CN105441359A8 - 一株地衣芽孢杆菌及其应用 - Google Patents
一株地衣芽孢杆菌及其应用 Download PDFInfo
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- CN105441359A8 CN105441359A8 CN201510956636.8A CN201510956636A CN105441359A8 CN 105441359 A8 CN105441359 A8 CN 105441359A8 CN 201510956636 A CN201510956636 A CN 201510956636A CN 105441359 A8 CN105441359 A8 CN 105441359A8
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- China
- Prior art keywords
- bacillus licheniformis
- denitrification
- nitrate
- application
- nitrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 59
- 239000002351 wastewater Substances 0.000 claims abstract description 19
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003546 flue gas Substances 0.000 claims abstract description 16
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 14
- 229910052799 carbon Inorganic materials 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 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 claims description 12
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- 230000008569 process Effects 0.000 claims description 8
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 229940074404 sodium succinate Drugs 0.000 claims description 5
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 abstract description 25
- 229910002651 NO3 Inorganic materials 0.000 abstract description 15
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 abstract description 15
- 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 abstract description 13
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 abstract description 10
- 230000012010 growth Effects 0.000 abstract description 10
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- 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 9
- 239000002609 medium Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 108020004465 16S ribosomal RNA Proteins 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
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- 239000001301 oxygen Substances 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
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- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 description 3
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
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- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 2
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- 235000013619 trace mineral Nutrition 0.000 description 2
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- 239000002912 waste gas Substances 0.000 description 2
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 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
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 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
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 108010025915 Nitrite Reductases Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
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- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 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 1
- 239000002689 soil Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 239000010918 textile wastewater Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/54—Nitrogen compounds
- B01D53/56—Nitrogen oxides
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
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- C02F3/02—Aerobic processes
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- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D2257/404—Nitrogen oxides other than dinitrogen oxide
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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
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
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- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
本发明公开了一株地衣芽孢杆菌及其应用,所述地衣芽孢杆菌(Bacillus licheniformis)B2,由中国典型培养物保藏中心保藏,其保藏编号是CCTCC NO:M 2015590,保藏日期为2015年10月8日。本发明提供的地衣芽孢杆菌生长温度范围广,且具有高效好氧反硝化能力,能应用于处理硝酸盐废水或含NOx的烟气。该菌株可在30~50℃下在24小时内对初始浓度为140mg/L硝酸盐氮去除率达99%以上,并且没有亚硝酸盐积累。该菌株的发现可解决温度波动对生物脱氮脱硝影响大的难题。
Description
一株地衣芽孢杆菌及其应用
技术领域
[0001]本发明属于环境工程、生物领域,涉及一株具有好氧反硝化性能的地衣芽孢杆菌 (Bacillus lichenif〇rmis)B2菌株及其在高温环境下脱除废水中脱除水中的硝酸根与亚 硝酸根的应用。
背景技术
[0002]农业生产方式发生了重大改变,农药、化肥大量使用且与日倶增。农药化肥施于农 作物上,其中10 %〜20 %附着在植物体上,其余80 %〜90 %散落在土壤、水体、大气中。农药 化肥通过地表径流或降水带入等方式进入地表水体,造成富营养化,甚至污染地下水。印染 和纺织废水、炼油废水、肉类加工和饲料生产废水等都含有高浓度的氨氮和硝酸盐。另外, 由于能源需求日益强劲,矿物燃料的燃烧、汽车尾气的排放量不断增加,大气中活性氮化合 物含量近年来迅速增长。氮污染不仅破坏生态系统,也严重危害到人体健康。
[0003]在众多处理硝酸氮废水的研宄中生物脱除法具有对环境影响程度轻、工艺简单等 优点,作为一种绿色高效的处理技术而越来越被重视。而在废气脱氮治理方法中,生物过滤 法作为一种低成本、高效率、无二次污染的方法,已成为大气污染控制领域热点之一。
[0004]最早提出好氧反硝化(aerobic denitrification)的是Robertson和Kuenen,他们 在实验室中观察到在氧气存在的条件下发生了反硝化现象。国内外学者在好氧反硝化领域 进行了广泛深入的研宄,证明好氧反硝化较之传统反硝化技术存在明显优势。近年来,不断 有好氧反硝化菌被分离出来,存在于副球菌属、假单胞菌属、产碱菌属、芽孢杆菌属等50多 个属。然而这些报道的好氧反硝化菌大多生长于常温环境下(25〜35°C),关于高温条件下 (40°C以上)的好氧反硝化菌报道较少,由于目前很多工业废水都超过40°C,高温菌可直接 处理高温废水,节省成本。另外,在30〜50°C均能保持高效反硝化效能的菌种更鲜有报道。 生物法的其中一个缺点是容易受温度波动的影响,因此,筛选分离在较广温度范围下(包括 高温)具有高效性能的好氧反硝化细菌的研究具有实际意义。
发明内容
[0005] 本发明之目的是提供一种在较广温度范围下(包括高温)具有高效好氧反硝化性 能的菌株及其用途。
[0006] 本发明的目的通过以下技术方案实现:
[0007] 本发明较广温度范围下具有高效好氧反硝化性能的菌株是地衣芽孢杆菌 (Bacillus licheniformis)B2,该菌株具有以下特征:
[0008] (1)菌落特征:菌落表面不光滑不透明,边缘不整齐;杆菌,菌体单个,成对或链状 排列;革兰氏染色阳性;芽孢椭圆形,中生;
[0009] (2)糖、醇类发酵试验显示为发酵型,接触酶、厌氧生长、V-P测定、硝酸盐还原、明 胶液化、淀粉水解、酪素水解等实验均为阳性,氧化酶、卵磷脂酶、吲哚、酪氨酸、苯丙氨酸脱 氨酶等实验均为阴性,可进行反硝化作用,能利用硝态氮或亚硝态氮生长;
[0010] ⑶该菌株的16S rDNA基因序列特征:其16S rDNA具有序歹!J表所示的核苷酸序列, 其16S rDNA具有序列的片段长度为1272bp,在GenBank中的登录号为n〇 . KT777466,与 GenBank数据库中已知细菌的16SrDNA进行BLAST比对,结果表明该菌株与地衣芽孢杆菌 (Bacillus licheniformis)的同源性为的%,其中与菌株Bacillus licheniformis strain AV12最相似,同源性达99%。
[0011] 应用MEGA软件采用Neighbor-Joining法构建16S rDNA系统发育树,确定其进化地 位,结合其形态特征和生理生化特征,该菌株最有可能是地衣芽孢杆菌属(Bacillus licheniformis),并命名为Bacillus licheniformis B2。
[0012]所述地衣芽孢杆菌用于含氮废水的脱氮处理,或用于含N〇x烟气脱硝处理。地衣芽 孢杆菌B2对温度耐受程度强,在30〜50°C条件下生长且具有高效的好氧反硝化能力,硝酸 盐去除率均2 99%,并且没有亚硝酸盐积累。此外,地衣芽孢杆菌B2对氧气的耐受性强,在 11 • 2mg/L溶解氧浓度下,硝酸盐去除率仍达98•7%,且没有亚硝酸盐积累。以上特性表明该 菌株能应用于温度波动大、氧气含量高的实际高温废水或烟气中,将含氮污染物还原为氮 气,达到脱氮的目的。
[0013]优选地,所述地衣芽孢杆菌B2用于30〜5〇°C含氮废水的高效好氧反硝化脱氮处 理。
[0014] 优选地,所述地衣芽孢杆菌B2用于30〜50°C烟气波动情况下的高效好氧反硝化脱 硝处理。
[0015]所述地衣芽孢杆菌B2脱氮、脱硝处理的条件为:利用葡萄糖、蔗糖或丁二酸钠为碳 源,控制C/N为6〜15,pH为7〜9,振荡转速为120〜200rpm。
[0016]优选地,所述C/N为9,pH为8,振荡转速为160rpm。
[0017]与现有技术相比,本发明具有如下有益效果:
[0018] (1)本发明提供的地衣芽孢杆菌生长温度范围广,且具有高效好氧反硝化能力,能 应用于处理硝酸盐废水或含N0x的烟气。
[0019] (2)该菌株可在30〜50°C下在24小时内对初始浓度为140mg/L硝酸盐氮去除率达 99%以上,并且没有亚硝酸盐积累。
[0020] (3)该菌株的发现可解决温度波动对生物脱氮脱硝影响大的难题。
[0021] 本发明所述地衣芽抱杆菌(Bacillus licheniformis)B2,由中国典型培养物保藏 中心保藏(简称CCTCC),其保藏编号是CCTCC N0:M 2015590,保藏日期为2015年1〇月8日,保 藏地址为中国武汉大学。
附图说明
[0022]图 1 为Bacillus licheniformis B2的系统发育树图。
[0023]图2不同碳源下菌Bacillus licheniformis B2的反硝化性能比较图。
[0024]图3不同C/N下菌Bacillus licheniformis B2的反硝化性能比较图。
[0025]图4不同转速下菌Bacillus licheniformis B2的反硝化性能比较图。
[0026]图5不同pH下菌Bacillus licheniformis B2的反硝化性能比较图。
[0027]图6不同温度下菌Bacillus licheniformis B2的反硝化性能比较图。
[0028]图7为烟气脱硝生物过滤装置示意图,进气口 1、雾化器增湿器2、温度控制器3,生 物滴滤塔4、恒温水浴锅5、质量流量计6、出气口 7。
[0029] 图8为在真实烟气环境下培养菌Bacillus licheniformis B2的反硝化性能图。
具体实施方式
[0030]下面结合具体实施例对本发明作进一步具体详细描述,但本发明的实施方式不限 于此,对于未特别注明的工艺参数,可参照常规技术进行。
[0031] 实施例1:菌种的分离、纯化及鉴定
[0032] 本研究向玉米叶水解液中接入不含碳源的反硝化培养基,初始温度为40°C, 160rpm的振荡培养箱中。培养24h后,以体积比为10%的接种量接入90mL无菌的含反硝化培 养基的玉米叶水解液中。重复上述步骤3天后发现菌液生长良好,并且脱氮率达到了85%以 上。经过富集培养后的菌液通过倍比稀释法后涂布平板以分离出单菌落。将菌液取lmL至装 有9mL无菌水的试管中,充分混合后得到HT1梯度的菌悬液,以此类推制备10 2〜1(T8不同梯 度的菌悬液,为了获得可以清晰观察的菌落形态,选取10 4、10'10 6三个梯度的菌悬液,各 取O.lmL涂布至已制备好的带有溴百里酚蓝(BTB)显色的反硝化固体培养基上,倒置放入40 °C恒温恒湿培养箱中。培养2天后,可见平板中长有单菌落,由于BTB指示剂的作用,发生反 硝化作用的菌落周围产生蓝色晕圈。从平板中挑取形态不同且周围有蓝色晕圈的单菌落, 在无菌的反硝化固体培养基中划线后放置在40°C恒温恒湿培养箱中培养,如此重复三次得 到纯菌落。
[0033]在复筛阶段将上述分离得到后的不同形态的单菌落分别接种于装有90ml的无菌 反硝化培养基中,在40°C,l6〇rpm条件下振荡培养对小时,取培养后的菌液测量ODsoo值以及 培养液中的硝态氮浓度包括硝酸盐氮和亚硝酸盐氮,考察菌株在24h内的生长状况和硝态 氮去除效果,最后筛选出一株优势菌株编号为B2,其24h内脱氮率大于95%。
[0034]将上述分离纯化得到的菌株进行生理生化鉴定及16S rDNA测序。PCR引物为通用 (下游引物)JCR扩增反应条件为:94°C预变性lmin,经过30个循环,56°C退火lmin,72°C扩 增2min,最后72°C延伸7min。得到的产物进行16S rDNA测序(上海英俊生物技术有限公司), 片段长度为1272bp。将测序结果在NCBI网站上进行BLAST,结果得出B2与Bacillus licheniformis同源性达99%,命名为Bacillus licheniformis B2(GenBank登录号为 KT7?7466)。利用MEGA软件进行多序列比,运用Neighbor-Joining法构建系统发育树,详见 图1。为了进一步确定鉴定结果的可靠性,请广东省微生物分析测试中心对该菌进行生理生 化鉴定,最终确定该菌为地衣芽抱杆菌Bacillus licheniformis。
[OO35] 实施例2:地衣芽抱杆菌(Bacillus licheniformis)B2反硝化性能测定
[0036] 1、不同碳源下Bacillus licheniformis B2的反硝化性能
[0037]考察菌株B2对不同碳源的利用情况,包括葡萄糖、蔗糖、乙酸钠、丁二酸钠、梓檬酸 钠这5种碳源。具体操作步骤如下:
[0038]将lOmL菌悬液接种于9〇mL反硝化培养基中,反硝化培养基成分如下(g/L) :Kn〇3 l;Na2HP〇4.7H2〇 0_8;KH2P〇4 l.5;MgS〇4.7H2〇 0_2;微量元素溶液2mL/L;pH 7.0。微量元 素溶液成份(g/L):EDTA 50.0;ZnS〇4 2.2;CaCl2 5.5;MnCl2*4H2〇 5.06;FeS〇4.7H2〇 5.0; (NH4)6M〇7〇2.4H2〇 1.1;CuS〇4.5H2〇 1.57;C〇C12.6H2〇 1_61。分别加入上述 5 种碳源,使 c/ N为12,于40°C、160rpm条件下振荡培养。
[0039] 取Oh和24h的样检测硝酸氮、亚硝酸氮、0D6qq,结果如图2所示,菌株对葡萄糖、蔗 糖、丁二酸钠的利用率都比较高,硝酸根去除率均超过98%,但丁二酸钠为碳源时培养液中 有轻微亚硝酸盐积累(1.5mg/L)。而乙酸钠作为碳源时,硝酸根去除率仅为1.09%。葡萄糖 为碳源时,细菌生长量最大,〇D6Q〇达1.99。这可能是由于玉米叶水解液含有大量糖类尤其是 葡萄糖,B2更倾向于利用葡萄糖生长。
[0040] 2、不同碳氮比下Bacillus licheniformis B2的反硝化性能
[0041] 将10mL菌悬液接种于90mL反硝化培养基中,反硝化培养基中KN〇3含量固定为lg/ L,改变葡萄糖含量使C/N为3、6、9、12、15,其他组分如上述实验,于160印111、40°(:条件下振荡 培养24h,以确定最优碳氮比。
[0042] 取Oh和24h的培养液检测硝酸氮、亚硝酸氮、0D6QQ,结果如图3所示,最佳C/N为9,菌 株此条件下硝酸根去除率达到100%,〇D6Q()达2.08。进一步加大葡萄糖含量并没有带来积极 作用。
[0043] 3、不同转速下Bacillus licheniformis B2的反硝化性能
[0044]接种10mL菌液进入90mL的反硝化液体培养基中,反硝化培养基中其他组分不变, 葡萄糖为3.12g/L,调节振荡培养箱转速分别为0、60、12〇、160、180、200印111,分别对应了缺 氧以及氧含量逐步增加的过程,温度均为40°C,培养24h,以考察在不同氧含量情况下,B2的 反硝化效果及生长状况。
[0045] 测定Oh和24h的硝酸氮、亚硝酸氮、0D6Q。,结果如图4所示,0D6QQ在160rpm下取得最 大值2.05。菌株在120〜200rpm条件下,反硝化率均超过99%,且没有亚硝酸盐积累,表明在 高氧含量的情况,B2也能进行高效脱氮。因此,B2在实际废水或废气治理中有广泛的应用前 景。
[0046] 4、不同pH条件下Bacillus licheniformis B2的反硝化性能
[0047] 接种1 OmL菌液进入90mL的反硝化液体培养基中,分别调节初始pH至6、7、8、9、10, 均以160rpm的转速振荡培养24h,以考察在不同pH条件下,菌株B2的反硝化效果及生长状 况。
[0048]测定Oh和24h的硝酸氮、亚硝酸氮、ODox),结果如图5所示,菌株在pH为7〜9条件下, 反硝化率均2 99.5%,表明B2在pH波动下也能进行高效好氧反硝化。在pH为8时,菌液浓度 最高。由于利用葡萄糖为碳源时,菌株会进一步分解葡萄糖导致pH的下降,因此偏碱性有利 于菌株的生长。
[0049] 5、不同温度Bacillus licheniformis B2的反硝化性能
[0050] 接种10mL菌液进入90mL的反硝化液体培养基中,分别在30°C、35°C、40°C、45°C、5〇 °〇和55°〇条件下,均以160rpm的转速振荡培养24h,以考察在不同温度下,菌株B2的反硝化 效果及生长状况。
[0051 ] 测定Oh和24h的硝酸氮、亚硝酸氮、0D6。。,结果如图6所示,虽然菌株在30°C下菌液 浓度最高,但在30〜50°C的条件下,反硝化效率均达到99%及以上且没有亚硝酸盐积累,表 明在上述温度范围内,菌株体内的硝酸盐还原酶和亚硝酸盐还原酶不受温度的影响,这种 情况是极为罕见的。B2可在较广的温度范围内(包括高温)进行高效脱氮,这对应用于温度 波动大的实际含氮废水或烟气具有深远意义。
[0052] 实施例3:地衣芽孢杆菌(Bacillus licheniformis)B2的实际烟气脱硝实验
[0053]利用生物滴滤塔进行高温环境下脱除N0X的实验。生物过滤系统如图7所示,进气 口 1、雾化器增湿器2、温度控制器3,生物滴滤塔4、恒温水浴锅5、质量流量计6、出气口 7。生 物滤塔挂膜期储液池内营养组分同实施例2,考察系统在实际烟气波动的情况下运行的稳 定性。
[0054]烟气流速为1L • mirT1,停留时间为lmin。为使B2始终占优势地位,每三天更换三分 之二含10 %新鲜B2的营养液。硝酸盐去除效率逐渐提高,达到80 %后,逐渐降低营养液中硝 酸盐的浓度并通入烟气,最终营养液中不加入硝酸盐。由于烟气波动较大,每天测定一次 N0X含量,7天计算一次平均值,结果如图8所示,N0X去除率由第一周的47.5 %上升至第6周的 92%,然后在第7周稍有下降,为88.6%,第8周又稍有上升。另外,从第5周开始系统基本稳 定运行,去除率维持在88〜92%之间,说明此生物滴滤塔在有氧和烟气波动条件下能有效 去除NOx。
[0055] 该实例证明在实际烟气波动条件下,地衣芽胞杆菌Bacillus licheniformis B2 具有高效的好氧反硝化性能,对N0X有较好的去除效果。
[0056] 实施例4:地衣芽孢杆菌(Bacillus licheniformis)B2用于水耕废水脱氮
[0057]将该菌用于某温室水耕废水的脱氮处理。废水流量为155m3/d,N03--N325mg/L, pH6.45,7jC温在30°C左右,处理工艺为生物曝气滤池。
[0058] 调节废水pH至8左右,将B2按0.5%的接种量添加到曝气生物池中,处理后平均 N〇3_-N为9• 8mg/L,去除率为97.0%,除去在缩短水力停留时间的时候出现短暂的去除率降 低之外,并没有发现过大的波动,并且没有亚硝酸盐积累。以投加地衣芽孢杆菌JH8(申请号 为201410137313 • 1)做对照组,其N03_-N去除率仅83 • 2%,出水亚硝酸盐浓度为0.5mg/L。由 于JH8适宜温度范围为45〜55°C,JH8投加至处理温度为30°C的生物曝气滤池系统中并不能 提高其处理能力。相反,B2的投加明显提高了系统的脱氮性能,该菌能应用于高硝氮废水的 处理。
[0059] 实施例5:地衣芽孢杆菌(Bacillus licheniformis)B2用于地下水脱氮处理 [0060] 将该菌用于某区域地下水的脱氮处理。水质中N03—-N浓度为2〇.50mg/L,N02—-N 0 • 52mg/L,NH4+-N 3 • 83mg/L,各项氮指标相对比较低;将该菌按〇 • 5%的接种量添加至逆流 式生物滤池中,稳定后测得地下水经处理后的N03—-N与N02_-N完全去除,NH/-N为0 • 〇7mg/L。 以投加地衣芽孢杆菌JH8(申请号为201410137313 • 1)作对照组,其处理后水质(N〇3_-N 0 • 5mg/L,N〇2_-N 0 • 1 lmg/L,NH4+-N 0.25mg/L)。经比较可见,B2在较低浓度氨氮、硝氮以及 亚硝氮的同步硝化反硝化速率要高于投加JH8的处理系统。因此,该菌对低浓度的氨氮与亚 硝酸氮也能起到较好脱除效果,可以用于地下水氮污染的治理。
[0061]上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的 限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化, 均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1. 一'株地衣牙抱杆菌,其特征在于,所述地衣芽抱杆菌(Bacillus licheniformis)B2, 由中国典型培养物保藏中心保藏,其保藏编号是CCTCC NO:M 2015590,保藏日期为2015年 10月8日。
2.权利要求1所述地衣芽孢杆菌的应用,其特征在于,所述地衣芽孢杆菌用于含氮废水 的脱氮处理,或用于含NOx烟气脱硝处理。
3.根据权利要求2所述的应用,其特征在于,所述地衣芽孢杆菌B2用于30〜50°C含氮废 水的好氧反硝化脱氮处理。
4.根据权利要求2所述的应用,其特征在于,所述地衣芽孢杆菌B2用于30〜50°C烟气波 动情况下的好氧反硝化脱硝处理。
5. 根据权利要求2或3或4所述的应用,其特征在于,所述地衣芽孢杆菌B2脱氮、脱?肖& 理的条件为:利用葡萄糖、蔗糖或丁二酸钠为碳源,控制C/N为6〜15,pH为7〜9,振荡转速为 120〜200rpm。
6. 根据权利要求5所述的应用,其特征在于,所述C/N为9。
7. 根据权利要求5或6所述的应用,其特征在于,所述pH为8。
8. 根据权利要求5或6所述的应用,其特征在于,所述振荡转速为160rpm。
9. 根据权利要求7所述的应用,其特征在于,所述振荡转速为160rpm。
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