CN113122465A - Preparation and preservation method of nitrobacteria dry powder microbial inoculum - Google Patents
Preparation and preservation method of nitrobacteria dry powder microbial inoculum Download PDFInfo
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- CN113122465A CN113122465A CN201911418215.4A CN201911418215A CN113122465A CN 113122465 A CN113122465 A CN 113122465A CN 201911418215 A CN201911418215 A CN 201911418215A CN 113122465 A CN113122465 A CN 113122465A
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- 241000108664 Nitrobacteria Species 0.000 title claims abstract description 88
- 239000000843 powder Substances 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 52
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000004321 preservation Methods 0.000 title claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 239000000725 suspension Substances 0.000 claims abstract description 24
- 238000005273 aeration Methods 0.000 claims abstract description 22
- 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 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 17
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 17
- 239000000661 sodium alginate Substances 0.000 claims abstract description 17
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 17
- 238000001291 vacuum drying Methods 0.000 claims abstract description 16
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 150000002148 esters Chemical class 0.000 claims abstract description 10
- 238000010926 purge Methods 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 8
- 230000001546 nitrifying effect Effects 0.000 claims description 17
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 13
- 229920002674 hyaluronan Polymers 0.000 claims description 13
- 229960003160 hyaluronic acid Drugs 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- -1 rhamnose ester Chemical class 0.000 claims description 11
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 8
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 8
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 8
- 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 claims description 6
- 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 claims description 6
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical compound OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 claims description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 claims 1
- 229930186217 Glycolipid Natural products 0.000 claims 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 claims 1
- 230000000694 effects Effects 0.000 abstract description 24
- 238000011084 recovery Methods 0.000 abstract description 8
- 230000007774 longterm Effects 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 44
- 210000004027 cell Anatomy 0.000 description 22
- 230000004083 survival effect Effects 0.000 description 14
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 13
- 230000004913 activation Effects 0.000 description 11
- 239000007853 buffer solution Substances 0.000 description 11
- 230000000813 microbial effect Effects 0.000 description 7
- 239000003223 protective agent Substances 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000003647 oxidation Effects 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229910000406 trisodium phosphate Inorganic materials 0.000 description 1
- 235000019801 trisodium phosphate Nutrition 0.000 description 1
Classifications
-
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- 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
Abstract
The invention relates to a method for preparing and preserving nitrobacteria dry powder microbial inoculum, which comprises the steps of dehydrating nitrobacteria suspension until the water content is higher than 80 percent, preparing a nitrobacteria concentrated solution A, adding sodium alginate, and reacting under the aeration condition; then dehydrating until the water content is 60-80% to prepare a nitrobacteria concentrated solution B, then adding sugar ester substances, mixing and purging with nitrogen; and then dehydrating until the water content is lower than 50%, preparing a nitrobacteria concentrated solution C, and carrying out vacuum drying to obtain the nitrobacteria dry powder microbial inoculum. The method realizes the preparation of nitrobacteria dry powder and room temperature preservation by vacuum drying, the prepared dry powder microbial inoculum is convenient to transport and use, and the activity of the bacteria after long-term preservation is recovered quickly and the activity recovery rate is high.
Description
Technical Field
The invention belongs to the field of sewage treatment, and particularly relates to a preparation and preservation method of a nitrobacteria dry powder microbial inoculum.
Background
The biological agent comprises liquid and solid, and the preservation method also comprises liquid and solid. Heterotrophic microorganisms capable of producing spores can be processed into resting cells to prepare dry powder microbial inoculum for preservation in a conidium stage, and the influence on the activity of the microbial inoculum is small. In the case of autotrophic microorganisms such as nitrifying bacteria, since no spores exist, the activity of the cells is reduced or even lost during the preparation of dry powder, and particularly, the activity of the cells is reduced or even lost with the lapse of time during the preservation. The existing nitrifying bacteria agent or bacterial strain is mostly liquid, the common preservation method is normal temperature liquid or ultra-low temperature freezing preservation, the preservation period of the bacteria preserved by the normal temperature liquid is short, the cost is additionally increased by the bacteria preserved by the low temperature freezing preservation, and the long-distance preservation and transportation are inconvenient in the application process.
At present, nitrobacteria are mostly frozen and preserved by direct ultralow-temperature liquid, the preservation activity is good, but the cost is high, and the preserved bacteria are inconvenient to transport. CN106554921A discloses a method for preserving nitrobacteria, which comprises adding a growth promoter during the culture of nitrobacteria, culturing until the growth stabilization period, collecting thallus, mixing with a preservation nutrient solution, controlling the water content to be 40-80%, adding a preserving agent, and preserving at low temperature. CN108486019A discloses a preservation method of nitrosobacteria, which comprises (1) preparing protective agent: adding 2-4 parts of sodium glutamate, 18-25 parts of trisodium phosphate and 1-3 parts of cysteine into 90-120 parts of sterile water, and uniformly mixing to obtain a protective agent; (2) adding thalli; (3) and (5) freezing and preserving. The methods all adopt low-temperature freezing preservation, the temperature is lower, the preservation cost is higher, and the preserved thalli are inconvenient to transport. CN103880168A discloses a long-term preservation and activity recovery method for aerobic nitrification granular sludge, which recovers normal treatment capacity 7 days after preservation, but the invention only aims at the aerobic granular sludge, and the preservation time is 180 days, which is not suitable for long-term preservation.
In order to ensure the activity of the cells during the preparation of the dry powder, a protecting agent may be added. CN106635803A discloses a preparation and preservation method of an anaerobic ammonium oxidation bacterium dry powder microbial inoculum, which mainly uses sodium alginate as a protective agent, is vacuumized and dried at 60 ℃ to obtain the anaerobic ammonium oxidation dry powder microbial inoculum, and the activity of the anaerobic ammonium oxidation dry powder microbial inoculum is recovered to 41 percent after the anaerobic ammonium oxidation dry powder microbial inoculum is stored for 30 days at 4 ℃. CN107557302A discloses that the activity recovery rate reaches 65.8% after an anaerobic ammonia oxidation dry powder microbial inoculum is obtained by taking dimethyl sulfoxide and glycerol as a mixed protective agent and drying the mixed protective agent at the temperature of-92 ℃ in a vacuum pumping manner and is preserved for 75 days at the temperature of 4 ℃. The anaerobic ammonium oxidation bacteria preserved in the form of dry powder in the patent have low activity recovery rate and short preservation period. However, the protective agents are not ideal for preparing autotrophic nitrifying bacteria dry powder.
Disclosure of Invention
The invention aims to provide a preparation and preservation method of nitrobacteria dry powder microbial inoculum, which realizes the preparation and room temperature preservation of nitrobacteria dry powder by vacuum drying, the prepared dry powder microbial inoculum is convenient to transport and use, the activity of the microbial inoculum after long-term preservation is fast to recover, and the activity recovery rate is high.
The invention provides a preparation method of a nitrobacteria dry powder microbial inoculum, which mainly comprises the following steps:
(1) dehydrating the nitrobacteria suspension until the water content is higher than 80%, preparing a nitrobacteria concentrated solution A, adding sodium alginate, and reacting under an aeration condition;
(2) dehydrating the mixture obtained in the step (1) until the water content is 60-80%, preparing a nitrobacteria concentrated solution B, adding sugar ester substances, mixing and purging with nitrogen;
(3) and (3) dehydrating the mixture obtained in the step (2) until the water content is lower than 50%, preparing a nitrobacteria concentrated solution C, and performing vacuum drying to obtain the nitrobacteria dry powder microbial inoculum.
In the invention, the nitrifying bacteria suspension in the step (1) can be obtained by culturing according to a conventional method in the field, and the substrate of the nitrifying bacteria suspension is ammonia nitrogen.
In the invention, in the step (1), the nitrifying bacteria suspension is preferably dehydrated to the water content of 85-95% to prepare the nitrifying bacteria concentrated solution A.
In the invention, the step (1) is preferably to add 0.1 to 1 mass percent of sodium alginate solution. The mass ratio of the sodium alginate to the nitrobacteria concentrated solution A is 1: 1000-10000.
In the invention, the aeration condition of the step (1) is to introduce air, and the aeration time is 6-24 h.
In the present invention, the sugar ester substance in step (2) includes at least one of rhamnose ester, trehalose ester, sophorolipid, sucrose ester, etc., preferably trehalose ester.
In the invention, the adding amount of the sugar ester substances in the step (2) accounts for 0.1-0.5% of the mass of the nitrobacteria concentrated solution B.
In the invention, nitrogen purging is carried out in the mixing process in the step (2), and the purging time is 30-180 min.
In the invention, the mixture obtained in the step (3) is dehydrated until the water content is 30-50%.
In the invention, hyaluronic acid or/and sodium hyaluronate are further added into the nitrobacteria concentrated solution C in the step (3), and the mass ratio of the hyaluronic acid or/and sodium hyaluronate to the nitrobacteria concentrated solution C is 1: 1000-10000. Preferably, 0.1 to 1 mass percent of hyaluronic acid solution or/and sodium hyaluronate solution is/are added.
In the invention, the vacuum drying in the step (3) is carried out under the conditions that the pressure is 1-2kPa and the temperature is 40-60 ℃.
In the invention, the dehydration adopts gravity settling, centrifugation or filtration and other modes.
In the invention, the water content refers to the percentage of the weight of the water contained in the nitrobacteria concentrated solution in the total weight of the concentrated solution.
The invention provides a method for preserving nitrobacteria, wherein nitrobacteria suspension is prepared into a dry powder microbial inoculum according to the method, and the dry powder microbial inoculum is vacuum-packaged and preserved at room temperature for standby, and can be preserved for 1 to 2 years generally.
The invention has the following beneficial effects:
according to the invention, before vacuum drying of nitrobacteria, sodium alginate and sugar ester substances are respectively added at different preparation stages, and different water contents and aeration conditions are combined, so that the tolerance capability of bacterial cells in the vacuum drying process can be improved, the cells are completely protected from inside to outside, the damage of vacuum and temperature to cell walls, enzymes, proteins and water is avoided, and the prepared dry powder microbial inoculum can be preserved at room temperature for a long time.
According to the invention, before vacuum drying, hyaluronic acid (sodium) is further added when the water content is dehydrated to be lower than 50%, so that the tolerance of the dry powder microbial inoculum in the vacuum drying process is further improved, and the cell survival rate is improved.
The dry powder preparation method and the preservation method thereof can protect the thallus from losing activity in the long-term room temperature preservation process, and have long preservation time and quick activity recovery. The vacuum drying method is adopted, the preservation cost is obviously reduced, the prepared dry powder nitrobacteria are convenient to preserve, and the preserved bacteria have high activity and are convenient to transport and use.
Detailed Description
The method and effects of the present invention will be described in further detail by 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 ammonium in water quality-distillation and titration method'; cell viability = (total number of cells-dead cells) ÷ total number of cells × 100%, measured using beckmann's flow cytometer CytoFLEX.
Example 1
(1) And (3) performing gravity settling on the nitrobacteria suspension, then removing supernatant to obtain nitrobacteria concentrated solution A with the water content of 85%, then adding a sodium alginate solution with the mass fraction of 0.1%, wherein the mass ratio of the sodium alginate to the nitrobacteria concentrated solution A is 1:1000, and introducing air for aeration for 8 hours.
(2) Dehydrating the mixture obtained in the step (1) by adopting a filtering mode until the water content is 60%, preparing a nitrobacteria concentrated solution B, then adding trehalose ester, wherein the adding amount of the trehalose ester accounts for 0.1% of the mass of the nitrobacteria concentrated solution B, and purging with nitrogen for 60min in the mixing process.
(3) And (3) centrifugally dewatering the mixture obtained in the step (2) until the water content is 30%, preparing a nitrobacteria concentrated solution C, and performing vacuum drying under the conditions that the pressure is 1kPa and the temperature is 40 ℃ to obtain the nitrobacteria dry powder microbial inoculum. Compared with the equivalent nitrobacteria suspension in the step (1), the obtained nitrobacteria dry powder has equivalent microbial quantity and 75% of cell survival rate.
The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 12 months, is washed by buffer solution and is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to more than 90 percent from the initial 9 percent after 20 days.
Example 2
(1) And (3) performing gravity settling on the nitrobacteria suspension, then removing supernatant to obtain a nitrobacteria concentrated solution A with the water content of 90%, then adding a sodium alginate solution with the mass fraction of 0.5%, wherein the mass ratio of the sodium alginate to the nitrobacteria concentrated solution A is 1:5000, and introducing air for aeration for 16 h.
(2) Dehydrating the mixture obtained in the step (1) by adopting a filtering mode until the water content is 70%, preparing a nitrifying bacteria concentrated solution B, adding rhamnose ester, wherein the adding amount accounts for 0.5% of the mass of the nitrifying bacteria concentrated solution B, and purging with nitrogen for 120min in the mixing process.
(3) And (3) centrifugally dewatering the mixture obtained in the step (2) until the water content is 40%, preparing a nitrobacteria concentrated solution C, and performing vacuum drying under the conditions that the pressure is 1.5kPa and the temperature is 45 ℃ to obtain the nitrobacteria dry powder microbial inoculum. Compared with the equivalent nitrobacteria suspension in the step (1), the obtained nitrobacteria dry powder has equivalent microbial quantity and 73 percent of cell survival rate.
The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 12 months, is washed by buffer solution and is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to more than 90 percent from the initial 8 percent after 22 days.
Example 3
(1) And (3) performing gravity settling on the nitrobacteria suspension, then removing supernatant to obtain a nitrobacteria concentrated solution A with the water content of 90%, then adding a sodium alginate solution with the mass fraction of 0.5%, wherein the mass ratio of the sodium alginate to the nitrobacteria concentrated solution A is 1:5000, and introducing air for aeration for 16 h.
(2) And (2) dehydrating the mixture obtained in the step (1) by adopting a filtering mode until the water content is 80%, preparing a nitrifying bacteria concentrated solution B, adding sucrose ester, wherein the adding amount accounts for 1% of the mass of the nitrifying bacteria concentrated solution B, and purging with nitrogen for 180min in the mixing process.
(3) And (3) centrifugally dewatering the mixture obtained in the step (2) until the water content is 50%, preparing a nitrobacteria concentrated solution C, and performing vacuum drying under the conditions that the pressure is 2kPa and the temperature is 55 ℃ to obtain the nitrobacteria dry powder microbial inoculum. Compared with the equivalent nitrobacteria suspension in the step (1), the obtained nitrobacteria dry powder has equivalent microbial quantity and 71 percent of cell survival rate.
The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after 12 months, is washed by buffer solution and is added into a 250mL reactor for aeration activation, and the removal rate of ammonia nitrogen is improved to more than 90 percent from the initial 7 percent after 25 days, which shows that the activity of the dry powder microbial inoculum of nitrobacteria prepared by the method of the invention is quickly recovered.
Example 4
The preparation method and the process are the same as example 1, except that the nitrobacteria concentrated solution C is prepared in the step (3), then hyaluronic acid with the mass fraction of 0.1% is added, the mass ratio of the added hyaluronic acid to the nitrobacteria concentrated solution C is 1:1000, and the mixture is uniformly mixed under stirring and then is dried in vacuum.
Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has equivalent microbial quantity and 85 percent of cell survival rate. The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 18 months, is washed by buffer solution, is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to more than 90% from the initial 11% after 17 days, thereby further improving the activity recovery effect.
Example 5
The preparation method and the process are the same as example 2, except that the nitrobacteria concentrated solution C is prepared in the step (3), then hyaluronic acid with the mass fraction of 0.5% is added, the mass ratio of the added hyaluronic acid to the nitrobacteria concentrated solution C is 1:5000, and vacuum drying is carried out after uniform mixing under stirring.
Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has equivalent microbial quantity and 83% of cell survival rate. The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 18 months, is washed by buffer solution, is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to more than 90% from the initial 10% after 20 days, thereby further improving the activity recovery effect.
Example 6
The preparation method and procedure were the same as in example 4, except that sodium hyaluronate was used instead of hyaluronic acid. Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has equivalent microbial quantity and 84 percent of cell survival rate. The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 18 months, is washed by buffer solution and is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to more than 90 percent from 11 percent after 18 days.
Example 7
The preparation method and procedure were the same as in example 5, except that sodium hyaluronate was used instead of hyaluronic acid. Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has equivalent microbial quantity and 82% of cell survival rate. The dry powder microbial inoculum is stored at room temperature after being vacuum-packed, is taken out after being stored at room temperature for 12 months, is washed by buffer solution and is added into a 250mL reactor for aeration activation, and the removal rate of ammonia nitrogen is improved to more than 90 percent from the initial 10 percent after 21 days.
Comparative example 1
The preparation method and procedure were the same as in example 1, except that sodium alginate was used in both step (1) and step (2). Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the cell survival rate of the obtained nitrobacteria dry powder is only 30 percent. After being preserved for 6 months at room temperature, the solution is taken out and washed by buffer solution, and then the solution is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to 30 percent from the initial 3 percent after 20 days. Therefore, in the preparation process of the nitrobacteria dry powder microbial inoculum, the effect is not ideal by only adding sodium alginate.
Comparative example 2
The preparation method and procedure were the same as in example 1, except that sugar esters were used in both step (1) and step (2). Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has the cell survival rate of only 40 percent. After being stored for 1 year at room temperature, the solution is taken out and washed by buffer solution, and then the solution is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is only improved to 40 percent from the initial 4 percent after 20 days. Therefore, only sugar esters are added in the preparation process of the nitrobacteria dry powder microbial inoculum, and the effect is not ideal.
Comparative example 3
The preparation method and the process are the same as example 1, except that the dehydration rate of the steps (1) and (2) is 85 percent. Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has the cell survival rate of only 59 percent. After being stored for 1 year at room temperature, the solution is taken out and washed by buffer solution, and then the solution is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is only improved to 55 percent from the initial 6 percent after 20 days.
Comparative example 4
The preparation and procedure were as in example 1, except that the dehydration rate in each step was 40%. Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has the cell survival rate of only 49 percent. After being stored for 1 year at room temperature, the solution is taken out and washed by buffer solution, and then the solution is added into a 250mL reactor for aeration activation, and the ammonia nitrogen removal rate is improved to 45 percent from the initial 5 percent after 20 days.
Comparative example 5
The preparation method and procedure were the same as in example 1, except that air was introduced in both steps (1) and (2). Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has the cell survival rate of only 59 percent.
Comparative example 6
The preparation method and procedure were the same as in example 1, except that nitrogen gas was introduced in both steps (1) and (2). Compared with the equivalent nitrobacteria suspension before the preparation of the dry powder, the obtained nitrobacteria dry powder has the cell survival rate of only 53 percent.
Claims (13)
1. A preparation method of a nitrobacteria dry powder microbial inoculum is characterized by comprising the following steps:
(1) dehydrating the nitrobacteria suspension until the water content is higher than 80%, preparing a nitrobacteria concentrated solution A, adding sodium alginate, and reacting under an aeration condition;
(2) dehydrating the mixture obtained in the step (1) until the water content is 60-80%, preparing a nitrobacteria concentrated solution B, adding sugar ester substances, mixing and purging with nitrogen;
(3) and (3) dehydrating the mixture obtained in the step (2) until the water content is lower than 50%, preparing a nitrobacteria concentrated solution C, and performing vacuum drying to obtain the nitrobacteria dry powder microbial inoculum.
2. The method of claim 1, wherein: dehydrating the nitrifying bacteria suspension until the water content is 85% -95% to prepare a nitrifying bacteria concentrated solution A.
3. The method of claim 1, wherein: adding 0.1-1% of sodium alginate solution by mass.
4. A method according to claim 1 or 3, characterized in that: the mass ratio of the sodium alginate to the nitrobacteria concentrated solution A in the step (1) is 1: 1000-10000.
5. The method of claim 1, wherein: the aeration condition in the step (1) is that air is introduced, and the aeration time is 6-24 h.
6. The method of claim 1, wherein: the sugar ester substance in the step (2) comprises at least one of rhamnose ester, trehalose glycolipid, sophorolipid and sucrose ester, preferably trehalose ester.
7. The method according to claim 1 or 6, characterized in that: the adding amount of the sugar ester substances in the step (2) accounts for 0.1-0.5% of the mass of the nitrobacteria concentrated solution B.
8. The method of claim 1, wherein: and (3) carrying out nitrogen purging in the mixing process in the step (2), wherein the purging time is 30-180 min.
9. The method of claim 1, wherein: and (4) dehydrating the mixture obtained in the step (3) until the water content is 30-50%.
10. The method of claim 1, wherein: adding hyaluronic acid or/and sodium hyaluronate into the nitrifying bacteria concentrated solution C in the step (3), wherein the mass ratio of the hyaluronic acid or/and sodium hyaluronate to the nitrifying bacteria concentrated solution C is 1: 1000-10000.
11. The method according to claim 1 or 10, characterized in that: adding 0.1-1% hyaluronic acid solution or/and sodium hyaluronate solution in the step (3).
12. The method of claim 1, wherein: the vacuum drying in the step (3) is carried out under the conditions that the pressure is 1-2kPa and the temperature is 40-60 ℃.
13. A preservation method of nitrifying bacteria, which is characterized in that a dry powder microbial inoculum of the nitrifying bacteria is prepared by the method of any one of claims 1 to 12, and the dry powder microbial inoculum is vacuum-packed and preserved at room temperature.
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