CN112725182B - Preparation and rejuvenation method of anaerobic ammonium oxidation bacteria dry powder microbial agent - Google Patents
Preparation and rejuvenation method of anaerobic ammonium oxidation bacteria dry powder microbial agent Download PDFInfo
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- CN112725182B CN112725182B CN201911031744.9A CN201911031744A CN112725182B CN 112725182 B CN112725182 B CN 112725182B CN 201911031744 A CN201911031744 A CN 201911031744A CN 112725182 B CN112725182 B CN 112725182B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 81
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 68
- 230000003647 oxidation Effects 0.000 title claims abstract description 67
- 239000000843 powder Substances 0.000 title claims abstract description 64
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 230000003716 rejuvenation Effects 0.000 title claims description 14
- 239000003795 chemical substances by application Substances 0.000 title description 3
- 230000000813 microbial effect Effects 0.000 title description 3
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 71
- 239000003223 protective agent Substances 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000004108 freeze drying Methods 0.000 claims abstract description 25
- 229960001484 edetic acid Drugs 0.000 claims abstract description 23
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 18
- 239000002244 precipitate Substances 0.000 claims abstract description 16
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 12
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 11
- 229930195725 Mannitol Natural products 0.000 claims abstract description 11
- 239000000594 mannitol Substances 0.000 claims abstract description 11
- 235000010355 mannitol Nutrition 0.000 claims abstract description 11
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000011616 biotin Substances 0.000 claims abstract description 10
- 229960002685 biotin Drugs 0.000 claims abstract description 10
- 235000020958 biotin Nutrition 0.000 claims abstract description 10
- 230000008569 process Effects 0.000 claims abstract description 7
- 239000008367 deionised water Substances 0.000 claims abstract description 6
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 4
- 229960001855 mannitol Drugs 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 35
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 26
- 239000000203 mixture Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- 229910021529 ammonia Inorganic materials 0.000 claims description 17
- 239000006228 supernatant Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- 239000004323 potassium nitrate Substances 0.000 claims description 13
- 235000010333 potassium nitrate Nutrition 0.000 claims description 13
- 239000002351 wastewater Substances 0.000 claims description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 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 claims description 5
- 238000007664 blowing Methods 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 238000005086 pumping Methods 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 241001453382 Nitrosomonadales Species 0.000 claims description 4
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000007599 discharging Methods 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims 1
- 229910052751 metal Inorganic materials 0.000 claims 1
- 239000002184 metal Substances 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 24
- 238000011084 recovery Methods 0.000 abstract description 16
- 238000003860 storage Methods 0.000 description 16
- 230000004083 survival effect Effects 0.000 description 12
- 239000010802 sludge Substances 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 6
- 238000007447 staining method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229910052564 epsomite Inorganic materials 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
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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
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention provides a preparation method of an anaerobic ammonium oxidation bacteria dry powder microbial inoculum, which adopts aqueous solution of medicaments of polyethylene glycol, mannitol, biotin, Ethylene Diamine Tetraacetic Acid (EDTA) and hydroxylamine as a protective agent, and the addition amount of the protective agent solution is 1-2 times of the mass of anaerobic ammonium oxidation bacteria precipitates. Calculated by 100mL of deionized water, the protective agent contains 0.8-1.0g of polyethylene glycol, 0.5-0.7g of mannitol, 0.2-0.4g of biotin, 0.6-0.8g of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.4-0.6g of hydroxylamine. The protective agent can protect anaerobic ammonium oxidation bacteria in the freeze drying treatment process and simultaneously can ensure that the microbial inoculum has higher activity recovery rate after being stored for a long time.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a method for preparing and rejuvenating an anaerobic ammonia oxidation bacterium dry powder microbial agent.
Background
The anammox technology has the advantages of high efficiency, energy conservation, environmental protection and the like, has attracted wide attention since the introduction, and researchers at home and abroad are dedicated to researching the industrial application of the anammox technology in the field of wastewater denitrification. The anaerobic ammonia oxidation reaction-based biological denitrification process is gradually replacing the traditional nitrification-denitrification biological denitrification process. The Dutch, Detden and DOKHAVEN sewage treatment plant is the first sewage treatment plant in the world to put anaerobic ammonia oxidation technology into practical production, and is started for 3.5 years. At present, most of practical engineering applications of anammox are located in Europe, and only China and Japan have practical engineering of anammox in operation. The actual engineering of partial anammox is shown in the table below.
Practical engineering of anammox
From the successfully operated anammox sewage treatment plants, it can be seen that the start-up time of a reactor using pure anammox sludge as sludge is obviously shorter than that of other sludge, so that the study on the storage and rejuvenation method of anammox sludge is of great importance to the engineering application and popularization of the anammox technology. The current method of more efficient storage of microorganisms is cryogenic storage, a storage technique that is particularly important for cryoprotectant selection. Commonly used protective agents are trehalose, dimethyl sulfoxide, mannitol, and the like. The current storage technology has the defects of strict requirements on storage conditions, high inactivation ratio of microbial inoculum and the like.
CN201710009335.3 discloses a method for preparing and preserving an anammox bacterium dry powder microbial inoculum, which mainly uses sodium alginate as a protective agent, is vacuumized and dried at 60 ℃ to obtain the anammox dry powder microbial inoculum, and the activity of the anammox dry powder microbial inoculum is restored to 41 percent after the anammox bacterium dry powder microbial inoculum is preserved for 30 days at 4 ℃. Wang Cai Hua et al (research on preservation method of anaerobic ammonium oxidation bacteria mixture) respectively investigated the preservation effect of anaerobic ammonium oxidation bacteria at 4 ℃ and normal temperature by using ammonium sulfate and ammonium chloride as preservation matrixes, and obtained that the preservation effect at 4 ℃ is the best by using ammonium sulfate as the matrix, and the activity retention rate of the rejuvenated thallus is 75.3%. CN201710992104.9 discloses that the anaerobic ammonia oxidation dry powder microbial inoculum is obtained by taking dimethyl sulfoxide and glycerol as a mixed protective agent, drying the mixed protective agent under vacuum at-92 ℃, and the activity recovery rate reaches 65.8 percent after the mixed protective agent is preserved for 75 days at 4 ℃. Generally, the activity retention rate and recovery rate of the anammox bacteria are weak after the anammox bacteria are prepared into powder.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for preparing and rejuvenating an anammox dry powder microbial inoculum, so as to protect anammox bacteria at a low temperature and ensure that the microbial inoculum has a high activity recovery rate after long-term storage.
The invention provides a preparation method of an anammox bacterium dry powder microbial inoculum, which takes aqueous solutions of five medicaments, namely polyethylene glycol, mannitol, biotin, Ethylene Diamine Tetraacetic Acid (EDTA) and hydroxylamine, as protective agents, and the addition amount of the protective agent solution is 1-2 times of the mass of anammox bacterium precipitates. The protective agent can protect anaerobic ammonium oxidation bacteria in the freeze drying treatment process and simultaneously can ensure that the microbial inoculum has higher activity recovery rate after being stored for a long time.
The invention provides a preparation method of an anaerobic ammonium oxidation bacterium dry powder microbial inoculum, which comprises the following steps:
(1) taking anaerobic ammonium oxidation bacteria to be preserved, centrifuging, and removing supernatant to obtain anaerobic ammonium oxidation bacteria precipitate;
(2) soaking the obtained anaerobic ammonium oxidation bacteria precipitate in potassium nitrate solution with nitrate nitrogen concentration of 10-20mg/L for 10-15min, centrifuging, and removing supernatant to obtain anaerobic ammonium oxidation bacteria precipitate treated with potassium nitrate;
(3) mixing the anaerobic ammonium oxidation bacteria sediment treated by potassium nitrate with a protective agent solution, uniformly oscillating on a vortex apparatus, and then blowing off by using nitrogen to remove dissolved oxygen in the mixture;
(4) and (4) freeze-drying the mixture obtained in the step (3) to obtain an anaerobic ammonia oxidizing bacteria dry powder microbial inoculum, carrying out vacuum pumping packaging on the anaerobic ammonia oxidizing bacteria dry powder microbial inoculum, and storing in a dark place for later use.
In the invention, the anaerobic ammonium oxidation bacteria are chemoautotrophic denitrogenation bacteria, carbon dioxide or carbonate is used as a carbon source, and NO is used2 --N is an electron acceptor, NH4 +Oxidation of-N to N2Mixed flora or activated sludge.
The operation conditions of the centrifugation in the step (1) are as follows: the rotating speed is 8000-; the centrifugation process may be performed a plurality of times, preferably 3 to 5 times. Preferably, 0.1-0.3mol/L Phosphate Buffer Solution (PBS) with pH of 7.0-7.2 is added into anaerobic ammonium oxidation bacteria during the centrifugal treatment. Adding phosphate buffer solution, mixing on a vortex, standing for 10-15min, and centrifuging to remove supernatant.
The protective agent solution in the step (3) contains 0.8-1.0g of polyethylene glycol, 0.5-0.7g of mannitol, 0.2-0.4g of biotin, 0.6-0.8g of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.4-0.6g of hydroxylamine based on 100mL of deionized water.
The freeze drying in the step (4) is to place the mixture in a vacuum freeze dryer, vacuumize the mixture until the pressure reaches 5-10 Pa, set the temperature for the first 10-12h to be-40 to-30 ℃, preferably-38 to-28 ℃, set the temperature for the second 10-12h to be-70 to-50 ℃, preferably-65 to-55 ℃, and dry the mixture. And (5) freeze-drying in the step (4) to obtain the dry powder microbial inoculum of the anammox bacteria, wherein the water content is 15-20%. The temperature for keeping in the dark in the step (4) is 3-5 ℃.
The invention provides a method for rejuvenating an anaerobic ammonia oxidation dry powder microbial inoculum, which comprises the following steps:
(1) inoculating the dry powder microbial inoculum into an anaerobic reactor, wherein the inoculation concentration is about 2000-3000mg/L, adding wastewater, and standing;
(2) stirring, controlling the temperature of the reactor at 30-35 ℃, normally feeding and discharging water in the device, and operating for 7-25 days to rejuvenate the anammox dry powder microbial inoculum.
The wastewater in the step (1) is preferably artificially prepared wastewater, contains ammonia nitrogen and nitrite nitrogen, has the ratio of 1 (1-2), and has the total nitrogen of about 80-120 mg/L. More preferably, the wastewater contains CaCl2·2H2O, EDTA ferrous sulfate, potassium dihydrogen phosphate and MgSO4·7H2O and the like. Wherein CaCl2·2H20.005-0.01g/L of O content 0.1-0.5g/L, EDTA content, 0.005-0.01g/L of ferrous sulfate content, 0.001-0.05g/L of potassium dihydrogen phosphate content and MgSO4·7H2The content of O is 0.1-0.3 g/L. The standing in the step (1) is preferably a standing for 20 to 30 hours.
Compared with the prior art, the invention has the following beneficial effects:
(1) by adding aqueous solution of polyethylene glycol, mannitol, biotin, Ethylene Diamine Tetraacetic Acid (EDTA) and hydroxylamine as mixed protective agents into anaerobic ammonium oxidation bacteria, synergistic action exists among the protective agents, and the bacterial cells are well protected in the freeze drying process;
(2) the dry powder microbial inoculum obtained by the invention can still obtain higher recovery rate of the activity of the thallus after long-time storage.
(3) The protective agent added in the invention has the advantages of low toxicity, high thallus utilization rate and no secondary pollution.
Detailed Description
The method and effects of the present invention will be described in detail with reference to examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The test materials used in the following examples were purchased from biochemical reagent stores unless otherwise specified.
In the embodiment of the invention, the ammonia nitrogen concentration is measured by GB7478-87 'determination of water quality-ammonium-distillation and titration method'; the nitrite nitrogen concentration is measured by GB7493-87 water quality-nitrite nitrogen determination-spectrophotometry; the total nitrogen concentration adopts GB 11894-89 'determination of water quality-total nitrogen-alkaline potassium persulfate digestion ultraviolet spectrophotometry'.
The anaerobic ammonium oxidation bacteria inoculated in the embodiment are anaerobic ammonium oxidation dry powder microbial inoculum prepared by the method, and the water inflow is artificial water distribution. The reactor used in the examples was a complete mixing anaerobic reactor, the top of which was equipped with a stirrer, and the feed water was pumped from the bottom of the reactor by a peristaltic pump. The effective volume of the reactor is 3.0L, the reactor is provided with a temperature control system, a pH control system and a temperature control system, and the sludge concentration (MLSS) of the anaerobic ammonia oxidation sludge inoculated in the reactor is about 2000 mg/L. The reactor is heated in water bath, the temperature of the reactor is controlled to be 30 +/-1 ℃, the concentration of dissolved oxygen is not more than 0.1mg/L, and the water conservancy retention time is 12 hours.
Example 1
The preparation method of the dry powder microbial inoculum comprises the following steps:
(1) taking the anaerobic ammonium oxidation bacteria to be preserved, measuring the activity of the anaerobic ammonium oxidation bacteria at the moment to be 0.2g N/g VSS/d, centrifuging the bacteria at 8000r/min for 15min, and discarding the supernatant; adding 0.1mol/L Phosphate Buffer Solution (PBS) with the pH value of 7.0 into the centrifuged anammox bacteria, resuspending on a vortex device, uniformly mixing, standing for 10min, centrifuging to remove supernatant, and repeating for 3 times to obtain an anammox bacteria precipitate;
(2) and soaking the obtained anaerobic ammonium oxidation bacteria sediment in a potassium nitrate solution with the nitrate nitrogen concentration of 15mg/L for resuspension for 10min, centrifuging, and removing supernatant to obtain the anaerobic ammonium oxidation bacteria sediment treated by potassium nitrate.
(3) Mixing the obtained anaerobic ammonium oxidation bacteria precipitate treated by potassium nitrate with a protective agent solution according to the mass ratio of 1:1, oscillating on a vortex apparatus, uniformly mixing, and then blowing off by using nitrogen to remove dissolved oxygen in the mixture; the protective agent solution is prepared by dissolving 0.8g of polyethylene glycol, 0.7g of mannitol, 0.2g of biotin, 0.8g of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.6g of hydroxylamine in 100mL of deionized water.
(4) Placing the mixture obtained in the step (3) in a vacuum freeze dryer, vacuumizing to 5Pa, setting the temperature to-35 ℃ in the first 12h and the temperature to-60 ℃ in the second 12h, and drying to obtain the dry powder microbial inoculum of the anaerobic ammonia oxidation bacteria with the water content of 15%; and (3) carrying out vacuum-pumping packaging on the anaerobic ammonium oxidation dry powder microbial inoculum obtained by freeze drying, and storing the anaerobic ammonium oxidation dry powder microbial inoculum in a dark place at the temperature of 4 ℃ for later use.
The microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anaerobic ammonium oxidation bacteria is 91% before the dry powder microbial inoculum is prepared, and the cell survival rate is 83% after the dry powder microbial inoculum is prepared, which shows that the protective effect of the protective agent on the bacteria in the freeze drying treatment is obvious.
A rejuvenation (activity recovery) experiment is carried out on the anaerobic ammonia oxidation dry powder microbial inoculum stored for 90 days, and the dry powder microbial inoculum is inoculated in an anaerobic reactor at the inoculation concentration of about 2000 mg/L. And (4) adding the manually prepared wastewater, and standing for 24 hours. The artificially configured wastewater contains ammonia nitrogen and nitrite nitrogen with the ratio of about 1:1.32, and the total nitrogen is about 100 mg/L. The wastewater contains CaCl2·2H2O (0.3 g/L), EDTA (0.00625 g/L), ferrous sulfate (0.00625 g/L), potassium dihydrogen phosphate (0.025 g/L) and MgSO4·7H2O（0.2g/L）。
After 24h, the stirring is started, the temperature of the reactor is controlled at 30 ℃, and the device normally feeds in and discharges water. The reactor is operated for 3 days, the total nitrogen of the effluent is measured to be gradually reduced to 85mg/L and shows a small descending trend, and the bacteria at the stage show denitrification activity gradually. When the reactor is operated to the 7 th day, the total nitrogen concentration of effluent can be reduced to 45mg/L, the total nitrogen removal rate reaches 55%, when the reactor is operated to the 10 th day, almost no nitrite nitrogen exists in the effluent of the reactor, the total nitrite nitrogen is 21mg/L, the activity of the anaerobic ammonium oxidation bacteria is measured to be about 0.08g N/g VSS/d, and the activity recovery rate reaches 40%. On the 11 th day, the concentration of the pollutants of the inlet water of the reactor is doubled, namely the total nitrogen of the inlet water is increased to 200 mg/L, the operation is carried out to the 15 th day, the total nitrogen of the outlet water is 51 mg/L, and the activity recovery rate of the anaerobic ammonium oxidation bacteria is about 74 percent. In the next 5 days, the total nitrogen of effluent is weakened, the total nitrogen of effluent is 32mg/L in the 20 th day, and the activity recovery rate of the anaerobic ammonium oxidation bacteria is basically maintained at about 84%.
Example 2
The preparation method of the dry powder microbial inoculum comprises the following steps:
(1) taking the anaerobic ammonia oxidation bacteria to be preserved, measuring the activity of the anaerobic ammonia oxidation bacteria at the moment to be 0.2g N/g VSS/d, centrifuging the bacteria at 9000r/min for 10min, and discarding supernatant; adding 0.1mol/L Phosphate Buffer Solution (PBS) with the pH value of 7.2 into the centrifuged anammox bacteria, resuspending on a vortex device, uniformly mixing, standing for 15min, centrifuging to remove supernatant, and repeating for 4 times to obtain an anammox bacteria precipitate;
(2) and soaking the obtained anaerobic ammonium oxidation bacteria sediment in a potassium nitrate solution with the nitrate nitrogen concentration of 20mg/L for resuspension for 15min, centrifuging, and removing supernatant to obtain the anaerobic ammonium oxidation bacteria sediment treated by potassium nitrate.
(3) Mixing the obtained anaerobic ammonium oxidation bacteria precipitate treated by potassium nitrate with a protective agent solution according to the mass ratio of 1:2, oscillating on a vortex machine, uniformly mixing, and then blowing off by using nitrogen to remove dissolved oxygen in the mixture; the protective agent solution is prepared by dissolving 1.0g of polyethylene glycol, 0.5g of mannitol, 0.4g of biotin, 0.6g of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.5g of hydroxylamine in 100mL of deionized water.
(4) Placing the mixture obtained in the step (3) in a vacuum freeze dryer, vacuumizing to the pressure of 7Pa, setting the temperature to-35 ℃ in the first 12h and the temperature to-60 ℃ in the second 12h, and drying to obtain the dry powder microbial inoculum of the anaerobic ammonia oxidation bacteria with the water content of 20%; and (3) carrying out vacuum-pumping packaging on the anaerobic ammonium oxidation dry powder microbial inoculum obtained by freeze drying, and storing the anaerobic ammonium oxidation dry powder microbial inoculum in a dark place at the temperature of 4 ℃ for later use.
The microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anammox bacteria is 90% before the dry powder microbial inoculum is prepared, and the cell survival rate of the anammox bacteria is 84% after the dry powder microbial inoculum is prepared, which shows that the protective effect of the protective agent on the bacteria is obvious in freeze drying treatment.
The activity recovery experiment of the anammox dry powder microbial inoculum stored for 90 days is carried out, the operation parameters and the recovery process are the same as those of the embodiment 1, the total nitrogen of the effluent water at the 20 th day is 33mg/L, and the activity recovery rate of the anammox bacteria is basically maintained at about 82%.
Example 3
The preparation method of the dry powder microbial inoculum comprises the following steps:
(1) taking anaerobic ammonium oxidation bacteria to be preserved, measuring the activity of the anaerobic ammonium oxidation bacteria at the moment to be 0.2g N/g VSS/d, centrifuging the bacteria at 10000r/min for 10min, and removing supernatant; adding 0.1mol/L Phosphate Buffer Solution (PBS) with the pH value of 7.2 into the centrifuged anammox bacteria, resuspending on a vortex device, uniformly mixing, standing for 12min, centrifuging to remove supernatant, and repeating for 5 times to obtain an anammox bacteria precipitate;
(2) and soaking the obtained anammox bacterium precipitate in a potassium nitrate solution with nitrate nitrogen concentration of 10mg/L for resuspension for 12min, centrifuging, and removing a supernatant.
(3) Mixing the obtained anaerobic ammonium oxidation bacteria precipitate with a protective agent solution according to the mass ratio of 1:1, oscillating on a vortex device, uniformly mixing, and then blowing off by using nitrogen to remove dissolved oxygen in the mixture; the protective agent solution is prepared by dissolving 0.9g of polyethylene glycol, 0.6g of mannitol, 0.3g of biotin, 0.7g of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.5g of hydroxylamine in 100mL of deionized water.
(4) Placing the mixture obtained in the step (3) in a vacuum freeze dryer, vacuumizing until the pressure is below 8Pa, setting the temperature for the first 10h to be-35 ℃, setting the temperature for the second 10h to be-60 ℃, and drying to obtain the dry powder microbial inoculum of the anaerobic ammonia oxidation bacteria with the water content of 18%; and (3) carrying out vacuum-pumping packaging on the anaerobic ammonia oxidation dry powder microbial inoculum obtained by freeze drying, and storing in a dark place at 4 ℃ for later use.
The microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anaerobic ammonium oxidation bacteria is 91% before the dry powder microbial inoculum is prepared, and the cell survival rate is 83% after the dry powder microbial inoculum is prepared, which shows that the protective effect of the protective agent on the bacteria in the freeze drying treatment is obvious.
The anaerobic ammonium oxidation dry powder microbial inoculum stored for 90 days is subjected to rejuvenation (activity recovery) experiments, the operation parameters and the recovery process are the same as those of the example 1, the inoculation concentration in the step (1) is 3000mg/L, and the wastewater contains CaCl2·2H2O (0.1 g/L), EDTA (0.03 g/L), ferrous sulfate (0.01 g/L), potassium dihydrogen phosphate (0.015 g/L) and MgSO4·7H2O (0.1 g/L). The total nitrogen of the effluent at the 20 th day is 29mg/L, and the activity recovery rate of the anaerobic ammonium oxidation bacteria is basically maintained at about 85 percent.
Comparative example 1
The difference from example 1 is only that no protective agent is added; the microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anaerobic ammonia oxidation bacteria is 91% before the dry powder microbial inoculum is prepared, and the cell survival rate is only 5% after the dry powder microbial inoculum is prepared, which shows that the protective agent plays an important role in freeze drying treatment of the bacteria.
Comparative example 2
The difference is that polyethylene glycol is taken as a single protective agent, and the addition amount is the same as that of the protective agent in the example 1; the microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anaerobic ammonia oxidation bacteria is 91% before the dry powder microbial inoculum is prepared, and the cell survival rate is only 43% after the dry powder microbial inoculum is prepared, which indicates that a single protective agent cannot meet the requirements of freeze-drying and storage of the microbial inoculum.
Comparative example 3
The difference is the same as that of example 1, only that polyethylene glycol (1.6 g) and ethylene diamine tetraacetic acid (1.5 g) are taken as composite protective agents, and the addition amount is the same as that of the protective agent in example 1; the microbial inoculum before and after freeze-drying and storage for 90 days is taken to carry out cell viability determination by using a flow cytometry-PI single staining method. The cell survival rate of the anaerobic ammonia oxidation bacteria is 91% before the preparation of the dry powder microbial inoculum, and the cell survival rate is only 54% after the preparation of the dry powder microbial inoculum, which indicates that the protective agent containing two medicament components can not meet the requirements of freeze-drying and storage of the bacteria.
Claims (12)
1. A preparation method of an anaerobic ammonium oxidation bacterium dry powder microbial inoculum is characterized in that aqueous solutions of five medicaments, namely polyethylene glycol, mannitol, biotin, ethylene diamine tetraacetic acid and hydroxylamine, are used as protective agents, and the addition amount of the protective agent solution is 1-2 times of the mass amount of anaerobic ammonium oxidation bacterium precipitates; the protective agent solution contains 0.8-1.0g of polyethylene glycol, 0.5-0.7g of mannitol, 0.2-0.4g of biotin, 0.6-0.8g of ethylene diamine tetraacetic acid and 0.4-0.6g of hydroxylamine based on 100mL of deionized water;
the preparation method comprises the following steps:
(1) taking anaerobic ammonium oxidation bacteria to be preserved, centrifuging, and removing supernatant to obtain anaerobic ammonium oxidation bacteria precipitate;
(2) soaking the obtained anaerobic ammonium oxidation bacteria precipitate in potassium nitrate solution with nitrate nitrogen concentration of 10-20mg/L for 10-15min, centrifuging, and removing supernatant to obtain anaerobic ammonium oxidation bacteria precipitate treated with potassium nitrate;
(3) mixing the anaerobic ammonium oxidation bacteria sediment treated by potassium nitrate with a protective agent solution, uniformly oscillating on a vortex apparatus, and then blowing off by using nitrogen to remove dissolved oxygen in the mixture;
(4) and (4) freeze-drying the mixture obtained in the step (3) to obtain an anaerobic ammonia oxidizing bacteria dry powder microbial inoculum, carrying out vacuum pumping packaging on the anaerobic ammonia oxidizing bacteria dry powder microbial inoculum, and storing in a dark place for later use.
2. The method for preparing the dry powder microbial inoculum according to the claim 1, wherein the operation conditions of the centrifugation in the step (1) are as follows: the rotating speed is 8000-10000r/min, and the time is 10-15 min.
3. The method for preparing a dry powder microbial inoculum according to claim 1, wherein 0.1-0.3mol/L phosphate buffer solution with pH of 7.0-7.2 is added into anaerobic ammonium oxidation bacteria in the centrifugal treatment process in the step (1).
4. The method for preparing the dry powder microbial inoculum according to the claim 1, wherein the freeze drying in the step (4) is to place the mixture in a vacuum freeze dryer, vacuumize the mixture until the pressure is 5 to 10 Pa, set the temperature to-40 to-30 ℃ for the first 10 to 12 hours, set the temperature to-70 to-50 ℃ for the second 10 to 12 hours, and dry the mixture.
5. The method for preparing the dry powder microbial inoculum according to the claim 1, wherein the freeze drying in the step (4) is to place the mixture in a vacuum freeze dryer, vacuumize the mixture until the pressure is 5 to 10 Pa, set the temperature to-38 to-28 ℃ for the first 10 to 12 hours, set the temperature to-65 to-55 ℃ for the second 10 to 12 hours, and dry the mixture.
6. The preparation method of the dry powder microbial inoculum according to claim 1, wherein the water content of the dry powder microbial inoculum of the anaerobic ammonium oxidation bacteria obtained by freeze-drying in the step (4) is 15-20%.
7. The method for preparing a dry powder microbial inoculum according to claim 1, wherein the temperature for storing in the dark in the step (4) is 3-5 ℃.
8. The rejuvenation method of the anaerobic ammonia oxidation dry powder microbial inoculum prepared by the preparation method of any one of claims 1 to 7 is characterized by comprising the following steps:
(1) inoculating the dry powder microbial inoculum into an anaerobic reactor, wherein the inoculation concentration is about 2000-3000mg/L, adding wastewater, and standing;
(2) stirring, controlling the temperature of the reactor at 30-35 ℃, normally feeding and discharging water, and running for 7-25 days to rejuvenate the anammox dry powder microbial inoculum.
9. The method for rejuvenating the anammox dry powder microbial inoculum according to claim 8, wherein the wastewater in the step (1) contains ammonia nitrogen and nitrite nitrogen in a ratio of 1 (1-2), and the total nitrogen is 80-120 mg/L.
10. The method for rejuvenating the anammox dry powder microbial inoculum according to claim 8 wherein the wastewater in step (1) contains CaCl2·2H2O, EDTA ferrous sulfate, potassium dihydrogen phosphate and MgSO4·7H2One or more of O metal salts.
11. The method of rejuvenating anammox dry powder microbial inoculum according to claim 10 wherein CaCl is added2·2H20.005-0.01g/L of O content, 0.005-0.01g/L of ferrous sulfate, 0.001-0.05g/L of monopotassium phosphate and MgSO 0.1-0.5g/L, EDTA content4·7H2The content of O is 0.1-0.3 g/L.
12. The method for rejuvenating the dry anammox inoculant according to claim 10 wherein the standing in step (1) is 20-30 hours.
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