CN107345208A - A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage - Google Patents
A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage Download PDFInfo
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- CN107345208A CN107345208A CN201710467140.3A CN201710467140A CN107345208A CN 107345208 A CN107345208 A CN 107345208A CN 201710467140 A CN201710467140 A CN 201710467140A CN 107345208 A CN107345208 A CN 107345208A
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- Prior art keywords
- sewage
- green alga
- phosphorus
- application
- nitrogen
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 239000010865 sewage Substances 0.000 title claims abstract description 34
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 30
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000011574 phosphorus Substances 0.000 title claims abstract description 27
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 27
- 238000000407 epitaxy Methods 0.000 title description 4
- 241000195493 Cryptophyta Species 0.000 claims abstract description 34
- 241000195628 Chlorophyta Species 0.000 claims abstract description 13
- 239000002351 wastewater Substances 0.000 claims abstract description 11
- 241000195627 Chlamydomonadales Species 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 241000304537 Tetradesmus Species 0.000 claims abstract description 5
- 241000719329 Trentepohlia Species 0.000 claims abstract description 5
- 241000180279 Chlorococcum Species 0.000 claims abstract description 4
- 241000196319 Chlorophyceae Species 0.000 claims abstract description 4
- 241000195655 Scenedesmaceae Species 0.000 claims abstract description 4
- 238000009395 breeding Methods 0.000 claims description 6
- 230000001488 breeding effect Effects 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 3
- 238000009360 aquaculture Methods 0.000 abstract 1
- 244000144974 aquaculture Species 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 26
- 239000000243 solution Substances 0.000 description 20
- 239000008367 deionised water Substances 0.000 description 12
- 229910021641 deionized water Inorganic materials 0.000 description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000012549 training Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241000304538 Tetradesmus acuminatus Species 0.000 description 4
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 4
- 235000010323 ascorbic acid Nutrition 0.000 description 4
- 229960005070 ascorbic acid Drugs 0.000 description 4
- 239000011668 ascorbic acid Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910002567 K2S2O8 Inorganic materials 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 235000018660 ammonium molybdate Nutrition 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 3
- 230000009191 jumping Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 235000010333 potassium nitrate Nutrition 0.000 description 3
- 239000004323 potassium nitrate Substances 0.000 description 3
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- KQROHCSYOGBQGJ-UHFFFAOYSA-N 5-Hydroxytryptophol Chemical compound C1=C(O)C=C2C(CCO)=CNC2=C1 KQROHCSYOGBQGJ-UHFFFAOYSA-N 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- 108010022172 Chitinases Proteins 0.000 description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000011609 ammonium molybdate Substances 0.000 description 2
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 2
- 229940010552 ammonium molybdate Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- UFMZWBIQTDUYBN-UHFFFAOYSA-N cobalt dinitrate Chemical compound [Co+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O UFMZWBIQTDUYBN-UHFFFAOYSA-N 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000000243 photosynthetic effect Effects 0.000 description 2
- IIQJBVZYLIIMND-UHFFFAOYSA-J potassium;antimony(3+);2,3-dihydroxybutanedioate Chemical class [K+].[Sb+3].[O-]C(=O)C(O)C(O)C([O-])=O.[O-]C(=O)C(O)C(O)C([O-])=O IIQJBVZYLIIMND-UHFFFAOYSA-J 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229910003208 (NH4)6Mo7O24·4H2O Inorganic materials 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria 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
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- -1 Polyethylene Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000003816 axenic effect Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid group Chemical class C(CC(O)(C(=O)O)CC(=O)O)(=O)O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- MEFBJEMVZONFCJ-UHFFFAOYSA-N molybdate Chemical compound [O-][Mo]([O-])(=O)=O MEFBJEMVZONFCJ-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/89—Algae ; Processes using algae
-
- 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
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
-
- 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
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
-
- 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
- C12N1/12—Unicellular algae; Culture media therefor
-
- 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
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
-
- 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
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of green alga, it is identified, it is under the jurisdiction of Chlorophyta (Chlorophyta), Chlorococcum guiding principle (Chlorophyceae), Chlorococcale (Chlorococcales), Shan Zao sections (Scenedesmaceae), four chain Trentepohlias (Tetradesmus), the entitled WU4 of algae strain, is deposited in China typical culture collection center, and deposit number is：CCTCC M 2017335, preservation date are：On June 14th, 2017.The Novel green algae can be used for removing the nitrogen and phosphorus in sewage, significant effect.Relative to prior art, Novel green algae of the present invention, applied to the removal of nitrogen and phosphorus in aquaculture wastewater under cryogenic conditions, application process is simple, and removal efficiency of nitrogen and phosphorus is high, significant effect.
Description
Technical field
The present invention relates to a kind of new low-temperature epitaxy algae and the biology with organic pollution in its Environment control
(2), the server end analyzes and processes to the answer data, judges whether answer person's handwriting is effective, if invalid.
Background technology
In recent years, under the vigorously supporting of agricultural sector, large-scale cultivation development in China's is swift and violent, effective guarantee consumer
The demand supply growing to meat product, brings huge industrial economy benefit；It is but big caused by large-scale cultivation accumulation
Sewage and random discharge are measured, great pollution is caused to ecological environment, while also easily triggers epidemic disease to propagate and spread, seriously
Threaten human health and social harmony is stable.Wherein pig excrement and sewage category high concentration organic sewage, the content of nitrogen and phosphorous is big, directly arranges
Body eutrophication can be induced by putting, and cause seriously to pollute to water body and ecological environment of soil, be national environmental protection department supervision
Emphasis be also prevention and control difficult point.Pig excrement and sewage middle and high concentration nitrogen, the content of phosphorus how are effectively reduced, it is relevant to make up to country
Defined discharge standard, ecological environment security is not only concerned, while be also that can pig-breeding industry the sustainable health hair of scale
The important restriction factor of exhibition, it has also become the hot subject that multi-crossed disciplines exploratory development urgently solves.
Green alga is a kind of photosynthetic autotrophs biology, have photosynthetic efficiency is high, growth cycle is short, breeding is fast, strong adaptability, training
The features such as simple is supported, it needs to absorb nitrogen, the nutriment such as phosphorus in environment in growth course, to complicated in compound body
Macromolecular organic matter, so as to reduce the content of the organic substances such as Water, phosphorus, reach the effect of purifying water body.
It is such as of the prior art green but the degraded that green alga is applied to nitrogen and phosphorus in sewage at present also has some defects
Algae is all the nitrogen phosphorus in degraded sewage under the normal temperature even hot environment of summer, and at low ambient temperatures, these chlorella growths
It is obstructed, nitrogen phosphorus ability step-down of degrading.
Technical scheme
Goal of the invention：
For above-mentioned technical problem, the invention provides a kind of Novel green algae, and by laboratory experiment, it is in pig excrement and sewage
Nitrogen, phosphorus organic matter have a significantly consumption degradation function, the algae and its using technology have important scientific research value and it is huge should
Use potentiality.
Technical scheme：
To reach the invention described above purpose, identified the invention discloses a kind of green alga, it is under the jurisdiction of Chlorophyta
(Chlorophyta), Chlorococcum guiding principle (Chlorophyceae), Chlorococcale (Chlorococcales), Shan Zao sections
(Scenedesmaceae), the curved sharp four chains algae (Tetradesmus acuminatus) in four chain Trentepohlias (Tetradesmus),
The entitled WU4 of algae strain, i.e. Classification And Nomenclature are：Tetradesmus acuminatus WU4, are deposited in China typical culture collection
Center, preservation address：Chinese Wuhan Wuhan Universitys, postcode：430072, deposit number is：CCTCC M 2017335, preservation
Date is：On June 14th, 2017.
The 18SrDNA nucleotides of the green alga is as shown in SEQ ID NO.1.
The condition of culture of the green alga is as follows：15 ± 2 degrees Celsius of cultivation temperature, culture matrix are BG11 culture mediums.
The green alga algae strain is by being sampled from high nitrogen and phosphorus content sanitary sewage water body, and gained is screened through culture of isolated.
Culture of isolated screening uses dilution spread method of scoring：Coating, line are that a kind of base of pure culture is obtained in microorganism
The classical way of plinth.Algae solution is subjected to proper ratio dilution, filtered using the filter membrane of 0.45 μm of filter sizes, with reference to sterile
Fluid nutrient medium washs filtration algae solution repeatedly, while uses flat board culture and picking single bacterium colony technology, so as to obtain axenic culture.
Present invention also offers the application that the green alga removes nitrogen in sewage, the temperature of application is 6-15 degrees Celsius, described
Sewage is the breeding wastewater containing high concentration N, phosphorus organic pollution, such as pig excrement and sewage etc..
Present invention also offers the application that the green alga removes phosphor in sewage, the temperature of application is 6-15 degrees Celsius, described
Sewage is the breeding wastewater containing high concentration N, phosphorus organic pollution, such as pig excrement and sewage etc..
Specific embodiment described herein is only to spirit explanation for example of the invention
Under warm environment, these chlorella growths are obstructed, nitrogen phosphorus ability step-down of degrading.Therefore, the present invention screens a kind of low temperature green alga, can be very
Good growth at low ambient temperatures, and nitrogen and phosphorus content in pig excrement and sewage can be removed under cryogenic, the significant effect of acquirement.
Technique effect
Relative to prior art, Novel green algae of the present invention has following technical advantage：
Novel green algae of the present invention, applied to the removal of nitrogen and phosphorus in pig excrement and sewage under cryogenic conditions, application process letter
Single, removal efficiency of nitrogen and phosphorus is high, significant effect.
Brief description of the drawings
Fig. 1 is the standard curve of total phosphorus in Method Measuring Total Quantity of Phosphorus；
Fig. 2 is clearance of the WU4 algaes strain of the present invention in pig excrement and sewage total phosphorus
Fig. 3 is the standard curve of total nitrogen in determination of total nitrogen content method；
Fig. 4 is clearance of the WU4 algaes strain of the present invention in pig excrement and sewage total nitrogen.
Embodiment
The present invention is further described below with reference to specific embodiment.
Involved reagent and culture medium prescription in embodiment：
(1) BG11 culture medium prescriptions：
Its constituent is：Contain 30g NaNO in 1L deionized waters3、0.8g K2HPO4、1.5g MgSO4·7H2O、
2.86g H3BO3、1.81g MnCl2·4H2O、0.22g ZnSO4·7H2O、0.39g Na2MoO·2H2O、0.08g CuSO4·
2H2O、0.05g Co(NO3)2·6H2O、1.36g CaCl2、2g Na2CO3, 0.3g ferric citrates, 0.3g citric acids,
0.05g EDTANa2。
(2)(1+1)H2SO4
(3) molybdate solution：Dissolve 13g ammonium molybdates (NH4)6MO7O24·4H2O is in 100ml deionized waters.Dissolving
0.35g potassium antimony tartrates KSbC4H4O7·1/2H2O is in 100ml deionized waters.It is in the case where being stirred continuously, ammonium molybdate solution is slow
Slowly it is added to 300ml (1+1) H2SO4In, add antimony tartrate potassium solution and be well mixed.Brown Glass Brown glass bottles and jars only, 4 DEG C of preservations.
(4) 10% ascorbic acid solutions：Dissolving 10g ascorbic acid is settled to 100ml with deionized water.Brown Glass Brown glass bottles and jars only, 4
DEG C preserve.
(5) 5%K2S2O8：Dissolve 5g K2S2O8, 100ml is settled to deionized water.
(6) phosphate stock solution：Weigh 0.2197gKH2PO4, add 5ml (1+1) H2SO4, it is settled to deionized water
1000ml。
(7) phosphate standard solution：10ml phosphate stock solution is taken to be diluted in 250ml volumetric flasks with deionized water
Graticule.Now match somebody with somebody.
(8) alkaline chitinase solution：Weigh 40g K2S2O8, 15gNaOH, 1000ml is settled to deionized water, is stored in
Polyethylene bottle.
(9) (1+9) hydrochloric acid
(10) potassium nitrate Standard Reserving Solution：Weigh 0.7218gKNO3, 1000ml is settled to deionized water.Add 2ml
Chloroform is protective agent, and 4 DEG C preserve.
(11) potassium nitrate standard solution：Stock solution is diluted 10 times with deionized water and obtained.
Concrete operation step：
(1) WU4 is separating obtained from sewage water body
Dilution spread method of scoring：Coating, line are that a kind of basic classical way of pure culture is obtained in microorganism, specifically
Step is as follows：It is acquired from high nitrogen and phosphorus content sanitary sewage water body with plankton net, the algae of collection is placed on equipped with training
In the sample bottle for supporting base.Adopting next water sample from field, impurity is many sometimes, so preliminary filtering need to be carried out to the water sample of collection
Processing.Because algae cell density than relatively low, therefore easily causes the failure being separately cultured in sample, in order to avoid directly to collection
The failure that sample is separately cultured, enrichment pretreatment first can be carried out to the sample of filtration treatment, add appropriate BG11 trainings thereto
Base is supported to accelerate the growth of target algae kind, the frustule in sample is reached the separation training for being advantageous to target algae kind after certain density
Support.Algae solution sample to be separated by enrichment culture is carried out gradient dilution with sterilized BG11 culture mediums, then flat board
It is coated with partition method and carries out algae kind separation.It is that 1% agar carries out high pressure steam sterilization to add content in the medium, after sterilizing terminates
When the temperature of nutrient solution it is cool to 50 degree or so when culture medium poured into sterile petri dish rapidly, that is, it is standby that solid medium is made
With.The algae solution of dilution is equably coated in the media surface of sterilizing with spreading rod, is then placed in low temperature and irradiance incubator
Culture, the best monoclonal algae kind of picking growing way, that is, completes the separation of new algae strain.Then sterile BG11 cultures are inoculated in
Enrichment and growth in base.
The above-mentioned enrichment algae solutions of 50ml are taken, 5000r/min, centrifuge 8min.Supernatant is removed, after then addition liquid nitrogen is fully ground,
It is placed in a clean centrifuge tube.400 μ l PlantZol are added, vibration mixes, and sample is suspended completely.Add 7.5 μ l RNase
A fully mixes into above-mentioned lysate.55 DEG C are incubated 15 minutes.Add isometric phenol-chloroform-isoamyl alcohol (25:24:1),
Vibration mixes, 12000rpm centrifugations 5min.Careful upper strata aqueous phase of drawing in clean centrifuge tube, add isometric phenol-chloroform-
Isoamyl alcohol (25:24:1) it is, fully reverse to mix.12000rpm centrifuges 5min, removes supernatant.Add 500 μ l 70% ethanol, whirlpool
Rotation vibration 5 seconds, 12000rpm centrifugation 5min, removes supernatant.1-2min is centrifuged again, and exhaust residual liquid.DNA precipitations are dried, are added
Enter 50 μ lTE, 65 DEG C of incubation 10min dissolving DNAs, during which flick hydrotropy for several times, 4 DEG C of preservations.
The primer sequence of 18SrDNA for expanding eucaryon green alga is：
- the ACCTGGTTGATCCTGCCAGTAG-3 of upstream 5 " " (SEQ ID NO.2)
- the ACCTTGTTACGACTTCTCCTTCCTCC-3 of downstream 5 " " (SEQ ID NO.3)
PCR reaction conditions：95 DEG C of denaturation 3min；95 DEG C of 30sec, 55 DEG C of 45sec, 72 DEG C of 1min, 32 circulations；Last 72
DEG C extension 10min.Amplified production holds up biology Co., Ltd of section by Nanjing and completes sequencing.
The 18SrDNA nucleotide sequences of the algae strain (WU4) are as shown in SEQ ID NO.1, by observing it under the microscope
Form and identification is compared in ncbi database, it is under the jurisdiction of Chlorophyta (Chlorophyta), Chlorococcum guiding principle
(Chlorophyceae), Chlorococcale (Chlorococcales), Shan Zao section (Scenedesmaceae), four chain Trentepohlias
(Tetradesmus) the curved sharp four chains algae (Tetradesmus acuminatus) in, the entitled WU4 of algae strain, i.e. Classification And Nomenclature are：
Tetradesmus acuminatus WU4, are deposited in China typical culture collection center, preservation address：Chinese Wuhan
Wuhan University, postcode：430072, deposit number is：CCTCC M 2017335, preservation date are：On June 14th, 2017.
(2) above-mentioned separated algae is cultivated in Nanjing Normal University's algae training room under 15 ± 2 degrees celsius with BG11
Liquid culture.
(3) hoggery sewerage is derived from Nanjing agriculture science institute pig farm.Pig-farm wastewater 0.45 μm of membrane filtration, mistake
The pig-farm wastewater of filter is used as experiment material.
The water quality condition of experiment hoggery sewerage
(4) this experiment was carried out 2 months in 2017, and temperature control is at 6-15 degrees Celsius.The pig manure for taking above-mentioned filtering good gives up
Water 40ml takes 5ml WU4 algae solutions to be added thereto in 100ml conical flasks, do three it is parallel.In order to which WU4 preferably adapts to pig manure
The environment of waste water, is repeatedly transferred.
The stand density (A750nm) of the WU4 of table 1 inoculations for the first time
Second of stand density (A750nm) transferred of the WU4 of table 2
The stand density (A750nm) of the WU4 of table 3 third time switchings
(5) survey it with the algae strain of third time switching and remove the content of nitrogen phosphorus in pig-farm wastewater.
(6) assay method of pig-farm wastewater total phosphorus is ammonium molybdate spectrophotometric method in this experiment.
1. taking 7 50ml color-comparison tubes, 0,0.5,1,3,5,10,15ml phosphate standard solution is added to it respectively,
25ml is settled to deionized water.Then 4ml 5% potassium persulfate solution is added to it, in one piece of gauze of mouth of pipe bag after jumping a queue
And tightened with rope.
2. above-mentioned colorimetric cylinder is put into large beaker, it is placed in high-pressure sterilizing pot and heats, 1.1Kg/cm is set2, 120 DEG C,
30min.When pressure indication table in pot subject to sterilization drops to zero, take out colorimetric cylinder and let cool.It is then diluted to 50ml graticules.
3. adding 1ml ascorbic acid solutions in the digestion solution 2. obtained to step respectively, mix.Add 2ml molybdates after 30s
Solution, after being stored at room temperature 15min after fully mixing, absorbance is determined at 700nm wavelength.Deduct the absorbance of blank test
Afterwards, and corresponding phosphorus content drawing curve (as shown in Figure 1).
4. water sampling 2ml, centrifuge 5000r/min, 3min.Supernatant 1ml is taken to be determined in 50ml color-comparison tubes with deionized water
Hold to 25ml.Then 4ml 5% potassium persulfate solution is added to it, is pricked in one piece of gauze of mouth of pipe bag after jumping a queue and with rope
Tightly.
5. with step 2..
6. because water sample contains color, (1+1) H of two parts of volumes of mixing is added to its digestion solution2SO4With a volume
10% ascorbic acid solution, mixed liquor will configure on the same day.After being stored at room temperature 15min after fully mixing, determined at 700nm wavelength
Absorbance.Phosphorus content is checked in from standard curve after deducting the absorbance of blank test.
The WU4 algaes strain of table 4 removes the efficiency (%) of total phosphorus in pig excrement and sewage
The strain of WU4 algaes removes the percentage of phosphorus in pig excrement and sewage, as a result as shown in Figure 2.
(7) assay method of pig-farm wastewater total nitrogen is potassium persulfate oxidation ultraviolet spectrophotometry in this experiment.
1. taking 8 25ml color-comparison tubes, adding 0,0.5,1,2,3,5,7,8ml potassium nitrate standard to it respectively uses
Liquid, and it is diluted to 10ml graticules with deionized water.
2. to above-mentioned color-comparison tube add 5ml alkaline chitinase, after jumping a queue one piece of gauze of mouth of pipe bag and with restrict
Tightened.Above-mentioned colorimetric cylinder is put into large beaker, is placed in high-pressure sterilizing pot and heats, 1.1Kg/cm is set2, 120 DEG C,
30min.When pressure indication table in pot subject to sterilization drops to most zero, colorimetric cylinder is taken out, is cooled to room temperature.
3. adding (1+9) hydrochloric acid 1ml, then 25ml graticules are diluted to deionized water.10min is stood after mixing, is used
10mm quartz colorimetric utensils survey its absorbance (A=A220-2 × A275) at 220nm and 275nm respectively.With the absorbance of correction
Draw standard curve (as shown in Figure 3).
4. water sampling 2ml, centrifuge 5000r/min, 3min.Supernatant 1ml is taken to be determined in 25ml color-comparison tubes with deionized water
Hold to 10ml.Operate below according to Specification Curve of Increasing step 2. -3..Then according to calibration absorbance, looked on calibration curve
Go out the nitrogen pool of response, then total nitrogen content is calculated with following equation.
Total nitrogen (mg/L)=m/v
In formula：M is the nitrogen content (μ g) checked in from standard curve；
V is taken volume of water sample (ml)
The WU4 algaes strain of table 5 removes the efficiency (%) of total nitrogen in pig excrement and sewage
The strain of WU4 algaes removes the percentage of nitrogen in pig excrement and sewage, as a result as shown in Figure 4.
Technical scheme is described in detail above-described embodiment, it should be understood that described above
Specific embodiment only of the invention, is not intended to limit the invention, it is all done in the spirit of the present invention any repair
Change or improve, should be included within the scope of the invention.
SEQUENCE LISTING
<110>Nanjing Normal University
<120>A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage
<130> 2017.06
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1668
<212> DNA
<213>Four chain Trentepohlias (Tetradesmus)
<400> 1
aactgcttat actgtgaaac tgcgaatggc tcattaaatc agttatagtt tatttggtgg 60
taccttacta ctcggataac cgtagtaatt ctagggctaa tacgtgcgta aatcccgact 120
tctggaaggg acgtatatat tagataaaag gccgaccgag ctttgctcga cccgcggtga 180
accatgatat cttcacgaag cgcatggcct tgagccggcg ctgttccatt caaatttctg 240
ccctatcaac tttcgatggt aggatagagg cctaccatgg tggtaacggg tgacggagga 300
ttagggttcg attccggaga gggagcctga gaaacggcta ccacatccaa ggaaggcagc 360
aggcgcgcaa attacccaat cctgatacgg ggaggtagtg acaataaata acaataccgg 420
gcatttcatg tctggtaatt ggaatgagta caatctaaat cccttaacga ggatccattg 480
gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt atatttaagt 540
tgttgcagtt aaaaagctcg tagttggatt tcgggtgggt tctagcggtc cgcctatggt 600
gagtactgct atggccttcc tttctgtcgg ggacgggctt ctgggcttca ctgtccggga 660
ctcggagtcg acgtggttac tttgagtaaa ttagagtgtt caaagcaggc ttacgccaga 720
atactttagc atggaataac acgataggac tctggcctat cttgttggtc tgtaggaccg 780
gagtaatgat taagagggac agtcgggggc attcgtattt cattgtcaga ggtgaaattc 840
ttggatttat gaaagacgaa ctactgcgaa agcatttgcc aaggatgttt tcattaatca 900
agaacgaaag ttgggggctc gaagacgatt agataccgtc gtagtctcaa ccataaacga 960
tgccgactag ggattggcga atgttttttt aatgacttcg ccagcacctt atgagaaatc 1020
aaagtttttg ggttccgggg ggagtatggt cgcaaggctg aaacttaaag gaattgacgg 1080
aagggcacca ccaggcgtgg agcctgcggc ttaatttgac tcaacacggg aaaacttacc 1140
aggtccagac atagtgagga ttgacagatt gagagctctt tcttgattct atgggtggtg 1200
gtgcatggcc gttcttagtt ggtgggttgc cttgtcaggt tgattccggt aacgaacgag 1260
acctcagcct gctaaatagt ctcagttgct ttttgcagct ggctgacttc ttagagggac 1320
tattggcgtt tagtcaatgg aagtatgagg caataacagg tctgtgatgc ccttagatgt 1380
tctgggccgc acgcgcgcta cactgatgca ttcaacaagc ctatccttga ccgaagggtc 1440
tgggtaatct ttgaaactgc atcgtgatgg ggatagatta ttgcaattat tagtcttcaa 1500
cgaggaatgc ctagtaagcg caagtcatca gcttgcgttg attacgtccc tgccctttgt 1560
acacaccgcc cgtcgctcct accgattggg tgtgctggtg aagtgttcgg attggcagct 1620
tagggtggca acacctcagg tctgccgaga agttcataaa ccctccca 1668
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
acctggttga tcctgccagt ag 22
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence
<400> 3
accttgttac gacttctcct tcctcc 26
Claims (8)
1. a kind of green alga, it is characterised in that identified, it is under the jurisdiction of Chlorophyta (Chlorophyta), Chlorococcum guiding principle
(Chlorophyceae), Chlorococcale (Chlorococcales), Shan Zao section (Scenedesmaceae), four chain Trentepohlias
(Tetradesmus), the entitled WU4 of algae strain, is deposited in China typical culture collection center, and deposit number is：CCTCC M
2017335, preservation date is：On June 14th, 2017.
2. green alga according to claim 1, it is characterised in that the 18SrDNA nucleotides of the green alga such as SEQ ID NO.1
It is shown.
3. green alga according to claim 1, it is characterised in that the condition of culture of the green alga is as follows：Cultivation temperature 15 ± 2
Degree Celsius, culture matrix is BG11 culture mediums.
4. green alga according to claim 1, it is characterised in that the green alga algae strain is dirty by being lived from high nitrogen and phosphorus content
Sampled in water water body, gained is screened through culture of isolated.
5. any one of the claim 1-4 green algas remove the application of nitrogen in sewage, it is characterised in that the temperature of the application is
6-15 degrees Celsius.
6. application according to claim 5, it is characterised in that the sewage is pig excrement and sewage or other breeding wastewaters.
7. any one of the claim 1-4 green algas remove the application of phosphor in sewage, it is characterised in that the temperature of the application is
6-15 degrees Celsius.
8. application according to claim 7, it is characterised in that the sewage is pig excrement and sewage or other breeding wastewaters.
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| CN104388315A (en) * | 2014-11-07 | 2015-03-04 | 山东大学 | Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof |
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| CN104388315A (en) * | 2014-11-07 | 2015-03-04 | 山东大学 | Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof |
| CN106754390A (en) * | 2016-12-30 | 2017-05-31 | 山东宝来利来生物工程股份有限公司 | The albuminiferous chlorella of one plant height and its cultural method and application |
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Cited By (2)
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