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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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 44
- 239000010865 sewage Substances 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 68
- 102000004190 Enzymes Human genes 0.000 claims abstract description 68
- 241000894006 Bacteria Species 0.000 claims abstract description 62
- 239000000243 solution Substances 0.000 claims abstract description 53
- 239000011259 mixed solution Substances 0.000 claims abstract description 32
- 230000001546 nitrifying effect Effects 0.000 claims abstract description 29
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000005406 washing Methods 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000002156 mixing Methods 0.000 claims abstract description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 11
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 11
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 11
- 239000000661 sodium alginate Substances 0.000 claims abstract description 11
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 11
- 241001052560 Thallis Species 0.000 claims abstract description 7
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000008367 deionised water Substances 0.000 claims abstract description 6
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 6
- 238000001179 sorption measurement Methods 0.000 claims abstract description 4
- 239000001110 calcium chloride Substances 0.000 claims abstract 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 32
- 239000002351 wastewater Substances 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 241000108664 Nitrobacteria Species 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 9
- 239000010802 sludge Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 239000000287 crude extract Substances 0.000 claims description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000003431 cross linking reagent Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 2
- 238000002525 ultrasonication Methods 0.000 claims description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 14
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000004327 boric acid Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000008188 pellet Substances 0.000 abstract 1
- 239000012047 saturated solution Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 17
- 239000000203 mixture Substances 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 238000012258 culturing Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 239000012535 impurity Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 238000004065 wastewater treatment Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000011573 trace mineral Substances 0.000 description 4
- 235000013619 trace mineral Nutrition 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 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 3
- 239000013543 active substance Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 241000195628 Chlorophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 210000002816 gill Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- Genetics & Genomics (AREA)
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Molecular Biology (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
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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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