CN113528369A - Preparation method and application of synchronous nitrification and denitrification bacterial agent - Google Patents
Preparation method and application of synchronous nitrification and denitrification bacterial agent Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000001360 synchronised effect Effects 0.000 title claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 title claims description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 70
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 50
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 17
- 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 16
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 16
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 16
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 16
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims abstract description 16
- 229960004853 betadex Drugs 0.000 claims abstract description 16
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 16
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 16
- 239000000661 sodium alginate Substances 0.000 claims abstract description 16
- 230000004913 activation Effects 0.000 claims abstract description 15
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 12
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000000725 suspension Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 14
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 13
- 229960002413 ferric citrate Drugs 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 239000011573 trace mineral Substances 0.000 claims description 5
- 235000013619 trace mineral Nutrition 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000007836 KH2PO4 Substances 0.000 claims 1
- 238000001816 cooling Methods 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 15
- 239000001301 oxygen Substances 0.000 abstract description 12
- 229910052760 oxygen Inorganic materials 0.000 abstract description 12
- 238000005516 engineering process Methods 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 10
- 239000008188 pellet Substances 0.000 abstract description 7
- 239000010865 sewage Substances 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 238000004065 wastewater treatment Methods 0.000 abstract description 3
- 239000000969 carrier Substances 0.000 abstract description 2
- 238000010276 construction Methods 0.000 abstract description 2
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 abstract 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 229910002651 NO3 Inorganic materials 0.000 description 24
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 23
- 239000000203 mixture Substances 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 11
- 238000004140 cleaning Methods 0.000 description 10
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 4
- 239000010802 sludge Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000002798 spectrophotometry method Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241001057811 Paracoccus <mealybug> Species 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 241000589597 Paracoccus denitrificans Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000006149 azo coupling reaction Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013065 commercial product Substances 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
- 238000006731 degradation reaction Methods 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- -1 iron ions Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- 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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- 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
- C02F3/30—Aerobic and anaerobic processes
- C02F3/302—Nitrification and denitrification treatment
- C02F3/305—Nitrification and denitrification treatment characterised by the denitrification
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- 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
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- 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
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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Abstract
The invention discloses a preparation method of a synchronous nitrification and denitrification microbial agent and application thereof in wastewater treatment, belonging to the technical field of denitrification microbial application. The technical method comprises the steps of embedding denitrifying bacteria into spheroids with sodium alginate and beta-cyclodextrin as carriers and ferric sulfate as a cross-linking agent at the temperature of 4 ℃, wherein the diameters of the spheroids are 2-5 mm, and the spheroids are cleaned by normal saline and then dried in the air to obtain the anaerobic denitrifying bacteria agent. The technology mainly comprises the following steps: activation of denitrifying bacteria, enlarged culture of denitrifying bacteria, preparation of immobilized pellets, and use conditions and effects of denitrifying bacteria agent. The raw materials required by the preparation method are environment-friendly and degradable, and the requirements on the preparation process and equipment are simple. The synchronous nitrification and denitrification microbial inoculum obtained by the invention has an obvious denitrification effect under the low-oxygen condition, can simultaneously nitrify and denitrify in the same reactor, overcomes the defects of the traditional process, reduces the capital construction and operation cost in the sewage treatment process, and has a better application prospect.
Description
Technical Field
The invention belongs to the technical field of wastewater treatment, relates to biological denitrification and bacteria immobilization technology, and particularly relates to a preparation method and application of a synchronous nitrification and denitrification microbial agent.
Background
The existing sewage treatment is taken off at presentThe nitrogen process mostly adopts the traditional activated sludge method, the total nitrogen removal is mostly realized through the ammoniation, nitrification and denitrification processes of microorganisms, the technology means that the denitrification process must be separately realized, namely the nitrification and denitrification are carried out in two different reactors, the nitrification liquid also needs to flow back, the process is complex and the operation cost is high, and meanwhile, the dominant bacteria are difficult to keep due to the fluidity and the suspension state of the bacteria when the operation reactors are debugged, and the dominant bacteria easily flow out along with the fluid, so that the debugging operation difficulty is large, and the period is long. With the most widely used A2And O is taken as an example, ammonia nitrogen is converted into nitrate through the nitrification of an aerobic tank, the mixed solution of the aerobic tank flows back to enter an anoxic tank, and the nitrate is converted into nitrogen by denitrifying microorganisms in the anoxic tank under the anaerobic or anoxic condition, so that the biological denitrification process of the sewage is completed. The process has high capital cost, complex design of sewage, sludge and a reflux system, large energy consumption and inconvenient management. In addition, the concentration of dissolved oxygen in the mixed liquid refluxed by the aerobic tank is higher, so that the anoxic denitrification effect is poor.
Analysis shows that the existing denitrification technology has unsatisfactory effect, the effluent hardly reaches the new discharge standard of total nitrogen, the traditional technology needs to be upgraded urgently, and the root cause of the problem of the existing sewage treatment system is the interference of dissolved oxygen to the denitrification process. Synchronous nitrification and denitrification refers to synchronous nitrification reaction and denitrification reaction in the same reactor. In which denitrification may be directly effected by nitration of the converted NO2-N without conversion of ammonia nitrogen to NO3N, reduced energy consumption, and reduced oxygen requirements. The simultaneous nitrification and denitrification in the same reactor can overcome the defects of the traditional process and save the capital construction and operation cost.
The immobilized microorganism technology is a biological technology which fixes specially selected microorganisms on a carrier selected for evidence so that the microorganisms are highly dense and keep biological activity and can rapidly proliferate in large quantities under proper conditions. Commonly used immobilization techniques are adsorption, entrapment, cross-linking and membrane entrapment. The technology is applied to wastewater treatment, can solve the problems that functional bacteria in a bioreactor are difficult to culture, low in concentration, difficult to become dominant bacteria and the like, and has the advantages of fast startup of the bioreactor, high treatment efficiency, strong impact resistance, easy separation of sludge and water and shortening of the time required by treatment.
The method utilizes immobilized microorganism technology, utilizes sodium alginate and beta-cyclodextrin to embed anaerobic denitrifying bacteria, and utilizes diffusion resistance of carriers and iron ions to oxygen to form an anoxic zone and an anaerobic zone inside particles, so that denitrifying bacteria can survive and metabolize, and the anaerobic denitrifying bacteria can obtain higher denitrifying effect even under aerobic conditions. The method has the advantages of high strain capacity, small influence on strain activity, and simple operation. The embedding material is natural high molecular polysaccharide, and the prepared immobilized cell ball has high strength, high reaction rate and high degradation efficiency.
Disclosure of Invention
The invention provides a preparation method of a synchronous nitrification and denitrification microbial agent and application thereof, aiming at the defects of high capital cost, complex design of sewage, sludge and a reflux system, large energy consumption, inconvenient management, large influence of oxygen on the traditional denitrification effect and the like in the traditional biological denitrification technology. The immobilized denitrifying bacteria pellet prepared by the method can prolong the retention time of denitrifying bacteria in a system, reduce and avoid the interference of external factors, particularly oxygen, on the bacteria, and can effectively improve the aerobic denitrification effect.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of a synchronous nitrification and denitrification bacterial agent comprises the following steps:
firstly, preparation of embedding strain suspension: under the anaerobic condition, the denitrifying bacteria strain preserved on the inclined plane is inoculated on an activation culture medium and cultured for 18-24h under the conditions of the temperature of 25-35 ℃ and the temperature of 130-; selecting strains on the activation culture medium, inoculating the strains on the fermentation culture medium, and carrying out culture for 20-28h under the conditions of 25-35 ℃ and 130-. And (3) centrifuging the bacterial liquid or the concentrated liquid for 15min at the rotating speed of 3000-5000 r/min, pouring out supernatant, centrifuging and washing the supernatant for 2-3 times by using normal saline, diluting the supernatant by using the normal saline until the OD600 value is about 0.3-0.5, and storing the supernatant at 0-10 ℃ for later use.
The activation medium is a liquid LB medium, and the specific formula is as follows: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast powder and 7.0-7.2 of pH.
The fermentation medium comprises the following specific components:
5-28 g/L of organic carbon source and MgSO4·7H2O0.1g/L，Na2HPO4·2H2O7.9g/L，KH2PO41.5g/L，KNO3/KNO22-6 g/L, 1mL/L of trace elements, adjusting the pH to be about 7.2, and sterilizing the culture medium before use.
Secondly, preparing strain immobilized beads: adding the prepared bacterial suspension into embedding agent, stirring and mixing uniformly, then adding the mixed solution into ferric citrate solution cross-linking agent drop by drop, crosslinking for 12-36h at 0-10 ℃, cleaning with sterile distilled water, and drying to obtain spherical aerobic denitrifying bacteria agent; the embedding medium is a mixed solution containing 1-4% by mass of sodium alginate and 1-4% by mass of beta-cyclodextrin, the sodium alginate and the beta-cyclodextrin are added into water, dissolved and mixed uniformly, sterilized at high temperature for 20min, and cooled to obtain the iron citrate cross-linking agent.
The denitrifying bacteria are anaerobic denitrifying bacteria which can be obtained by purchasing or manual screening, the obtained Paracoccus denitirichicans is facultative anaerobic denitrifying bacteria, and the volume ratio of the strain suspension to the embedding medium is 1 (10-50). The CaCl is2The mass concentration of the solution cross-linking agent is 1-4%. The grain size of the small spherical aerobic denitrifying bacteria agent is 2-5 mm.
The aerobic denitrifying bacteria agent prepared by the preparation method of the aerobic denitrifying bacteria agent and the application of the aerobic denitrifying bacteria agent in denitrification.
Comprises the following specific steps
Cleaning the prepared spherical microbial inoculum with distilled water, pouring the cleaned spherical microbial inoculum into a conical flask containing a denitrification culture medium (or nitrate-containing wastewater) according to the volume ratio of 1 (5-15) (the volume ratio of the aerobic denitrification microbial inoculum to the denitrification culture medium), placing the conical flask into a constant-temperature shaking table, carrying out open aerobic culture at the temperature of 28-30 ℃ and under the condition of 180r/min under 150-And (3) measuring the contents of nitrate and nitrite in the sample by a nitrogen coupling spectrophotometry to obtain the denitrification effect. The formulation of the denitrification medium used in the above experiment was as follows: 0.35g of NH4Cl、1.50g KNO33.0g of glucose, 7.04g of Na2HPO4、1.46g KH2PO4、0.06g MgSO4And 0.06mL of trace elements are dissolved in 600mL of sterile water and are uniformly mixed to obtain the denitrification culture medium.
The invention has the beneficial effects that: the spherule aerobic denitrifying bacteria agent prepared by the method isolates bacteria from an oxygen-containing culture medium (or aqueous solution), and the inside of the gel spherule of the aerobic denitrifying bacteria agent has the limitation on oxygen diffusion, so that an aerobic area in a large environment and an anoxic and anaerobic area in an internal microenvironment are formed, anaerobic denitrification of paracoccus denitrificans is facilitated, and high denitrification effect under the condition of oxygen existence is ensured. Meanwhile, the sodium alginate adopted by the invention can keep higher activity of microorganisms, and has the characteristics of large adsorption capacity, good mechanical strength, biodegradability and high biological activity; the beta-cyclodextrin can reduce the hydrogen bonding effect between molecules, thereby reducing the strength of the blend membrane, improving the elongation of the blend membrane and having good hygroscopicity. The combination of the two makes the prepared gel pellet have higher strength and activity.
The ferric citrate adopted by the method as a cross-linking agent is beneficial to improving the barrier property, the mechanical property and the water resistance of the film, and the gel property of sodium alginate and beta-cyclodextrin is enhanced, wherein ferric ions can effectively block oxygen.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are intended for purposes of illustration and explanation only and are not intended to limit the scope of the invention; wherein, the anaerobic denitrifying bacteria Paracoccus denitirichicans is a commercial product.
Example 1
A preparation method of a synchronous nitrification and denitrification bacterial agent comprises the following steps:
1. preparation of embedded strain suspension: under the anaerobic condition, inoculating the denitrifying bacteria strain preserved on the inclined plane on an activation culture medium, and culturing for 25h at the temperature of 25-35 ℃ and under the condition of 130-; selecting strains on the activation culture medium, inoculating the strains on the fermentation culture medium, and culturing for 24h under the conditions of 25-35 ℃ and 130-. Centrifuging the bacterial solution or concentrated solution at rotation speed of 3800r/min for 15min, removing supernatant, washing with normal saline for 3 times, diluting with normal saline to OD600 value of about 0.3, and storing at 4 deg.C.
2. Preparing a strain immobilized pellet: and (3) mixing the bacterial suspension prepared in the step (1): adding the mixture into an embedding agent according to a proportion of 40, stirring and mixing uniformly, then adding the mixed solution into a ferric citrate solution cross-linking agent drop by drop, sealing the mixture at the temperature of 4 ℃, placing the mixture in a refrigerator for cross-linking for 24 hours, and then cleaning and drying the mixture by using sterile distilled water to obtain a spherical aerobic denitrifying bacteria agent with the diameter of 2-3 mm; the embedding medium is a mixed solution containing sodium alginate with the mass fraction of 2% and beta-cyclodextrin with the mass fraction of 2%, the sodium alginate and the beta-cyclodextrin are added into water, dissolved and mixed uniformly, sterilized at high temperature for 20min, and cooled to obtain the ferric citrate solution with the mass concentration of 2%.
3. Step 2, the application of the spherical aerobic denitrifying bacteria agent in the aspect of denitrification specifically comprises the following steps:
the invention uses a denitrification culture medium to verify the application effect of the aerobic denitrifying bacteria agent, and the formula of the denitrification culture medium is as follows: 0.35g NH4Cl、1.50g KNO33.0g of glucose, 7.04g of Na2 HPO4、1.46g KH2 PO4、0.06g MgSO4And 0.06mL of trace elements are dissolved in 600mL of sterile water and are uniformly mixed to obtain the denitrification culture medium. Cleaning the prepared spherule aerobic denitrifying bacteria agent with distilled water, adding the cleaned spherule aerobic denitrifying bacteria agent into a denitrifying culture medium according to the volume ratio of 1:10 (the aerobic denitrifying bacteria agent to the denitrifying culture medium), then placing the conical flask into a constant-temperature shaking table, carrying out open aerobic culture at the temperature of 28 ℃, carrying out 150r/min rotation speed, taking samples every 4h, and measuring the content of nitrate and nitrite in the samples.
As shown in Table 1, the initial nitrate concentration was 330.7mg/L, and the nitrate removal rate was gradually increased as the nitrate concentration gradually decreased as the reaction proceeded, i.e., 23%, 50%, 86% and 98% for 12h, 16h, 20h and 24h, respectively.
Example 2
1. Preparation of embedded strain suspension: under the anaerobic condition, inoculating the denitrifying bacteria strain preserved on the inclined plane on an activation culture medium, and culturing for 28h at the temperature of 25-35 ℃ and the temperature of 130-; selecting strains on the activation culture medium, inoculating the strains on the fermentation culture medium, and culturing for 24h under the conditions of 25-35 ℃ and 130-. Centrifuging the bacterial solution or concentrated solution at 4000r/min for 10min, removing supernatant after centrifugation, washing with normal saline for 3 times, diluting with normal saline to OD600 value of about 0.3, and storing at 4 deg.C.
2. Preparing a strain immobilized pellet: and (3) mixing the bacterial suspension prepared in the step (1): adding the mixture into an embedding agent in a ratio of 20, stirring and mixing uniformly, then adding the mixed solution into a ferric citrate solution cross-linking agent drop by drop, sealing the opening of the; the embedding medium is a mixed solution containing 3 mass percent of sodium alginate and 1 mass percent of beta-cyclodextrin, the sodium alginate and the beta-cyclodextrin are added into water, dissolved and mixed uniformly, sterilized at high temperature for 20min, and cooled to obtain the ferric citrate solution with the mass concentration of 2%.
3. Step 2, the application of the spherical aerobic denitrifying bacteria agent in the aspect of denitrification specifically comprises the following steps:
cleaning the prepared spherulitic aerobic denitrifying bacteria agent with distilled water, adding the cleaned spherulitic aerobic denitrifying bacteria agent into a conical flask containing a denitrifying culture medium (the formula is the same as that in example 1) according to the volume ratio of 1:10, placing the conical flask in a constant-temperature shaking table, carrying out open aerobic culture at the temperature of 28 ℃, rotating at 150r/min, taking samples every 4 hours, and measuring the content of nitrate and nitrite in the samples.
As shown in Table 1, the initial nitrate concentration was 330.07mg/L, and the nitrate removal rate was gradually increased as the nitrate concentration gradually decreased as the reaction proceeded, i.e., 15%, 36%, 65% and 98% for 12h, 16h, 20h and 24h, respectively.
Example 3
1. Preparation of embedded strain suspension: under the anaerobic condition, inoculating the denitrifying bacteria strain preserved on the inclined plane on an activation culture medium, and culturing for 28h at the temperature of 25-35 ℃ and the temperature of 130-; selecting strains on the activation culture medium, inoculating the strains on the fermentation culture medium, and culturing for 24h under the conditions of 25-35 ℃ and 130-. Centrifuging the bacterial solution or concentrated solution at 4000r/min for 15min, removing supernatant after centrifugation, washing with normal saline for 3 times, diluting with normal saline to OD600 value of about 0.3, and storing at 4 deg.C.
2. Preparing a strain immobilized pellet: and (3) mixing the bacterial suspension prepared in the step (1): adding the mixture into an embedding agent in a proportion of 50, stirring and mixing uniformly, then adding the mixed solution into a ferric citrate solution cross-linking agent drop by drop, sealing the mixture at the temperature of 4 ℃, placing the mixture in a refrigerator for cross-linking for 24 hours, and then cleaning and drying the mixture by using sterile distilled water to obtain a spherical aerobic denitrifying bacteria agent with the diameter of 2-3 mm; the embedding medium is a mixed solution containing 4 mass percent of sodium alginate and 2 mass percent of beta-cyclodextrin, the sodium alginate and the beta-cyclodextrin are added into water, dissolved and mixed uniformly, sterilized at high temperature for 20min, and cooled to obtain the ferric citrate solution with the mass concentration of 2%.
3. Step 2, the application of the spherical aerobic denitrifying bacteria agent in the aspect of denitrification specifically comprises the following steps:
cleaning the prepared spherulitic aerobic denitrifying bacteria agent with distilled water, adding the cleaned spherulitic aerobic denitrifying bacteria agent into a conical flask containing a denitrifying culture medium (the formula is the same as that in example 1) according to the volume ratio of 1:10, placing the conical flask in a constant-temperature shaking table, carrying out open aerobic culture at the temperature of 28 ℃, rotating at 150r/min, taking samples every 4 hours, and measuring the content of nitrate and nitrite in the samples.
As shown in Table 1, the initial nitrate concentration was 330.07mg/L, and the nitrate removal rate was gradually increased as the nitrate concentration gradually decreased as the reaction proceeded, i.e., 18%, 39%, 52% and 97% for 12h, 16h, 20h and 24h, respectively.
Example 4
1. Preparation of embedded strain suspension: under the anaerobic condition, inoculating the denitrifying bacteria strain preserved on the inclined plane on an activation culture medium, and culturing for 28h at the temperature of 25-35 ℃ and the temperature of 130-; selecting strains on the activation culture medium, inoculating the strains on the fermentation culture medium, and culturing for 24h under the conditions of 25-35 ℃ and 130-. Centrifuging the bacterial solution or concentrated solution at 4000r/min for 10min, removing supernatant after centrifugation, washing with normal saline for 3 times, diluting with normal saline to OD600 value of about 0.3, and storing at 4 deg.C.
2. Preparing a strain immobilized pellet: and (3) mixing the bacterial suspension prepared in the step (1): adding the mixture into an embedding agent in a ratio of 20, stirring and mixing uniformly, then adding the mixed solution into a ferric citrate solution cross-linking agent drop by drop, sealing the mixture at the temperature of 4 ℃, placing the mixture in a refrigerator for cross-linking for 24 hours, and then cleaning and drying the mixture by using sterile distilled water to obtain a spherical aerobic denitrifying bacteria agent with the diameter of 3-4 mm; the embedding medium is a mixed solution containing 3 mass percent of sodium alginate and 1 mass percent of beta-cyclodextrin, the sodium alginate and the beta-cyclodextrin are added into water, dissolved and mixed uniformly, sterilized at high temperature for 20min, and cooled to obtain the ferric citrate solution with the mass concentration of 2%.
3. Step 2, the application of the spherical aerobic denitrifying bacteria agent in the aspect of denitrification specifically comprises the following steps:
cleaning the prepared spherulitic aerobic denitrifying bacteria agent with distilled water, adding the cleaned spherulitic aerobic denitrifying bacteria agent into a conical flask containing a denitrifying culture medium (the formula is the same as that in example 1) according to the volume ratio of 1:10, placing the conical flask in a constant-temperature shaking table, carrying out open aerobic culture at the temperature of 28 ℃, rotating at 150r/min, taking samples every 4 hours, and measuring the content of nitrate and nitrite in the samples.
As shown in Table 1, the initial nitrate concentration was 330.07mg/L, and the nitrate removal rate was gradually increased as the nitrate concentration gradually decreased as the reaction proceeded, i.e., 17%, 57%, 91% and 98% were obtained for 12h, 16h, 20h and 24h, respectively.
Example 5
1. The denitrifying bacteria suspension was prepared in the same manner as in example 1.
2. The denitrifying bacteria solution was divided into two equal portions, one portion was used for producing the strain-immobilized beads according to step 2 of example 1, and the other portion was stored under refrigeration.
3. A denitrification medium was prepared in the same manner as in example 1.
4. Two 200mL Erlenmeyer flasks, numbered I, II, were prepared.
Code I: cleaning the prepared spherulitic aerobic denitrifying bacteria agent with distilled water, adding the cleaned spherulitic aerobic denitrifying bacteria agent into a conical flask containing a denitrifying culture medium (the formula is the same as that in example 1) according to the volume ratio of 1:10 (the volume ratio of the aerobic denitrifying bacteria agent to the denitrifying culture medium is 50 mL), then placing the conical flask into a constant-temperature shaking table, carrying out open aerobic culture at 28 ℃, rotating at 150r/min, taking samples every 4h, and respectively measuring the contents of nitrate and nitrite in the samples by using an ultraviolet spectrophotometry method and a diazo coupling spectrophotometry method.
Number II: adding free denitrifying bacteria solution with the same amount as the number I into a conical flask containing 50mL denitrifying culture medium, then placing the conical flask into a constant-temperature shaking table for open aerobic culture at the temperature of 28 ℃, rotating at the speed of 150r/min, taking samples every 4h, and respectively measuring the content of nitrate and nitrite in the samples.
5. The reduction capacity of the spherule synchronous nitrification and denitrification bacteria agent to the nitrate state is obviously higher than that of the free state denitrification bacteria liquid through determination, the total nitrogen removal rate reaches 90 percent, and the free state denitrification bacteria liquid is only 18.8 percent. Experiments prove that the denitrification effect of the globular synchronous nitrification and denitrification bacteria agent is obviously improved in the presence of oxygen.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
TABLE 1
Claims (8)
1. A preparation method of a synchronous nitrification and denitrification bacterial agent is characterized by comprising the following steps:
firstly, preparation of embedding strain suspension: under the anaerobic condition, inoculating denitrifying bacteria strain preserved on the inclined plane on an activation culture medium for culturing for 18-24h, selecting the strain on the activation culture medium, inoculating on a fermentation culture medium, and carrying out culture for 20-28h under the condition of 25-35 ℃ to obtain denitrifying bacteria liquid or concentrated liquid for enlarged culture. And (3) centrifuging the bacterial liquid or the concentrated liquid for 15min at the rotating speed of 3000-5000 r/min, pouring out supernatant, centrifuging and washing the supernatant for 2-3 times by using normal saline, diluting the supernatant by using the normal saline until the OD600 value is 0.3-0.5, and sealing and storing the supernatant at 0-10 ℃ for later use.
The activation culture medium is specifically a liquid LB culture medium: 10g/L of peptone, 5g/L of sodium chloride, 10g/L of yeast powder and 7.0-7.2 of pH.
The fermentation medium comprises the following specific components:
5-30 g/L of organic carbon source and MgSO4·7H2O0.1g/L，Na2HPO4·2H2O7.9g/L，KH2PO41.5g/L，KNO3/KNO21-6 g/L of trace elements and 1mL/L of trace elements, adjusting the pH to be about 7.2, and sterilizing the culture medium before use.
Secondly, preparing strain immobilized beads: adding the prepared bacterial suspension into embedding agent according to the volume ratio of the strain suspension to the embedding agent of 1 (30-50), stirring and mixing uniformly, then adding the mixed solution into ferric citrate solution cross-linking agent drop by drop, carrying out cross-linking reaction for 8-36h at 0-10 ℃, washing with distilled water, and drying to obtain spherical aerobic denitrifying bacteria agent; wherein the embedding medium is a mixed solution containing 1-4% by mass of sodium alginate and 1-4% by mass of beta-cyclodextrin, and is prepared by adding sodium alginate and beta-cyclodextrin into water, dissolving, mixing, sterilizing at high temperature for 20min, and cooling.
2. The preparation method of the synchronous nitrification and denitrification microbial inoculum according to claim 1, which is characterized in that: in the first step, the denitrifying bacteria are anaerobic denitrifying bacteria.
3. The preparation method of the synchronous nitrification and denitrification microbial inoculum according to claim 1, which is characterized in that: the OD600 value of the denitrifying bacteria liquid or concentrated solution is about 0.3, and the sealing and refrigerating temperature is lower than 4 ℃.
4. The preparation method of the synchronous nitrification and denitrification microbial inoculum according to claim 1, which is characterized in that: in the second step, the volume ratio of the strain suspension to the embedding medium is 1: (10-40).
5. The preparation method of the synchronous nitrification and denitrification microbial inoculum according to claim 1, which is characterized in that: in the second step, the cross-linking agent is ferric citrate solution, and the mass concentration of the cross-linking agent is 2-4%.
6. The preparation method of the synchronous nitrification and denitrification microbial inoculum according to claim 1, which is characterized in that: in the second step, the grain size of the small spherical aerobic denitrifying bacteria agent is 2-3 mm.
7. The anaerobic denitrifying bacteria agent prepared by the preparation method of the synchronous nitrification and denitrification bacteria agent according to any one of claims 1 to 6.
8. Use of the simultaneous nitrification and denitrification bacterial agent as defined in claim 7 in denitrification.
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| CN115536138B (en) * | 2022-08-26 | 2024-05-14 | 龙海市雷尔生物科技有限公司 | Cyclodextrin cross-linked chitosan embedded nitrifying bacteria wastewater treatment agent and preparation method thereof |
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