CN109161513A - One plant of Sphingobacterium and its application - Google Patents
One plant of Sphingobacterium and its application Download PDFInfo
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- CN109161513A CN109161513A CN201811346777.8A CN201811346777A CN109161513A CN 109161513 A CN109161513 A CN 109161513A CN 201811346777 A CN201811346777 A CN 201811346777A CN 109161513 A CN109161513 A CN 109161513A
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- sphingobacterium
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- 241001136275 Sphingobacterium Species 0.000 title claims abstract description 75
- 244000005700 microbiome Species 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 16
- 239000002351 wastewater Substances 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims description 18
- 230000015556 catabolic process Effects 0.000 claims description 15
- 238000006731 degradation reaction Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 239000010865 sewage Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 238000009472 formulation Methods 0.000 claims description 8
- 239000003094 microcapsule Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 5
- 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 description 3
- 241000685602 Sphingobacterium sp. Species 0.000 claims description 3
- 239000000661 sodium alginate Substances 0.000 claims description 3
- 235000010413 sodium alginate Nutrition 0.000 claims description 3
- 229940005550 sodium alginate Drugs 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 230000007613 environmental effect Effects 0.000 claims description 2
- 230000002906 microbiologic effect Effects 0.000 claims description 2
- 239000011162 core material Substances 0.000 claims 3
- 239000000463 material Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 19
- 238000012545 processing Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 26
- 239000001963 growth medium Substances 0.000 description 13
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 10
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 8
- 239000000701 coagulant Substances 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000000813 microbial effect Effects 0.000 description 6
- 229910002651 NO3 Inorganic materials 0.000 description 5
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 5
- 101150004639 nirK gene Proteins 0.000 description 5
- 101150027124 nirS gene Proteins 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 5
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 4
- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 101100490994 Aeromonas hydrophila amoA gene Proteins 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 101100490996 Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298) amoA2 gene Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229910052564 epsomite Inorganic materials 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000001546 nitrifying effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Substances [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 235000013619 trace mineral Nutrition 0.000 description 3
- 239000011573 trace mineral Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 108010025915 Nitrite Reductases Proteins 0.000 description 2
- 241000108664 Nitrobacteria Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 101150083634 amoA gene Proteins 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000012851 eutrophication Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 238000006396 nitration reaction Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000009938 salting Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229910019629 (NH4)6Mo7O2·4H2O Inorganic materials 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001344672 Liberibacter crescens BT-1 Species 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 239000000149 chemical water pollutant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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- 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
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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Abstract
The invention discloses one plant of Sphingobacterium and its applications, belong to bioengineering, field of environment engineering technology.One plant of Sphingobacterium of the invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2018, deposit number is CGMCC No.16596, the bacterial strain has heterotrophic nitrification, aerobic denitrification characteristic, it can be applied to nitric wastewater processing, river regulation field, it is greater than 90% and no pollution to the environment in the removal rate to ammonia nitrogen, achieve the effect that quick bio denitrogenation, restore the ability of the river self-purification, is of great significance in environment water treatment field.
Description
Technical field
The present invention relates to one plant of Sphingobacterium and its applications, belong to bioengineering, field of environment engineering technology.
Background technique
Ammonia-nitrogen content is exceeded in river, lake frequently results in water eutrophication, and dissolved oxygen in water is reduced, this has become China
One of the main reason for water pollution.Ammonia nitrogen is mainly derived from the use of farmland fertilizer, the animal excreta that aquaculture generates in water body
Just sewage etc., these polluters enter lake river by rainwash and bring a large amount of nitrate nitrogen and ammonia nitrogen into, cause water body
Deterioration and water ecological environment structural damage.
In traditional biological denitrificaion theory, ammonia nitrogen is gradually converted to nitrite nitrogen, nitre state through Autotrophic nitrification, nitrite bacteria
Nitrogen, then be converted to nitrogen from environment through denitrifying bacterium effect and remove.Due to actual environment complexity, the two parts are true
It is than relatively difficult to achieve in the ecosystem.
Facultative heterotrophic nitrification be not only only capable made from aerobic denitrifying bacteria nitrifying process and denitrification at process it is synchronous carry out at
To be possible, and growth rate is much larger than autotrophic bacterium, substantially reduces growth cycle,.The product of nitrifying process can be directly becoming
The substrate of denitrification process avoids nitration product accumulation to the inhibiting effect of nitration reaction, greatly improves biological denitrificaion efficiency.
Whole process is able to maintain soda acid relative equilibrium, the product of denitrification can supplement ambient basicity, to be to be maintained at pH
Within a certain range.Most of facultative heterotrophic nitrification and aerobic denitrifying bacteria environmental suitability are strong, are suitble to administer pollution in wide area
Waters.
Currently, researcher is also seldom for the research of facultative heterotrophic nitrification and aerobic denitrifying bacteria, and only grind
Studying carefully also mostly is to focus on its mechanism of degradation.Therefore, physio-biochemical characteristics to facultative heterotrophic nitrification and aerobic denitrifying bacteria and de-
Nitrogen efficiency carries out further investigation and is necessary, and is worth with important theory and actual application.
Summary of the invention
The first purpose of the invention is to provide one plant of Sphingobacterium (Sphingobacterium sp.), in 2018
October 17 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
No.16596, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
A second object of the present invention is to provide the microorganism formulations containing Sphingobacterium.
In one embodiment of the invention, in the microorganism formulation Sphingobacterium viable bacteria content >=1 ×
109cfu/g。
In one embodiment of the invention, the microorganism formulation is bio-microcapsule.
In one embodiment of the invention, the preparation method of the bio-microcapsule are as follows: (1) by active carbon 10-
50g/L, chitosan 5-50g/L, sodium alginate 10-50g/L, which are weighed, to be dissolved and is uniformly mixed, and coagulant liquid is obtained；(2) by 50-
100g solid-state thallus accesses in coagulant liquid, and obtaining cell concentration is 1 × 108-1×1011The microorganism coagulant liquid of cfu/ml；(3)
Microorganism coagulant liquid is instilled into the CaCl that mass concentration is 1-50g/L according to 15-25 drop/minute speed2In solution, obtain micro-
It is biological active granulated；(4) microbial activity particle is crosslinked 24 hours at room temperature in 5% glutaraldehyde solution；(5) by (4)
After microbial activity particle after middle crosslinking is impregnated 24-48 hours with sterile water, proliferation obtains the micro- glue of Sphingobacterium BT1 biology
Capsule.
A second object of the present invention is to provide the applications of the Sphingobacterium.
In one embodiment of the invention, the application includes degradation river, lake pollution object or sewage treatment etc..
In one embodiment of the invention, the application is handled nitric wastewater.
In one embodiment of the invention, it is described to nitric wastewater carry out processing be by the Sphingobacterium BT1
It is thrown in nitric wastewater by 10~30mg of final concentration thallus/L sewage.
In one embodiment of the invention, the Sphingobacterium BT1 is thrown to water in the form of bio-microcapsule
In body.
The utility model has the advantages that the bacterial strain has heterotrophic nitrification, aerobic denitrification special the present invention provides one plant of Sphingobacterium
Property, it can be applied to nitric wastewater processing, river regulation field, be greater than 90% and to environment without dirt in the removal rate to ammonia nitrogen
Dye achievees the effect that quick bio denitrogenation, restores the ability of the river self-purification, is of great significance in environment water treatment field.
Biomaterial preservation
One plant of Sphingobacterium (Sphingobacterium sp.) is preserved in China Microbiological on October 17th, 2018
Culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.16596, and preservation address is court of Beijing
The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica.
Detailed description of the invention
Fig. 1 is Sphingobacterium BT1 growth and ammonia nitrogen degradation curve；
Fig. 2 is Sphingobacterium BT1 to nitrate nitrogen degradation change curve；
Fig. 3 is Sphingobacterium BT1 to nitrous nitrogen degradation change curve；
Fig. 4 is the gel electrophoresis figure of amoA gene verifying；Wherein, Marker:DL2000 (100bp~2000bp)；1: base
Because of segment: amoA；
Fig. 5 is the gel electrophoresis figure of nirK gene verifying；Wherein, Marker:DL2000 (100bp~2000bp)；1: base
Because segment is nirS；2: genetic fragment nirK.
Specific embodiment
Heterotrophic nitrification culture medium (g/L): (NH4)2SO4 0.47,C4H4Na2O45.62, Vickers salting liquid 50ml, C/N=
10, pH=7.0, agar 2%.
Vickers salting liquid (g/L): K2HPO45.0, MgSO4·7H2O 2.5, NaCl 2.5, MnSO4·4H2O 0.05,
FeSO4·7H2O 0.05。
Aerobic denitrification culture medium: nitrate culture-medium: KNO32g, sodium citrate 5g, K2HPO41g, KH2PO41g,
MgSO4·7H2O 0.2g, trace element solution 2mL, supplement distilled water to 1L, pH7.2-7.5
Nitrite culture medium: NaNO22g, sodium citrate 5g, K2HPO41g, KH2PO41g, MgSO4·7H2O
0.2g, trace element solution 2mL, supplement distilled water to 1L, pH7.2-7.5
Trace element solution: EDTA 50.0g, ZnSO42.2g, CaCl2·2H2O 5.5g, MnCl2·4H2O 5.06g,
FeSO4·7H2O 5.0g, (NH4)6Mo7O2·4H2O 1.1g, CuSO4·5H2O 1.57g, CoCl2·6H2O 1.61g is mended and is steamed
Distilled water is to 1L, pH 6.0
Embodiment 1
The activated sludge in certain stable period of landfill leachate aerobic fermentation tank is as separation mud.In heterotrophic nitrification
On culture medium, separation is carried out using dilution a mixing flat board method and obtains purebred Sphingobacterium BT1 bacterial strain.Extract bacterial strain sphingol bar
The genome of bacterium BT1 carries out 16S rDNA sequencing.Sequencing result is similar to the known array progress in Genbank database
Degree comparison, the results show that bacterial strain Sphingobacterium BT1 and Sphingobacterium (Sphingobacterium) sequence homology are most
Height is accredited as Sphingobacterium.
The measurement of 2 Sphingobacterium of embodiment (Sphingobacterium) Sphingobacterium BT1 ammonia nitrogen degradation ability
After Sphingobacterium (Sphingobacterium) Sphingobacterium BT1 is activated enrichment culture, it is suspended to pipette 1ml
Liquid, without in the heterotrophic nitrification culture medium of agar, at 30 DEG C, is cultivated 48 hours to containing 100ml under the conditions of 160rpm/min.Often
Culture solution was taken every 8 hours.Part culture solution is taken to measure the growth curve of Sphingobacterium BT1 at 600nm, then remaining culture
Liquid detects the indexs such as filtrate ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and COD through 0.22 μm of filtering with microporous membrane.As a result such as table 1 and Fig. 1 institute
Show.
Variation of the 1 Sphingobacterium BT1 of table to ammonia nitrogen degradation rate
By table 1OD600The growth curve of the available bacterial strain of index.Bacterial strain only needs to enter by the lag phase of 8h
Exponential phase, in for 24 hours, OD value has reached 1.349 from 0.01, enters stationary phase afterwards for 24 hours, with common nitrobacteria growth cycle
It compares, shortens 24-48 hours.
In 40h, ammonia nitrogen is down to 4.49mg/L from 90.21mg/L, and ammonia nitrogen degradation rate reaches 95.01%, total nitrogen from
93.77mg/L is down to 5.11mg/L, and the degradation rate of total nitrogen is 94.65%.
It should be noted that nitrous nitrogen content builds up to 0.3mg/L from 0mg/L in 48 hours, nitrate nitrogen is dropped from 0.6mg/L
Down to 0.05mg/L, show that Sphingobacterium BT1 has accumulation nitrite nitrogen and degradation nitrate nitrogen ability, thus it is presumed that sheath
Ammonia alcohol bacillus BT1 may have the ability of aerobic denitrification.
Compared with common nitrobacteria or anaerobic denitrifying bacteria, Sphingobacterium BT1 has both heterotrophic nitrification and becomes reconciled
Oxygen denitrification function, nitrifying process and denitrification process can carry out simultaneously, and the product of nitrification can be directly as denitrifying substrate.
As it can be seen from table 1 Sphingobacterium BT1 growth cycle is short, plateau can reach in 24 hours, growth rate is much larger than certainly
Support nitrifier；Environment-adapting ability is strong, strong to the tolerance of ammonia nitrogen in high density.
The measurement of 3 Sphingobacterium of embodiment (Sphingobacterium) BT1 aerobic denitrification ability
(1) denitrification capability of Sphingobacterium BT1 is screened and is characterized using nitrate culture-medium.
After Sphingobacterium (Sphingobacterium) BT1 is activated enrichment culture, 1ml suspension is pipetted to containing
100ml at 30 DEG C, is cultivated, often without in the nitrate culture-medium or nitrite culture medium of agar under the conditions of 160rpm/min
Culture solution was taken to measure bacterial concentration (OD respectively every 8 hours600), nitrate nitrogen, nitrite nitrogen.
2 Sphingobacterium BT1 of table, which degrades to nitrate nitrogen, to be changed
Since table 2 and Fig. 2 it is found that Sphingobacterium BT1 was grown the 46th hour, reached peak value at 80 hours.100
In hour, nitrate nitrogen drops to 133.32mg/L from 240.42mg/L, and nitrate nitrogen degradation rate is 44.5%, and nitrite is tired from 0
Product shows that Sphingobacterium BT1 is preferable to the removal rate of nitrate nitrogen, has stronger denitrification function to 40.59mg/L.
(2) denitrification capability of Sphingobacterium BT1 is screened and is characterized using nitrite culture medium.In nitric acid
On the basis of salt culture medium, potassium nitrate therein is replaced with into sodium nitrite.
3 Sphingobacterium BT1 of table changes nitrous nitrogen degradation
As can be seen that strain is grown the 27th hour after inoculation since table 3 and Fig. 3, reached peak at 68 hours.?
In 100 hours, nitrous nitrogen content is down to 375.97mg/L from 439.86mg/L, and content of nitrite has dropped 14.5%, nitre state
Nitrogen is down to 53.13mg/L from 80.27mg/L, and content has dropped 33.8%.
The effect of comprehensive Sphingobacterium BT1 degradation NO3-N and NO2-N, it can be deduced that, Sphingobacterium BT1 with
When nitrate or nitrite are nitrogen source, a 25-40 hours incubation period is had, the logarithmic growth phase of bacterial strain is that 12-22 is small
When, stationary phase and decline phase are initially entered after 30 hours.Sphingobacterium BT1 can utilize merely nitrate or nitrite
As nitrogen source, achieve the effect that denitrogenation, to prove that Sphingobacterium BT1 has stronger denitrification function.
The measurement of 4 Sphingobacterium of embodiment (Sphingobacterium) Sphingobacterium BT1 aerobic denitrification ability
(1) being verified using PCR whether there is nitrification function gene amoA in Sphingobacterium BT1.Primer sequence is as follows:
amoA1F(5'-GGGGTTTCTACTGGTGGT-3')；
amoA2R(5’-CCCCTCKGSAAAGCCTTCTTC-3’)
Reaction condition: 95 DEG C of 3min；94 DEG C of 1min, 54.5 DEG C of 45s, 72 DEG C of 1min, 35 circulations；72 DEG C, 10min；PCR
Product is detected using 0.8% agarose gel electrophoresis, as a result as shown in Figure 4:
PCR product is the band of 491bp size, consistent with amoA gene order through being sequenced, it is possible thereby to confirm sphingol
There are amoA genes in bacillus BT1, have nitrification function.
(2) denitrification function gene Nir:Nir gene is divided into two kinds, is nirK and nirS respectively.NirK gene encodes Cu
Type nitrite reductase, another nirS gene Codocyte pigment reductase.It should be noted that both encoding genes
It will not exist simultaneously in same strain bacterium.Being verified using PCR whether there is nirK and nirS gene in Sphingobacterium BT1.Draw
Object sequence is as follows:
nirS-cd3aF(5’-GTSAACGTSAAGGARACSGG)
nirS-R3cd(5’-GASTTCGGRTGSGTCTTGA)
nirK-F1aCu(5’-ATCATGGTSCTGCCGCG)
nirK-R3Cu(5’-GCCTCG ATCAGRTTGTGGTT)
Reaction condition: 95 DEG C of 10min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72 DEG C, 10min；PCR is produced
Object is detected using 0.8% agarose gel electrophoresis, as a result as shown in Figure 5.
It can be seen from the figure that nirS gene is not present in bacterial strain Sphingobacterium BT1, there are nirK genes.Show
There are Cu type nitrite reductase in Sphingobacterium BT1, the aerobic denitrification function that genotype is showed with it is coincide.
Embodiment 5
It is anti-according to 1% volume ratio investment DN after Sphingobacterium BT1 is cultivated 24 hours on heterotrophic nitrification culture medium
It answers in device, DN reactor is efficient denitrification reactor, inside has the building of the cores such as water distribution system and three phase separator, and set Gao Jing
Than > 2, controllable parameter HRT, when HRT is set as 48h, reactor operating parameter is as follows:
The above parameter shows that Sphingobacterium BT1 can efficiently remove ammonia nitrogen in practical applications, within 48 hours,
Ammonia-nitrogen content is reduced to 35mg/L from 2000mg/L, and removal rate has reached 40.9mg/L*h, meanwhile, total nitrogen (TN) content exists
It is reduced to 200mg/L, COD to be reduced to 700mg/L, the degradation rate difference of total nitrogen and COD from 8000mg/L from 2500mg/L in 48h
92% and 91.25% are reached.DO is remained at less than 1mg/L, this shows concentration requirement of the Sphingobacterium BT1 to dissolved oxygen
It is low, it is adaptable, especially suitable for administering eutrophication, high ammonia nitrogen water body.
Application of the 6 bacterial strain Sphingobacterium BT1 of embodiment in terms of nitric wastewater processing
In being tested using Sphingobacterium BT1 to the processing of Nanjing sewage treatment plant nitric wastewater, according to the dense of 20ppm
Degree is in aerobic tank front end stream plus Sphingobacterium BT1 bacterium solution.In 48 hours, ammonia-nitrogen content is reduced to 5mg/L, ammonia nitrogen from 20mg/L
Removal rate has reached 75%, and day processing sewage quantity can reach 55000-60000m3, show that Sphingobacterium BT1 is big for handling
When scale sewage, the ammonia nitrogen concentration in nitric wastewater can be effectively reduced.
Application of the 7 bacterial strain Sphingobacterium BT1 of embodiment at microbial immobilized aspect
By Sphingobacterium BT1 bacterial strain in conjunction with bio-carrier, it is prepared into one kind and is suitable for river water body and can slowly release
The immobilized microorganism put.
The step of preparing immobilization bacterial strain are as follows:
The fermented and cultured of bacterial strain；Sphingobacterium is carried out in mechanical agitation type fermentor by the way of liquid fermentation
The pure culture of BT1, culture medium are heterotrophic nitrification fluid nutrient medium.After progress pure culture reaches 12-48 hours at 30 DEG C, obtain
The Sphingobacterium BT1 pure culture bacterium solution of high concentration, bacterium solution count plate is 1 × 108-1×1011cfu/ml。
The preparation of solid-state thallus: after strain fermentation, centrifugation removal fermentation liquid obtains solid-state thallus.
The preparation and immobilization of bio-carrier: (1) by active carbon 10-50g/L, chitosan 5-50g/L, sodium alginate 10-
50g/L, which is weighed, to be dissolved and is uniformly mixed, and coagulant liquid is obtained.(2) 50-100g solid-state thallus is accessed in coagulant liquid, obtains thallus
Concentration is 1 × 108-1×1011The microorganism coagulant liquid of cfu/ml.(3) by microorganism coagulant liquid according to 15-25 drop/minute speed
Degree instills the CaCl that mass concentration is 1-50g/L2In solution, microbial activity particle is obtained.(4) by microbial activity particle in
It is crosslinked at room temperature with 1:50 24 hours in 5% glutaraldehyde solution.(5) by the microbial activity particle nothing after crosslinking in (4)
After bacterium water impregnates 24-48 hours, proliferation obtains Sphingobacterium BT1 bio-microcapsule.
Up to 85% or more, bacterium survival rate is greater than burst size of the Sphingobacterium BT1 bio-microcapsule of acquisition in 30 days
5%, living bacteria count reaches 10 after verifying9cfu/g。
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>one plants of Sphingobacteriums and its applications
<160> 6
<170> PatentIn version 3.3
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<213>artificial sequence
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ggggtttcta ctggtggt 18
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gtsaacgtsa aggaracsgg 20
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gcctcgatca grttgtggtt 20
Claims (10)
1. one plant of Sphingobacterium (Sphingobacterium sp.) is preserved in China Microbiological bacterium on October 17th, 2018
Kind preservation administration committee common micro-organisms center, deposit number are CGMCC No.16596, and preservation address is Beijing's southern exposure
The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica.
2. the microorganism formulation containing Sphingobacterium described in claim 1.
3. microorganism formulation according to claim 2, which is characterized in that viable bacteria content >=1 of Sphingobacterium ×
109CFU/g。
4. microorganism formulation according to claim 2, which is characterized in that the microorganism formulation is bio-microcapsule.
5. microorganism formulation according to claim 4, which is characterized in that the bio-microcapsule includes shell and shell
Internal portion core material；Case material includes but is not limited to sodium alginate gel；Enclosure interior core material includes but is not limited to that calcium chloride is molten
Liquid is dispersed with 1 × 10 in the enclosure interior core material8~1 × 1011The cell of Sphingobacterium described in CFU/mL.
6. Sphingobacterium described in claim 1 is in terms of chemical industry, environmental area degradation river, lake pollution object or sewage treatment
Application.
7. application according to claim 6, which is characterized in that the sewage is nitric wastewater.
8. a kind of sewage water treatment method, which is characterized in that Sphingobacterium described in claim 1 is pressed 10~30mg of final concentration
Thallus/L sewage is thrown in sewage.
9. according to the method described in claim 8, it is characterized in that, the Sphingobacterium is launched in the form of bio-microcapsule
Into water body.
10. the appliance arrangement that bacterial strain described in application claim 1 carries out sewage treatment.
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| CN109825450A (en) * | 2019-01-17 | 2019-05-31 | 广东博沃特生物科技有限公司 | One plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its application |
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