CN114107102B - Acinetobacter and application thereof - Google Patents
Acinetobacter and application thereof Download PDFInfo
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- CN114107102B CN114107102B CN202111386106.6A CN202111386106A CN114107102B CN 114107102 B CN114107102 B CN 114107102B CN 202111386106 A CN202111386106 A CN 202111386106A CN 114107102 B CN114107102 B CN 114107102B
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- 241000589291 Acinetobacter Species 0.000 title claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 abstract description 7
- 239000001632 sodium acetate Substances 0.000 abstract description 7
- 235000017281 sodium acetate Nutrition 0.000 abstract description 7
- 230000001580 bacterial effect Effects 0.000 abstract description 5
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 3
- 241000588624 Acinetobacter calcoaceticus Species 0.000 abstract description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 abstract 1
- 238000011081 inoculation Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 24
- 241000894006 Bacteria Species 0.000 description 19
- 230000000694 effects Effects 0.000 description 18
- 239000001963 growth medium Substances 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- 239000012153 distilled water Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 239000002689 soil Substances 0.000 description 11
- 230000001360 synchronised effect Effects 0.000 description 10
- 239000011573 trace mineral Substances 0.000 description 10
- 235000013619 trace mineral Nutrition 0.000 description 10
- 239000000725 suspension Substances 0.000 description 9
- 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 8
- 244000005700 microbiome Species 0.000 description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 229910002651 NO3 Inorganic materials 0.000 description 7
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 229910001868 water Inorganic materials 0.000 description 6
- 230000001546 nitrifying effect Effects 0.000 description 5
- 239000010865 sewage Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- -1 C/N Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000004323 potassium nitrate Substances 0.000 description 4
- 235000010333 potassium nitrate Nutrition 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 241001064871 Acinetobacter oleivorans Species 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002798 spectrophotometry method Methods 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 2
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000001712 DNA sequencing Methods 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
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- C02F3/30—Aerobic and anaerobic processes
- C02F3/302—Nitrification and denitrification treatment
- C02F3/303—Nitrification and denitrification treatment characterised by the nitrification
-
- 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
- C02F3/30—Aerobic and anaerobic processes
- C02F3/302—Nitrification and denitrification treatment
- C02F3/305—Nitrification and denitrification treatment characterised by the denitrification
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- 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
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
-
- 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
Abstract
The invention relates to acinetobacter (Acinetobacter calcoaceticus) KX-3 with a preservation number of CGMCC No.23119 and a nucleotide sequence shown as SEQ ID NO. 1. The strain can remove 76.7 percent of ammonia nitrogen and 67.2 percent of NO under the conditions of sodium acetate as a carbon source, bacterial strain inoculation amount of 0.5 percent, C/N=12, pH=10.0 and temperature of 15 DEG C 3 ‑ . Provides good strain resources for biological denitrification.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to acinetobacter and application thereof.
Background
At present, due to the influence of human activities, the ammonia nitrogen index of urban river and relatively open water areas is not high. The ecological environment problems caused by this have had a significant impact on the human home environment and urban image. Traditionally, nutrients are removed primarily by biological treatment, and biological nutrient removal processes require replacement of anaerobic/aerobic conditions, making the treatment system complex and energy-consuming. Microbial remediation of water environment becomes a hot spot field of current research, and the removal of ammonia nitrogen, nitrite and nitrate in sewage water is gradually improved to a new level along with the research of nitrifying bacteria and denitrifying bacteria.
Biological denitrification mainly depends on two stages of a nitrification process and a denitrification process, but the growth and treatment capacity of microorganisms involved in the two processes can be influenced by various aspects of pH, temperature, dissolved oxygen, C/N, inoculum size and the like. Since temperature is a major factor affecting microbial activity, it is difficult to achieve efficient and stable biological denitrification at low temperatures. According to the existing research, the proper temperature range of denitrification is 25-35 ℃, and denitrification is inhibited below 25 ℃. The cryoprotectant has been widely studied and practically applied in various fields such as environmental engineering and biological application due to its different physiological structures, active enzyme activity at low temperature and wide temperature range of adaptability. The separation and screening of the denitrifying function bacteria with cold resistance at low temperature is significant for the field of microbial denitrification.
Disclosure of Invention
The invention aims to solve the technical problem of providing acinetobacter and application thereof, and fills the blank of denitrification of the existing low-temperature microorganisms.
The preservation number of the acinetobacter (Acinetobacter oleivorans) KX-3 is CGMCC No.23119, and the nucleotide sequence of the acinetobacter is shown as SEQ ID No. 1.
The screening method of the acinetobacter (separation method of low-temperature-resistant synchronous heterotrophic nitrification-aerobic denitrification microorganism strain under alkaline conditions) comprises the following steps:
(1) Enrichment of strains: mixing soil and sterile water to obtain suspension, and then shaking and culturing 1mL of suspension in an enrichment medium at 10-15 ℃ for 2-3 days;
(2) Adding the enrichment culture solution obtained in the step (1) into a denitrification culture medium, and carrying out shaking culture for 46-50h at the temperature of 10-15 ℃ to obtain an enrichment solution;
(3) Adding the enriched liquid obtained in the step (2) into a denitrification culture medium, carrying out shaking culture at 10-15 ℃, and testing NO 3 - Is not limited by the removal efficiency of (2); repeating the step until NO 3 - The removal efficiency of the catalyst reaches more than 80 percent;
(4) Taking NO from step (3) 3 - Diluting the suspension with the removal rate reaching more than 80% of stability, and obtaining the product with the removal rate of 10 -1 ～10 -8 Spread on bromothymol blue medium, and cultured at 15deg.C until visible strain appearsPeripheral bluing colonies;
(5) And (3) picking out the blue-changed colonies growing in the step (4) on a solid LB culture medium plate by using an inoculating loop, and repeatedly streaking for 3-5 times until single colonies appear.
(6) Determination of the denitrification capability of the strain: picking the single colony purified in the step (5) into a denitrification liquid culture medium by using a sterile inoculating loop, oscillating at 15 ℃ and 150r/min, and measuring the denitrification capacity of the strain every 4 hours;
(7) Determination of the nitrifying ability of the strain: selecting the step (6) to remove NO efficiently 3 - The denitrifying bacteria are transferred into a nitrifying medium to further test NH 4 + -removal capacity of N.
The preferred mode of the above steps is as follows:
the volume ratio of the soil to the sterile water in the step (1) is 1:9.
The soil in the step (1) is soil in a Antarctic Lasmann hilly area.
The formula of the enrichment medium in the step (1) is 5.0g of peptone, 5.0g,NaCl 30.0g,pH 7.0 +/-0.2 of yeast and 1L of distilled water.
The denitrification culture medium in the steps (2) and (3) comprises 1.3g of sodium acetate, 0.722g of potassium nitrate, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, pH 7.0+/-0.2 and 1L of distilled water. Trace elements: 10.0g of EDTA, 0.2g of zinc sulfate, 1.2g of manganese chloride tetrahydrate, 1.0g of ferrous sulfate heptahydrate, 0.5g of copper sulfate pentahydrate, 0.3g of cobalt chloride hexahydrate, 0.2g of sodium molybdate dihydrate, 0.1g of calcium chloride, pH 7.0+/-0.2 and 1L of distilled water.
The oscillation in the steps (1) - (3) is 130-150r/min.
The formula of the bromothymol blue culture medium in the step (4) comprises 1.3g of sodium acetate, 0.722g of potassium nitrate, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, 20g of agar, 1ml of 1% bromothymol blue, 7.0+/-0.2 of pH and 1L of distilled water.
The nitrifying culture medium in the step (7) comprises 1.3g of sodium acetate, 0.382g of ammonium chloride, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, 7.0+/-0.2 of pH value and 1L of distilled water.
The invention relates to an application of acinetobacter in biological denitrification at low temperature.
Acinetobacter (Acinetobacter oleivorans) KX-3 of the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23119 in the year 8 and the day 5 of 2021.
Advantageous effects
(1) According to the invention, soil in a Lasmann hilly area of a south pole 36 th scientific investigation site is selected as a strain source, and an appropriate culture medium is matched, so that the low-temperature-resistant synchronous heterotrophic nitrification-aerobic denitrification bacteria are obtained through enrichment and aerobic preliminary screening of aerobic strains, the same strain has a good denitrification effect at a low temperature, and the defect that the effect of the traditional nitrification-denitrification bacteria is poor at the low temperature is overcome.
The enrichment and domestication method for the low-temperature-resistant synchronous heterotrophic nitrification-aerobic denitrification bacteria is simple, easy to operate, short in domestication period and obviously improved in screening efficiency.
(2) The low-temperature-resistant synchronous heterotrophic nitrification-aerobic denitrification bacteria domesticated by the method can directly take NO 3-or NH4+ as an electron acceptor for high-efficiency denitrification, and the removal rate of NO 3-N and NH4+ -N reaches 67.2% and 76.7%, so that compared with the traditional denitrification and dephosphorization process, the tolerance of microorganisms at low temperature is greatly improved, and the method has great economic benefit and environmental benefit.
(3) According to the invention, soil from a 36 th-order south scientific investigation site Lasmann hilly area is selected as a strain source, and is matched with a proper culture medium, and synchronous heterotrophic nitrification-aerobic denitrification bacteria with good denitrification effect are obtained through aerobic strain enrichment, domestication, primary screening and culture, wherein the bacteria is Acinetobacter oleivorans CGMCC No.23119, and has good low-temperature denitrification effect under alkaline conditions.
Drawings
FIG. 1 is a scanning electron microscope image of KX-3 strain.
FIG. 2 shows the pairs of NH and KX-3 of 48h strain at different temperatures 4 + -N and NO 3 - -a removal effect of N.
FIG. 3 shows the pairs of NH and KX-3 of 48h strain at different pH values 4 + -N and NO 3 - -a removal effect of N.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Example 1
Enrichment and isolation of strains
(1) Strain sources: soil samples were from the soil of the returned Lasman hilly area under the 36 th scientific investigation of south Pole in China. Mixing soil samples with sterile water according to a volume ratio of 1:9 are put into a 250ml conical flask according to the proportion, and are oscillated for 1h at 15 ℃ and 120r/min to ensure that the soil and the sterile water are evenly distributed.
(2) Preparing enrichment medium: 5.0g of peptone, 5.0g,NaCl 30.0g,pH 7.0 +/-0.2 of yeast and 1L of distilled water.
(3) 5mL of the soil suspension mixed with sterile water in the step (1) is added into 100mL of sterilized enrichment medium, and the mixture is oscillated for 48 hours at 15 ℃ and 150r/min.
(4) Domestication of low-temperature-resistant aerobic denitrifying bacteria: preparing a denitrification medium: 1.3g of sodium acetate, 0.722g of potassium nitrate, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, pH 7.0+/-0.2 and 1L of distilled water. Trace elements: 10.0g of EDTA10.2 g of zinc sulfate, 1.2g of manganese chloride tetrahydrate, 1.0g of ferrous sulfate heptahydrate, 0.5g of copper sulfate pentahydrate, 0.3g of cobalt chloride hexahydrate, 0.2g of sodium molybdate dihydrate, 0.1g of calcium chloride, pH 7.0+/-0.2 and 1L of distilled water. 10mL of the enrichment liquid in the step (3) is added into 100mL of sterilized denitrification medium, and the mixture is oscillated for 48h at 15 ℃ and 150r/min. 10ml of the suspension was then added to fresh sterilized denitrification medium. And oscillating again at 15 ℃ for 48 hours at 150r/min. And measuring NO3 by spectrophotometry at the end of the reaction - -a removal effect of N. Repeating the above operation until NO3 - The removal effect of the N reaches more than 80% of stability.
(5) Low temperature resistant aerobic denitrifying bacteria primary screening: preparing bromothymol blue culture medium: 1.3g of sodium acetate, 0.722g of potassium nitrate, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, 20g of agar, 1ml of 1% bromothymol blue, pH 7.0+/-0.2 and 1L of distilled water. After autoclaving at 121 ℃, solid plates were made. Taking 1ml of liquid acclimation culture medium of the last period, diluting the bacterial liquid into 10 according to 10 times dilution method -1 —10 -8 0.1ml of the gradient bacterial suspension is coated on a denitrification solid medium containing bromothymol blue, and the suspension is uniformly coated by a sterile coater. Culturing in a biochemical incubator at 15 ℃ until clear colonies are grown. Single colonies of bromothymol blue medium changing from yellow to blue were selected, and streaking purification was continued on bromothymol blue solid medium, and this was repeated 3-5 times until single colonies appeared in isolation. Each single bacterium is named as A bacterium, B bacterium, C bacterium and the like. This is the initial strain with denitrification capability.
(6) Low temperature resistant aerobic denitrifying bacteria re-screening: transferring the strain with denitrification capability obtained in the step (5) into 100ml of sterilized denitrification medium by using a sterile inoculating loop, sampling every 4h, and measuring NO3 by using a spectrophotometry - -a removal effect of N. Obtaining the strain with high-efficiency aerobic denitrification capability.
(7) Determination of the nitrifying ability of the strain: inoculating the high-efficiency aerobic denitrification strain obtained in the step (6) into a heterotrophic nitrification culture medium. Heterotrophic nitrification medium: 1.3g of sodium acetate, 0.382g of ammonium chloride, 0.877g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 2ml of trace elements, 7.0+/-0.2 of pH and 1L of distilled water. Oscillating at 15 deg.c and 150r/min. NH was measured every 4h by Nahner reagent spectrophotometry 4 + -a removal effect of N.
(8) Strain preservation: preparation of purified preservation culture medium, peptone 10.0g, yeast extract 5.0g,NaCl 1.0g,20g agar, pH 7.0-7.2, and distilled water 1L. Sterilizing at 121deg.C for 30min. The synchronous heterotrophic nitrification-aerobic denitrification bacteria with the number of KX-3 are obtained through streak separation culture. It was streaked on a plate and kept in a-80℃refrigerator with 50% glycerol for further use.
(9) Identification of species
Reference is made to the "Berger's Manual of bacteria identification" and the "manual of identification of the common bacterial System".
Colony morphology characterization: acinetobacter (Acinetobacter calcoaceticus) KX-3 colony is round, and has clean, white and smooth edges.
16S rDNA sequence determination:
16S rDNA sequencing was performed by Shanghai Paeno Biotechnology Co. TaKaRaLysis Buffer for Microorganism to Direct PCR (Code No. 9164) and centrifuging to obtain supernatant as template, and reacting at 80deg.C for 15min; the target fragment was amplified by PCR using TaKaRa 16S rDNA Bacterial Identification PCR Kit (Code No. RR176). The PCR reaction system (50. Mu.L) was 1. Mu.L of template DNA, 25. Mu.L of PCR Premix, 0.5. Mu.L of Forward Primer (20 pmol/ul), 0.5. Mu.L of Reverse Primer2 (20 pmol/ul) and 23. Mu.L of 16S-free H2O. PCR reaction conditions were 94℃for 5min;30 cycles of 94℃1min,55℃1min,72℃1.5min;72 ℃ for 5min; the desired fragment was recovered by cutting with Takara MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0 (Code No. 9762); DNA sequencing was performed using SEQ Forward, SEQ International and SEQ Reverse as primers. After sequencing, the strain 16SrDNA sequence is obtained, and is shown as SEQ ID NO. 1.
Example 2
The following experiment was used to verify the beneficial effects of the present invention.
The steps of the synchronous heterotrophic nitrification-aerobic denitrification effect of the strain at different temperatures are as follows:
(1) Configured with NO 3 - -N and NH 4 + Weighing 0.288g KNO of simulated sewage with N as nitrogen source 3 With 0.153g NH 4 Cl)，0.615g CH 3 COONa、0.439gKH 2 PO 4 、0.20gMgSO 4 ·7H 2 O and 2mL of trace elements are dissolved in 1L of deionized water, and 100mL of prepared solution is respectively taken in a 150mL conical flask.
(2) Inoculating 0.5mL of enrichment culture medium suspension in logarithmic phase (OD 600 not less than 0.8) into the conical flask, and standing in water bath at 15, 25, 30, 35 and 40deg.CCulturing in a warm shaker, and monitoring NO every 4h 3 - -N and NH 4 + -N content.
As shown in FIG. 2, strain KX-3 was isolated from NO at 48h at various temperatures 3 - -N and NH 4 + Synchronous removal of N-As can be seen from FIG. 2, NO3 - -N and NH 4 + The N removal rate decreases gradually with increasing temperature, NO3 - -N and NH 4 + N reaches a maximum at 15℃of 62.35% and 70.6%, respectively.
The steps of the synchronous heterotrophic nitrification-aerobic denitrification effect of the strain under different pH values are as follows:
(1) Configured with NO 3 - -N and NH4 + Weighing 0.288g KNO of simulated sewage with N as nitrogen source 3 With 0.153g NH 4 Cl)，0.615g CH 3 COONa、0.439g KH 2 PO4、0.20g MgSO 4 ·7H 2 O and 2mL of trace elements are dissolved in 1L of deionized water, and 100mL of prepared solution is respectively taken in a 150mL conical flask.
(2) Inoculating 0.5mL of enrichment culture medium suspension in logarithmic phase (OD 600 is greater than or equal to 0.8) into the conical flask, culturing in water bath constant temperature shaker at 15deg.C with different solution pH= 5.0,6.0,7.0,8.0,9.0,10.0,11.0, and monitoring NO every 4h 3 - -N and NH4 + -N content.
As shown in FIG. 3, strain KX-3 was shown to be NO at 48h at various pH' s 3 - -N and NH 4 + Synchronous removal of N-As can be seen from FIG. 3, NO3 - -N and NH 4 + Increasing NO with increasing pH 3 - N removal efficiency reaches a maximum of 65.20% at pH 10. NH (NH) 4 + N reaches a maximum of 75.3% at pH 11. This shows a good effect of simultaneous denitrification of the strain KX-3 under alkaline conditions.
Comparative example 1
The sludge in the secondary sedimentation tank of the Songjiang sewage treatment plant is used as a strain source, and the strain obtained by screening is also good in denitrification effect and reaches about 80% through the same process and method as in the case. But the optimal reaction temperature is 30 ℃. And the strain KX-3 still has good denitrification effect at low temperature. This is probably due to the fact that the microorganism operating temperature in the Songjiang sewage plant is mostly medium temperature, and the strain is adapted without low temperature environment. And the sample source of the strain KX-3 is the south extremely cold region, and the microorganism has stronger adaptability to low temperature.
SEQUENCE LISTING
<110> university of east China
2, 1
<120> A Acinetobacter and use thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1420
<212> DNA
<213> 1
<400> 1
cttacacatg cagtcgagcg gagagaggta gcttgctacc gatcttagcg gcggacgggt 60
gagtaatgct taggaatctg cctattagtg ggggacaaca tttcgaaagg aatgctaata 120
ccgcatacgt cctacgggag aaagcagggg atcttcggac cttgcgctaa tagatgagcc 180
taagtcggat tagctagttg gtggggtaaa ggcctaccaa ggcgacgatc tgtagcgggt 240
ctgagaggat gatccgccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtggggaa tattggacaa tgggcggaag cctgatccag ccatgccgcg tgtgtgaaga 360
aggccttatg gttgtaaagc actttaagcg aggaggaggc tactttagtt aatacctaga 420
gatagtggac gttactcgca gaataagcac cggctaactc tgtgccagca gccgcggtaa 480
tacagagggt gcaagcgtta atcggattta ctgggcgtaa agcgcgcgta ggcggctaat 540
taagtcaaat gtgaaatccc cgagcttaac ttgggaattg cattcgatac tggttagcta 600
gagtgtggga gaggatggta gaattccagg tgtagcggtg aaatgcgtag agatctggag 660
gaataccgat ggcgaaggca gccatctggc ctaacactga cgctgaggtg cgaaagcatg 720
gggagcaaac aggattagat accctggtag tccatgccgt aaacgatgtc tactagccgt 780
tggggccttt gaggctttag tggcgcagct aacgcgataa gtagaccgcc tggggagtac 840
gggtcgcaag actaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt 900
ggtttaattc gatgcaacgc gaagaacctt acctggcctt gacatagtaa gaactttcca 960
gagatggatt ggtgccttcg ggaacttaca tacaggtgct gcatggctgt cgtcagctcg 1020
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttttcctt atttgccagc 1080
gagtaatgtc gggaacttta aggatactgc cagtgacaaa ctggaggaag gcggggacga 1140
cgtcaagtca tcatggccct tacggccagg gctacacacg tgctacaatg gtcggtacaa 1200
agggttgcta cctagcgata ggatgctaat ctcaaaaagc cgatcgtagt ccggattgga 1260
gtctgcaact cgactccatg aagtcggaat cgctagtaat cgcggatcag aatgccgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtt tgttgcacca 1380
gaagtagcta gcctaactgc aaagagggcg gtaccacggt 1420
Claims (2)
1. An acinetobacter, which is characterized in thatAcinetobacter oleivorans) KX-3 has a preservation number of CGMCC No.23119, and the nucleotide sequence of 16SrDNA is shown as SEQ ID NO. 1.
2. Use of acinetobacter according to claim 1 for biological denitrification, wherein said nitrogen is NH 4 + -N and NO 3 - -N。
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| CN106987547A (en) * | 2017-06-09 | 2017-07-28 | 重庆大学 | One plant of Acinetobacter bauamnnii and its application |
| CN107338197A (en) * | 2017-04-22 | 2017-11-10 | 中国烟草总公司福建省公司 | The acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade |
| CN110819560A (en) * | 2019-11-07 | 2020-02-21 | 北京师范大学 | Preparation and application method of composite microbial inoculum for efficiently treating chemical wastewater |
| CN111909867A (en) * | 2020-07-21 | 2020-11-10 | 广东石油化工学院 | Heterotrophic nitrification-aerobic denitrification bacterium and culture method and application thereof |
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| CN107338197A (en) * | 2017-04-22 | 2017-11-10 | 中国烟草总公司福建省公司 | The acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade |
| CN106987547A (en) * | 2017-06-09 | 2017-07-28 | 重庆大学 | One plant of Acinetobacter bauamnnii and its application |
| CN110819560A (en) * | 2019-11-07 | 2020-02-21 | 北京师范大学 | Preparation and application method of composite microbial inoculum for efficiently treating chemical wastewater |
| CN111909867A (en) * | 2020-07-21 | 2020-11-10 | 广东石油化工学院 | Heterotrophic nitrification-aerobic denitrification bacterium and culture method and application thereof |
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