CN109897804B - Zoebelia with nitrification and denitrification functions and application thereof - Google Patents
Zoebelia with nitrification and denitrification functions and application thereof Download PDFInfo
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- CN109897804B CN109897804B CN201910234181.7A CN201910234181A CN109897804B CN 109897804 B CN109897804 B CN 109897804B CN 201910234181 A CN201910234181 A CN 201910234181A CN 109897804 B CN109897804 B CN 109897804B
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 94
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 claims abstract description 23
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims abstract description 23
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000004321 preservation Methods 0.000 claims description 7
- 241000235035 Debaryomyces Species 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 238000000034 method Methods 0.000 abstract description 17
- 241000752227 Zobellella Species 0.000 abstract description 13
- 238000009360 aquaculture Methods 0.000 abstract description 13
- 244000144974 aquaculture Species 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 10
- 230000006872 improvement Effects 0.000 abstract description 5
- 230000009467 reduction Effects 0.000 abstract description 2
- 241000607598 Vibrio Species 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 11
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000002131 composite material Substances 0.000 description 9
- 108020004465 16S ribosomal RNA Proteins 0.000 description 7
- 229910002651 NO3 Inorganic materials 0.000 description 7
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- 241000251468 Actinopterygii Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 125000001477 organic nitrogen group Chemical group 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 241001569440 Zobellella sp. Species 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
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- 230000001954 sterilising effect Effects 0.000 description 5
- 235000013619 trace mineral Nutrition 0.000 description 5
- 239000011573 trace mineral Substances 0.000 description 5
- 241000238557 Decapoda Species 0.000 description 4
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- 238000009472 formulation Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 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 4
- 229960000999 sodium citrate dihydrate Drugs 0.000 description 4
- 229940074404 sodium succinate Drugs 0.000 description 4
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 3
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- 238000005516 engineering process Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241001057811 Paracoccus <mealybug> Species 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000752219 Zobellella taiwanensis Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- 239000003344 environmental pollutant Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000011146 sterile filtration Methods 0.000 description 2
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- GUBGYTABKSRVRQ-DCSYEGIMSA-N Beta-Lactose Chemical compound OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-DCSYEGIMSA-N 0.000 description 1
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- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 229920004934 Dacron® Polymers 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins 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
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- MZNYWPRCVDMOJG-UHFFFAOYSA-N N-(1-naphthyl)ethylenediamine dihydrochloride Chemical compound [Cl-].[Cl-].C1=CC=C2C([NH2+]CC[NH3+])=CC=CC2=C1 MZNYWPRCVDMOJG-UHFFFAOYSA-N 0.000 description 1
- 241000080590 Niso Species 0.000 description 1
- 108010025915 Nitrite Reductases Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 239000008272 agar Substances 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
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
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- 238000012851 eutrophication Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
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- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000009344 polyculture Methods 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a Znobel bacterium with nitrification and denitrification functions and application thereof. Zobellella sp 2G-5, deposited in the Guangdong province microbial culture collection center, with the address of No. 59 Securium Dacrooki, No. 100 Yuanlu, Vibrio, Guangzhou, the date of deposit being 2019, 03, 06 days, and the number of deposit being GDMCCNo.60606. The Zubeierella 2G-5 can rapidly and efficiently remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and total nitrogen in a water body, the removal effect on the three inorganic nitrogen sources and the total nitrogen is over 98 percent, no other inorganic nitrogen sources are obviously accumulated in the process, no secondary pollution is caused, and the bacterium has great application value in the improvement of culture water quality, such as reduction of the harm of the ammonia nitrogen, the nitrite nitrogen, the nitrate nitrogen and the like to a culture object in the aquaculture process.
Description
Technical Field
The invention belongs to the technical field of microbial application and environmental engineering, and particularly relates to a strain of denitrifying bacteria of Torber zeylanica with heterotrophic nitrification and aerobic denitrification functions and application thereof in improvement of culture water quality.
Background
With the general application of the intensive aquaculture mode, part of feed is converted into ammonia nitrogen in the metabolic processes of digestion, absorption and the like of cultured animals in the culture process and then is discharged into the culture environment, and further converted into nitrite and nitrate, so that the nitrogen content in the culture water is too high. Over-high nitrogen in the water body can cause eutrophication of the water body, mass propagation of algae, consumption of dissolved oxygen in the water body, and fishy smell of the water, so that various aquatic animals such as fish and the like die in large quantities. Such as excess ammonia and nitrite, are major sources of disease in aquaculture animals such as fish, shrimp, etc. Research shows that excessive nitrite in the culture water body mainly influences the respiration of fishes and shrimps to reduce the affinity of hemoglobin in fish blood and hemocyanin in shrimp blood to oxygen, thereby generating toxicity to aquaculture animals. Excessive ammonia nitrogen (mainly NH) in aquaculture water 4 + And non-ionized NH 3 Both forms) can cause the immune activity of fish and shrimp to be forced, and can also destroy the skin, stomach and intestinal mask of the cultured animals, cause the bleeding of the body surface and internal organs, and cause the death of acute poisoning. Therefore, people pay more and more attention to finding an efficient aquaculture water quality modifier for economically and effectively removing excessive nitrogen pollutants in aquaculture water.
At present, the treatment technology for excessive nitrogen in water mainly comprises chemical, physical and biological methods, wherein the biological denitrification treatment technology overcomes the defects of the physical and chemical denitrification method, has the advantages of simple operation, wide application range, good treatment effect, basically no secondary pollution and the like, and is widely applied in recent years. Microbial denitrification is a method for researching more in biological denitrification, traditionally, denitrification process is realized under anaerobic condition, in recent years, with the deep research on denitrifying bacteria, some bacteria are found to have heterotrophic nitrification and denitrification functions under aerobic condition, and the bacteria have unique advantages in removing ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in water body, can finally reduce ammonia nitrogen or nitrate nitrogen into gas or assimilate the bacteria to be separated from aquaculture water body, have no residue, do not cause secondary pollution, improve and maintain aquaculture ecological balance, reduce disease occurrence and play an important role in aquaculture water quality improvement.
Aerobic denitrifying bacteria have diversity in physiology, biochemistry and phylogeny. Research has shown that aerobic denitrifying bacteria are mainly distributed in the genera of Pseudomonas (Pseudomonas), Alcaligenes (Alcaligenes), Paracoccus (Paracoccus), Bacillus (Bacillus), and the like, and related documents and patents are more. Regarding the bacterial strain with denitrification effect in the genus Zobellella (Zobellella), only 2 inventions are invented in the currently granted countries, and patent 201510316321.7 (application No. 2015.06.10) discloses a strain of Zobellella maritima (Zobellella sp.) CYT1-8, which has the highest similarity to the model bacterium Zobellella denitificatina ZD1 and the similarity of 16S rRNA of 99.04%, and the related bacterial strain can rapidly remove nitrate and nitrite in a water body under aerobic conditions, but can accumulate a large amount of nitrite in the nitrate removal process. Patent 201310100699.4 (application date 2013.08.07) discloses an aerobic denitrifying bacterium, namely, Zobellella sp NKDN-01, which has the highest similarity with a model bacterium, namely, Zobellella taiwanensis ZT1, has a high removal rate on high-ammonia-nitrogen wastewater, and also has a certain nitrate nitrogen removal effect, but the removal effect on nitrite nitrogen is not mentioned, and in the patent, when the bacterium is used for treating artificial high-ammonia-nitrogen wastewater, the trace nitrite nitrogen is increased finally.
Disclosure of Invention
The invention aims to provide a strain of Zobellella (Zobellella sp.)2G-5 with heterotrophic nitrification and aerobic denitrification functions, which is preserved in the culture collection center (GDMCC) of Guangdong province, and is addressed to No. 59 Securium Dacron of No. 100 Xieli of Zyguyu district, Guangdong province, with the preservation date of 2019, 03 and 06 days, and with the preservation number of GDMCCNo.60606.
The invention separates and obtains the Zubeiella 2G-5 with heterotrophic nitrification and aerobic denitrification functions, and strains are respectively inoculated with NH 4 + -N，NO 2 - -N and NO 3 - Culturing in a culture medium with-N as a unique nitrogen source at 28 ℃ under shaking at 180rpm, taking out the culture solution to measure the OD 600 of the culture solution when culturing for 0, 12, 18, 24, 36 and 48 hours respectively, taking 2.0mL of the culture solution at the same time, centrifuging, measuring ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the supernatant, and measuring total nitrogen in the supernatant and thalli, wherein the result shows that the Zubel 2G-5 is applied to (NH) in the water body respectively 4 ) 2 SO 4 、NaNO 2 And KNO 3 All have quick and efficient removal effect. Culturing the strain in a culture medium with nitrite nitrogen as a unique nitrogen source for 48 hours, and then culturing NO 2 - The removal rates of N and total nitrogen are 99.00% and 99.60%, wherein 27.08% of the inorganic nitrogen is assimilated into bacterial organic nitrogen; in a culture medium with nitrate nitrogen as a unique nitrogen source, the strain is cultured for 48 hours and then NO is added 3 - The removal rates of N and total nitrogen are 99.58% and 99.84%, wherein 29.72% of inorganic nitrogen is assimilated into bacterial organic nitrogen; in a culture medium with ammonia nitrogen as a unique nitrogen source, the strain is cultured for 48 hours and then subjected to NH treatment 4 + The removal rates of-N and total nitrogen were 98.72% and 99.80%, of which 26.35% of the inorganic nitrogen was assimilated into microbial organic nitrogen. When the bacterial strain 2G-5 is researched to remove ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in a water body, the obvious accumulation of inorganic nitrogen in other forms is not detected in the process.
Therefore, the second purpose of the invention is to provide the application of the debaryomyces 2G-5 in removing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen. Such as in the preparation of preparations for removing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen.
Preferably, the method is applied to the denitrification of the water body containing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen and the improvement of water quality.
The ammonia nitrogen is ammonium sulfate, the nitrite nitrogen is sodium nitrite, and the nitrate nitrogen is potassium nitrate.
Preferably, the application is the application of inoculating the debaryomyces terreus 2G-5 into a culture medium which takes nitrite nitrogen, nitrate nitrogen or ammonia nitrogen as a unique nitrogen source for growth and denitrification.
The third object of the present invention is to provide a live denitrifier (e.g., water quality improver) having nitrification and denitrification functions, which contains as an active ingredient, Zoerella 2G-5.
Patent 201510316321.7 (application date 2015.06.10) discloses a strain of Zobellella maritima (Zobellella sp.) CYT1-8, which is the highest in similarity with the model bacterium Zobellella densificans ZD1 and has the 16S rRNA similarity of 99.04%, the strain involved in the patent can rapidly remove nitrate and nitrite in water under aerobic conditions, but a large amount of nitrite is accumulated in the nitrate removing process, but the strain involved in the invention has no obvious nitrite accumulation in the nitrate removing process, so that the activity of nitrite reductase in the denitrification process of the strain is presumed to be higher, nitrite reduced from nitrate can be rapidly further reduced into nitrogen-containing gas, and the strain has greater application potential in improving the water quality of actual culture water.
The Zubesella 2G-5 can quickly and efficiently remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and total nitrogen thereof in a water body, the removal effect on the three inorganic nitrogen sources and the total nitrogen is more than 98%, no other inorganic nitrogen sources are obviously accumulated in the process, no secondary pollution is caused, and the bacterium has great application value in the improvement of aquaculture water quality, such as reduction of harm of the ammonia nitrogen, the nitrite nitrogen, the nitrate nitrogen and the like to aquaculture objects in the aquaculture process.
Zobelllla sp.2g-5 was deposited at the guangdong province collection of microorganisms (GDMCC) in 2019, 03 and 06 days, and the deposition address: building 59 of first fure Zhonglu 100 college of overseas district of Guangdong province, Guangzhou city, with the preservation number GDMCCNo: 60606.
drawings
FIG. 1 is the colony morphology of Bordetella parapertussis 2G-5 on a solid plate;
FIG. 2 is a photograph of Therebel bacterium 2G-5 under a light microscope (100X);
FIG. 3 is a view of a Jolbert cellGrowth tendency and NO removal of bacterium 2G-5 2 - -a capability map of N;
FIG. 4 shows the growth trend of Klebsiella pneumoniae 2G-5 and NO elimination 3 - -a capability map of N;
FIG. 5 shows the growth tendency and NH removal of 2G-5 of Bessella sp 4 + -N capacity diagram.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
1. enrichment, separation, purification and preservation of 2G-5 from Zobellella sp
Enrichment culture:
preparing an enrichment culture medium, wherein the formula is as follows: sodium succinate 2.5g, sodium citrate dihydrate 2.5g, (NH) 4 ) 2 SO 4 0.66g,KNO 3 1.01g,NaNO 2 0.69g，K 2 HPO 4 1g，KH 2 PO 4 1g，MgSO 4 ·7H 2 0.2g of O, 7.4 of pH, and the volume is adjusted to 1000mL, and the mixture is subpackaged into 250mL triangular bottles with each bottle being 100 mL. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Then 1 percent (mass fraction) of the composite carbon source and 0.2 percent (mass fraction) of the composite trace elements are added after sterile filtration. A composite carbon source: 6.9g of D-glucose, 6.9g of D-fructose, 6.9g of D-lactose, 9.5g of sodium acetate, 6.4mL of lactic acid with the mass fraction of 90%, 7g of mannitol, 7mL of absolute ethyl alcohol, 6.3mL of glycerol, 4.8g of sodium benzoate and 4.6g of salicylic acid, dissolving in 500mL of water, and filtering and sterilizing by using a 0.22 mu m filter membrane. And (3) mixing trace element liquid: ZnSO 4 ·7H 2 O 0.1g，MnCl 2 ·4H 2 O 0.4g，H 3 BO 3 1.24g，CoCl 2 ·2H 2 O 0.5g，CuCl 2 ·2H 2 O 0.5g，NiSO 4 ·6H 2 O 0.02g，NaMoO 4 ·2H 2 O 0.4g，KI 0.2g，CaCl 2 5g，FeSO 4 ·7H 2 O1.1 g, EDTA-Na 10g, dissolved in 1000mLH 2 O, pH 7.4, 0.22 μm filter.
Taking 10mL of Shantou south Australian island fish and shrimp polyculture pond water sample, inoculating into a conical flask filled with the enrichment medium, culturing at 28 ℃ and 180rpm for 5d, and enriching denitrifying bacteria. Transferring 1mL of the incubated culture into a new conical flask filled with enrichment medium, culturing for 5d at 28 ℃ and 180rpm, performing secondary enrichment, and performing the third enrichment culture.
Separation, purification and preservation
For the above-mentioned three enrichment cultures, a solid plate was coated by dilution coating method (the solid culture medium used was agar added with 1.7% (mass fraction) based on the enrichment medium formulation); visually observing the colony morphology, streaking and purifying, picking the purified colony to 5mL liquid culture medium containing Duchenne tubes (enrichment medium, but separating three inorganic nitrogen sources), incubating at 28 ℃ and observing the gas production condition, and finding that the bacterial strain 2G-5 can convert nitrite into a gas product. And (5) carrying out expanded culture on the purified strains and planting glycerinum tubes and freeze-dried tubes.
Morphological characteristics
The morphological characteristics of the strain 2G-5 are as follows: gram-negative bacteria; the colony color is milky white and translucent (see figure 1); the bacterial colony is circular, the edge is regular, and the surface is wet and smooth; the cells were short rods in shape (see FIG. 2). The strains 2G-5 can be respectively expressed in (NH) 4 ) 2 SO 4 、NaNO 2 And KNO 3 Growth in medium as sole nitrogen source.
16S rDNA identification of 2G-5 by 2P-Bessella 2 (Zobellella sp.)
The genome DNA of the strain 2G-5 is extracted, a PCR product is obtained by amplifying a bacterial 16S rRNA gene amplification universal primer 27F/1492R, and the PCR product is sent to the Shanghai Meiji biological medicine science and technology Limited company (Guangzhou division) for sequence sequencing, and the sequencing result (the nucleotide sequence of the 16S rRNA is shown as SEQ ID NO. 1) is subjected to homology comparison analysis with the 16S rDNA sequence in an EzBioCloud website database, and the result analysis shows that the similarity of the sequence with the 16S rRNA gene sequence of the model bacterium Zobella densitifica 1 is the highest and is 98.43 percent, the similarity with the two model bacteria Z.taiwanensis ZTT 1 and Z.aerosile JC2671 is 97.36 percent, and the similarity with the model bacterium Z.endophica 59N8 is 97.21 percent. Based on the result analysis, the strain is preliminarily identified to be Zobellollasp, named as Zobellella (Zobellella) 2G-5, which is preserved in Guangdong province microorganism strain preservation center in 03 and 06 months in 2019, and is addressed to No. 59 building of Xieli Zhonglu No. 100 college in Guangzhou city, Guangzhou province, and the preservation number is GDMCCNo.60606.
Denitrogenation performance of 3-P-BEI (Zobellella) 2G-5
Inoculating 2G-5 of Beehmeria nobilis into liquid medium [ enriched medium formula, but with NO respectively 2 - -N、NO 3 - -N and NH 4 + N is the only nitrogen source, in particular:
with NO 2 - Liquid medium formulation with N as sole nitrogen source: sodium succinate 2.5g, sodium citrate dihydrate 2.5g, NaNO 2 0.69g，K 2 HPO 4 1g，KH 2 PO 4 1g，MgSO 4 ·7H 2 0.2g of O, 7.4 of pH, constant volume to 1000mL, 121 ℃, 20min, high temperature and high pressure sterilization, and then 1 percent (mass fraction, formula as above) of the sterile filtered composite carbon source and 0.2 percent (mass fraction, formula as above) of the composite trace element are added.
With NO 3 - Liquid medium formulation with N as sole nitrogen source: sodium succinate 2.5g, sodium citrate dihydrate 2.5g, KNO 3 1.01g，K 2 HPO 4 1g，KH 2 PO 4 1g，MgSO 4 ·7H 2 0.2g of O, 7.4 of pH, constant volume to 1000mL, 121 ℃, 20min, high temperature and high pressure sterilization, and then 1 percent (mass fraction, formula as above) of the sterile filtered composite carbon source and 0.2 percent (mass fraction, formula as above) of the composite trace element are added.
By NH 4 + Liquid medium formulation with N as sole nitrogen source: sodium succinate 2.5g, sodium citrate dihydrate 2.5g, (NH) 4 ) 2 SO 4 0.66g，K 2 HPO 4 1g，KH 2 PO 4 1g，MgSO 4 ·7H 2 0.2g of O, 7.4 of pH, constant volume to 1000mL, 121 ℃, 20min, high temperature and high pressure sterilization, then 1 percent of composite carbon source (mass fraction, formula as above) after sterile filtration and 0.2 percent of composite trace element (mass fraction, formula as above) are addedUpper)]，
Performing shake culture at 28 deg.C and 180rpm for 0, 12, 18, 24, 36, and 48 hr, respectively, sampling, collecting part of the sample for detecting OD 600 value, centrifuging the other part of the culture solution, collecting supernatant, and quantitatively detecting NO 2 - 、NO 3 - 、NH 4 + And total nitrogen concentration (TN-N), and total nitrogen concentration in the cells (CN-N) was also measured. The results are shown in FIGS. 3,4 and 5, and the pairs of the Znobacter 2G-5 and NO in the water body 2 - -N、NO 3 - -N、NH 4 + The N and the total nitrogen in the N have good removal effect. The strain is cultured for 48 hours in a culture medium taking nitrite nitrogen as a unique nitrogen source to NO 2 - The removal rates of N and total nitrogen were 99.00% and 99.60%, with 27.08% of the inorganic nitrogen being assimilated into bacterial organic nitrogen; in a culture medium with nitrate nitrogen as a unique nitrogen source, the strain is cultured for 48 hours and then NO is added 3 - The removal rates of N and total nitrogen are 99.58% and 99.84%, wherein 29.72% of inorganic nitrogen is assimilated into bacterial organic nitrogen; in a culture medium with ammonia nitrogen as a unique nitrogen source, the strain is cultured for 48 hours and then treated with NH 4 + The removal rates of-N and total nitrogen were 98.72% and 99.80%, of which 26.35% of the inorganic nitrogen was assimilated into microbial organic nitrogen. And when the strain is used for removing ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in a water body, no obvious accumulation of inorganic nitrogen in other forms is detected in the process. The Zubeilbert 2G-5 is a strain with nitrification and denitrification functions, has a high removal effect on ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in a water body, and does not accumulate other harmful nitrogenous products in the process. In view of this, the Zolbertia zeylanica 2G-5 has great potential in the practical application of improving the culture water quality, removing main nitrogen pollutants in the culture water body and guaranteeing the healthy growth of aquatic animals.
The experimental determination method comprises the following steps: NO 2 - -N is spectrophotometrically with N- (1-naphthyl) -ethylenediamine dihydrochloride; NO 3 - N measuring the concentration of nitrate nitrogen by adopting an ultraviolet spectrophotometry; NH 4 + -N is spectrophotometrically using sodium reagent; the total nitrogen is digested by alkaline potassium persulfate and is subjected to ultraviolet spectrophotometry.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> Zubeiella having both nitrification and denitrification functions and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1419
<212> DNA
<213> Tolberella (Zobellella sp.)
<400> 1
caacccactc ccatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgca 60
acattctgat ttgcgattac tagcgattcc gacttcacgg agtcgagttg cagactccga 120
tccggactac gacgcgcttt ttgggattcg cttaccatcg ctggttcgcc gccctctgta 180
cgcgccattg tagcacgtgt gtagccctac ccgtaagggc catgatgact tgacgtcgtc 240
cccaccttcc tccggtttat caccggcagt ctcctttgag ttcccgaccg aatcgctggc 300
aacaaaggac aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 360
gacgacagcc atgcagcacc tgtatgtaag ttcccgaagg cacccatcca tctctggaaa 420
gttcttacta tgtcaagggt aggtaaggtt cttcgcgttg catcgaatta aaccacatgc 480
tccaccgctt gtgcgggccc ccgtcaattc atttgagttt taaccttgcg gccgtactcc 540
ccaggcggtc aacttaacgc gttagctccg gaacccgcgc tcaaatggca cagactccaa 600
gttgacatcg tttacagcgt ggactaccag ggtatctaat cctgtttgct ccccacgctt 660
tcgcacctga gcgtcagtct ttggccaggg ggccgccttc gccactggta ttccttccga 720
tctctacgca tttcaccgct acaccggaaa ttctaccccc ctctccaaga ctctagcctg 780
ccagttccaa atgcagttcc caggttgagc ccggggcttt cacatctggc ttaacagacc 840
gcctgcgtgc gctttacgcc cagtcattcc gattaacgct tgcaccctcc gtattaccgc 900
ggctgctggc acggagttag ccggtgcttc ttctgtgggt aacgtcacag taagtgggta 960
ttcgccactc acctttcctc cccactgaaa gtgctttaca acccgaaggc cttcttcaca 1020
cacgcggcat ggctgcatca gggtttcccc cattgtgcaa tattccccac tgctgcctcc 1080
cgtaggagtc tgggccgtgt ctcagtccca gtgtggctgg tcatcctctc agaccagcta 1140
gagatcgtcg ccttggtgag ccattacctc accaaccagc taatctcact tgggctcatc 1200
caatcgcgca aggcccgaag gtcccctgct ttcccccgta gggcgtatgc ggtattagcc 1260
gtcgtttcca acggttatcc ccctcgactg ggcagatacc caagccttac tcacccgtcc 1320
gccgctcgtc agcaaagtag caagctaccc tctgttaccg ctcgacttgc atgtgttagg 1380
cctgccgcca gcgttcaatc tgagccatga tcaaactct 1419
Claims (7)
1. Beehella tabescensZobellellasp, 2G-5, deposited at GDMCC, 03/06, 2019, at the location: building No. 59 of Xieli Midland No. 100 Hospital, Guangdong province, Guangzhou City, the preservation number is GDMCC No: 60606.
2. the application of the debaryomyces terreus 2G-5 in removing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen from a water body.
3. The application of claim 2, wherein the water containing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen is denitrified by using the debaryomyces tabescens 2G-5 to improve the water quality.
4. The use of claim 2, wherein the use is the use of debaryomyces 2G-5 inoculated into a culture medium with nitrite nitrogen, nitrate nitrogen or ammonia nitrogen as the sole nitrogen source for growth and denitrification.
5. The application of the debaryomyces terreus 2G-5 of claim 1 in preparing a preparation for removing ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen.
6. A viable denitrification agent having nitrification and denitrification functions, characterized by comprising the bacterium nobilex 2G-5 according to claim 1 as an active ingredient.
7. The live denitrogenation agent according to claim 6, wherein the live denitrogenation agent is a water quality improver.
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| CN113336337B (en) * | 2021-06-04 | 2022-04-12 | 中国科学院重庆绿色智能技术研究院 | Novel integrated denitrification device and application |
| CN113699057B (en) * | 2021-07-06 | 2023-01-20 | 广州大学 | Rhodococcus toonapus with heterotrophic nitrification-aerobic denitrification function and application thereof |
| CN113308410B (en) * | 2021-07-08 | 2022-05-31 | 山东绿邦生物科技有限公司 | Denitrifying bacterium for efficient denitrification and application thereof |
| CN113800652B (en) * | 2021-10-28 | 2022-07-26 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Salt-tolerant aerobic denitrifying bacterium and application of coupling activated carbon thereof in strengthening water body pollution treatment |
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