solution containing 2% by weight of anhydrous CaCl2And the pH value of the saturated boric acid solution is adjusted to 6.5-7.0 in advance.
6. The method of claim 1, wherein in step S4, the mixed solution A and the carrier solution are mixed at a mass ratio of 1: 1-5.
7. The method for preparing an immobilized enzyme for denitrification of wastewater according to claim 1, wherein the obtaining of nitrifying bacteria and denitrifying bacteria comprises the steps of:
s1-1, respectively inoculating sludge in sewage plant treatment to nitrobacteria and denitrifying bacteria culture media for strain culture; s1-2, respectively carrying out centrifugal separation on the culture solution, and removing supernatant to obtain concentrated thallus of nitrobacteria and denitrifying bacteria; s1-3, merging the obtained thalli of the nitrifying bacteria and the denitrifying bacteria, adding a buffer solution to re-suspend the concentrated thalli, and washing for multiple times to obtain a thalli re-suspension solution for later use.
8. The method for preparing the immobilized enzyme for denitrification of sewage according to claim 1, wherein the sample inoculated to the culture medium of nitrifying bacteria is cultured for 25-35 days under the condition of pH 7.0-8.0, and the sample inoculated to the culture medium of denitrifying bacteria is cultured for 7-15 days under the conditions of dissolved oxygen of less than 0.5 mg/L, pH 6.5-7.5 and culture temperature of 25-35 ℃.
9. An immobilized enzyme for denitrification of wastewater, wherein the immobilized enzyme is prepared by the method of any one of claims 1-8.
10. Use of the immobilized enzyme of claim 9 for denitrification of wastewater.
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Cited By (3)
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CN113716686A (en) * | 2021-08-24 | 2021-11-30 | 北京工业大学 | Device and method for quickly starting deep denitrification of landfill leachate by coupling short-cut denitrification with anaerobic ammonia oxidation |
CN114044925A (en) * | 2021-12-06 | 2022-02-15 | 西安交通大学 | Preparation method and application of polyvinyl alcohol gel material |
CN114349104A (en) * | 2021-12-20 | 2022-04-15 | 北京恩菲环保技术有限公司 | Compound activated carbon, preparation method and application thereof |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1111677A (en) * | 1994-02-01 | 1995-11-15 | 住友化学工业株式会社 | Process for production of amide compounds using microorganism |
JPH0928373A (en) * | 1995-07-24 | 1997-02-04 | Mitsubishi Electric Corp | Nitrous acid reductase and its production |
CN103013973A (en) * | 2012-12-17 | 2013-04-03 | 常州大学 | Method for preparing aerobiotic nitrifier immobilizing vector |
CN103805591A (en) * | 2014-03-07 | 2014-05-21 | 徐州工程学院 | Method for co-immobilization of nitrite bacteria-denitrifying bacteria |
CN108486006A (en) * | 2018-03-22 | 2018-09-04 | 山东省科学院生态研究所 | A kind of preparation method of greasy filth oil degradation complex enzyme and application |
CN109351177A (en) * | 2018-12-05 | 2019-02-19 | 大连理工大学 | It is a kind of using nitrous oxide as the microorganism method of denitration of primary product |
CN110482715A (en) * | 2019-09-04 | 2019-11-22 | 黄河三角洲京博化工研究院有限公司 | A method of synchronous nitration denitrification denitrogenation is realized using immobilized microorganism technique |
-
2020
- 2020-04-25 CN CN202010335921.9A patent/CN111454935A/en active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1111677A (en) * | 1994-02-01 | 1995-11-15 | 住友化学工业株式会社 | Process for production of amide compounds using microorganism |
JPH0928373A (en) * | 1995-07-24 | 1997-02-04 | Mitsubishi Electric Corp | Nitrous acid reductase and its production |
CN103013973A (en) * | 2012-12-17 | 2013-04-03 | 常州大学 | Method for preparing aerobiotic nitrifier immobilizing vector |
CN103805591A (en) * | 2014-03-07 | 2014-05-21 | 徐州工程学院 | Method for co-immobilization of nitrite bacteria-denitrifying bacteria |
CN108486006A (en) * | 2018-03-22 | 2018-09-04 | 山东省科学院生态研究所 | A kind of preparation method of greasy filth oil degradation complex enzyme and application |
CN109351177A (en) * | 2018-12-05 | 2019-02-19 | 大连理工大学 | It is a kind of using nitrous oxide as the microorganism method of denitration of primary product |
CN110482715A (en) * | 2019-09-04 | 2019-11-22 | 黄河三角洲京博化工研究院有限公司 | A method of synchronous nitration denitrification denitrogenation is realized using immobilized microorganism technique |
Non-Patent Citations (1)
Title |
---|
张德华: "《蛋白质与酶工程》", 30 September 2015, 合肥工业大学出版社 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113716686A (en) * | 2021-08-24 | 2021-11-30 | 北京工业大学 | Device and method for quickly starting deep denitrification of landfill leachate by coupling short-cut denitrification with anaerobic ammonia oxidation |
CN113716686B (en) * | 2021-08-24 | 2023-03-14 | 北京工业大学 | Method for rapidly starting deep denitrification of landfill leachate by coupling short-cut denitrification with anaerobic ammonia oxidation |
CN114044925A (en) * | 2021-12-06 | 2022-02-15 | 西安交通大学 | Preparation method and application of polyvinyl alcohol gel material |
CN114349104A (en) * | 2021-12-20 | 2022-04-15 | 北京恩菲环保技术有限公司 | Compound activated carbon, preparation method and application thereof |
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