CN110656059B - Pseudomonas strain YG8, seed liquid and preparation method and application thereof - Google Patents
Pseudomonas strain YG8, seed liquid and preparation method and application thereof Download PDFInfo
- Publication number
- CN110656059B CN110656059B CN201810695307.6A CN201810695307A CN110656059B CN 110656059 B CN110656059 B CN 110656059B CN 201810695307 A CN201810695307 A CN 201810695307A CN 110656059 B CN110656059 B CN 110656059B
- Authority
- CN
- China
- Prior art keywords
- nitrogen
- strain
- pseudomonas
- removal rate
- ammonia 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.)
- Expired - Fee Related
Links
- 241000589516 Pseudomonas Species 0.000 title claims abstract description 46
- 239000007788 liquid Substances 0.000 title claims description 15
- 238000002360 preparation method Methods 0.000 title description 3
- 241000894006 Bacteria Species 0.000 claims abstract description 33
- 241000589755 Pseudomonas mendocina Species 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 11
- 239000002351 wastewater Substances 0.000 claims abstract description 11
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 44
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 39
- 230000001580 bacterial effect Effects 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 28
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 18
- 239000001632 sodium acetate Substances 0.000 claims description 18
- 235000017281 sodium acetate Nutrition 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000012258 culturing Methods 0.000 claims description 15
- 239000000725 suspension Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 229940074404 sodium succinate Drugs 0.000 claims description 6
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- 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 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 abstract description 105
- 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 62
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 abstract description 55
- 239000002054 inoculum Substances 0.000 abstract description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 78
- 229910052757 nitrogen Inorganic materials 0.000 description 39
- 230000000694 effects Effects 0.000 description 34
- 230000012010 growth Effects 0.000 description 33
- 230000008569 process Effects 0.000 description 27
- 230000002829 reductive effect Effects 0.000 description 19
- 238000011081 inoculation Methods 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 238000005070 sampling Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 230000003698 anagen phase Effects 0.000 description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 8
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 8
- 239000002028 Biomass Substances 0.000 description 7
- 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
- 241001052560 Thallis Species 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 230000009935 nitrosation Effects 0.000 description 7
- 238000007034 nitrosation reaction Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 241000588986 Alcaligenes Species 0.000 description 4
- 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 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 101710144092 Periplasmic nitrate reductase Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 230000001546 nitrifying effect Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000004323 potassium nitrate Substances 0.000 description 4
- 235000010333 potassium nitrate Nutrition 0.000 description 4
- 239000010865 sewage Substances 0.000 description 4
- 235000010288 sodium nitrite Nutrition 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241001057811 Paracoccus <mealybug> Species 0.000 description 3
- 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 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 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 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 101150109980 napA gene Proteins 0.000 description 3
- ZNCPFRVNHGOPAG-UHFFFAOYSA-L sodium oxalate Chemical compound [Na+].[Na+].[O-]C(=O)C([O-])=O ZNCPFRVNHGOPAG-UHFFFAOYSA-L 0.000 description 3
- 229940039790 sodium oxalate Drugs 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 2
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 108090000913 Nitrate Reductases Proteins 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012851 eutrophication Methods 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000001814 pectin Substances 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- 235000010987 pectin Nutrition 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 description 1
- 101710104136 3-hydroxyanthranilate 3,4-dioxygenase 2 Proteins 0.000 description 1
- 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 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000588624 Acinetobacter calcoaceticus Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000588813 Alcaligenes faecalis Species 0.000 description 1
- 241000005847 Alcaligenes sp. HN-S Species 0.000 description 1
- 101100453791 Alkalimonas amylolytica kefB gene Proteins 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 241001245216 Bacillus hwajinpoensis Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 101100186433 Dictyostelium discoideum napA gene Proteins 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 239000007818 Grignard reagent Substances 0.000 description 1
- 241001343354 Halenia corniculata Species 0.000 description 1
- 241000588754 Klebsiella sp. Species 0.000 description 1
- 241000016659 Klebsiella sp. Y6 Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins 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
- 241000219000 Populus Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 241000084225 Raoultella sp. Species 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 101100348562 Synechocystis sp. (strain PCC 6803 / Kazusa) nhaS5 gene Proteins 0.000 description 1
- 101150016368 TAD1 gene Proteins 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940005347 alcaligenes faecalis Drugs 0.000 description 1
- 101150090396 aphA gene Proteins 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 241001205446 bacterium YG-8 Species 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 101150081397 dps gene Proteins 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000021384 green leafy vegetables Nutrition 0.000 description 1
- 150000004795 grignard reagents Chemical class 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 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
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—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
- C12N1/20—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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses a Pseudomonas strain YG8, wherein the Pseudomonas strain YG8(Pseudomonas mendocina YG8) is heterotrophic nitrification-aerobic denitrification bacterium and is preserved in Guangdong province microorganism strain preservation center, the preservation time is 3-21 days in 2018, and the preservation number is GDMCC NO: 60340. the pseudomonas strain YG8 has high removal rate for ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, can provide inoculum resources for biological treatment of complex nitrogen-containing wastewater, and has great application value.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a pseudomonas strain YG8, a seed solution, a preparation method and an application thereof.
Background
With the rapid development of social economy and the aggravation of urbanization, the prevention and treatment of water pollution is more and more emphasized by people. How to solve the increasingly serious water eutrophication phenomenon becomes an important aspect for treating water pollution. Eutrophication of water harms the survival of aquatic organisms, affects the ecological environment, and increases the difficulty and cost of water treatment. The hypermethemoglobinemia is easily caused by the overhigh concentration of nitrate in drinking water.
The biological denitrification technology is widely applied due to the characteristics of economy, high efficiency, easy operation, no secondary pollution and the like. The traditional theory considers that the nitrification is that nitrifying bacteria convert ammonia nitrogen into nitrate nitrogen under aerobic conditions; the denitrification is that the denitrifying bacteria convert nitrate nitrogen into nitrogen under the anoxic condition. Since the nitrification process and the denitrification process are different in environmental conditions, it is necessary to increase the cost and decrease the denitrification efficiency.
Since the discovery of Heterotrophic Nitrification-Aerobic Denitrification bacteria Paracoccus pantopus (ATCC 35512), a Heterotrophic Nitrification-Aerobic Denitrification process (HN-AD) is proposed to break through the traditional biological Denitrification concept, namely, the Heterotrophic Nitrification-Aerobic Denitrification bacteria convert ammonia nitrogen into nitrate nitrogen under Aerobic conditions, and the nitrate nitrogen can be used as a substrate under anoxic conditions and further converted into nitrogen. From now on, nitrification and denitrification can be carried out in the same system, and novel biological denitrification technology is innovatively developed. With the continuous and deep research on the denitrification theory of wastewater, the remarkable denitrification effect of HN-AD bacteria makes people pay attention to the function of HN-AD bacteria in nitrogen circulation and the potential value of HN-AD bacteria in wastewater treatment.
The heterotrophic nitrification-aerobic denitrification bacteria discovered at present mainly comprise Bacillus (Bacillus), Alcaligenes (Alcaligenes), Paracoccus (Paracoccus) and Pseudomonas (Pseudomonas), however, nitrite nitrogen has certain toxicity to microorganisms and can inhibit the growth and denitrification performance of the strains, so that the heterotrophic nitrification-aerobic denitrification bacteria which can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen with high removal rate are fewer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the pseudomonas strain YG8 which has high removal rate on ammonia nitrogen, nitrate nitrogen and nitrite nitrogen.
In order to solve the technical problem, the invention adopts the following technical scheme:
the Pseudomonas strain YG8, wherein the Pseudomonas strain YG8(Pseudomonas mendocina YG8) is heterotrophic nitrification-aerobic denitrification bacterium, and is preserved in Guangdong province collection of microorganism strains, and the preservation unit address is as follows: the preservation time of the No. 59 building 5 of the No. 100 college of the Pistan-Turcz Zhonglu, Guangzhou city is 3 months and 21 days in 2018, and the preservation number is GDMCC NO: 60340.
as a general inventive concept, the invention also provides a seed solution of Pseudomonas bacterial strain YG8, which is prepared by activated culture of the Pseudomonas bacterial strain YG 8.
As a general inventive concept, the present invention also provides a method for preparing the seed liquid of the Pseudomonas strain YG8, comprising the following steps:
inoculating pseudomonas strain YG8 into LB culture medium, culturing to logarithmic phase, centrifuging the obtained bacterial suspension to remove supernatant, washing, and adding sterile water to obtain seed liquid of pseudomonas strain YG 8.
In one embodiment, the culturing process comprises: performing shaking culture at 25-35 ℃ and 100-180 rpm for 12-18 h.
In one embodiment, the LB medium comprises: peptone 10 g.L -1 5 g.L yeast extract -1 ，NaCl 10g·L -1 ，pH 7.0～7.5。
In one embodiment, sterile water is added to the bacterial liquid OD 600 0.65 to 0.80.
As a general inventive concept, the present invention also provides an application of the above Pseudomonas strain YG8 in the treatment of nitrogen-containing wastewater.
In one embodiment, the nitrogen-containing wastewater contains NH 4 + 、NO 3 - And NO 2 - One or more of (a).
In one embodiment, the nitrogen-containing wastewater also contains a carbon source.
In one embodiment, the carbon source is one or more of sodium citrate, sodium succinate and sodium acetate.
Compared with the prior art, the invention has the advantages that:
the heterotrophic nitrification-aerobic denitrification Pseudomonas YG8(Pseudomonas mendocina YG8) has high removal rate for ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, can provide inoculum resources for biological treatment of complex nitrogen-containing wastewater, and has great application value. The ammonia nitrogen removal rate reaches over 99.99 percent within 24 hours, and nitrate nitrogen and nitrite nitrogen are not accumulated; the removal rate of nitrate nitrogen reaches 96.77 percent, and almost no nitrite nitrogen is accumulated; the removal rate of nitrite nitrogen reaches 91.74%, and nitrate nitrogen is not accumulated.
Specifically, in the heterotrophic nitrification process, sodium acetate is used as a carbon source, C/N is 12, and 50-200 mg.L -1 The ammonia nitrogen concentration range, the ammonia nitrogen removal rate is increased along with the increase of the ammonia nitrogen concentration; at 200 mg.L -1 When the method is used, the ammonia nitrogen removal rate reaches 98.05 percent to the maximum; the nitrogen concentration of nitrate is 50 mg.L -1 When the concentration of nitrogen nitrate in YG8 is 50 mg.L, the removal rate of nitrogen nitrate is 97.76% at most -1 In the process, the removal rate of the nitrite nitrogen is 90.43% at most, so that the nitrite nitrogen has small toxic effect on pseudomonas YG8, and the growth of the strain and the nitrite nitrogen removal performance are not inhibited basically in a certain concentration range.
In addition, compared with other heterotrophic nitrification-aerobic denitrification bacteria, YG8 has a faster growth rate and YG8 has a higher removal rate of nitrate nitrogen, so that YG8 can remove the nitrogen pollution source in the water body with higher efficiency. And the ammonia nitrogen concentration which the strain tolerates reaches 500 mg.L -1 。
Drawings
FIG. 1 is a colony morphology of Pseudomonas YG8 of the present invention.
FIG. 2 is a transmission electron microscope image of Pseudomonas YG8 of the present invention.
FIG. 3 is a 16S rDNA amplification electrophoretogram of Pseudomonas YG8 of the present invention.
FIG. 4 is a phylogenetic tree of Pseudomonas YG8 according to the present invention.
FIG. 5 shows the PCR amplification electrophoresis of the YG8napA gene of Pseudomonas bacteria of the present invention.
FIG. 6 is a graph showing the aerobic denitrification performance of Pseudomonas YG8 using potassium nitrate as the sole nitrogen source.
FIG. 7 is a graph showing the aerobic denitrification performance of Pseudomonas YG8 with sodium nitrite as the sole nitrogen source.
FIG. 8 is a graph showing the heterotrophic nitrification-denitrification performance of Pseudomonas YG8 using ammonium sulfate as the sole nitrogen source according to the present invention.
FIG. 9 is a graph showing the effect of carbon source on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 10 is a graph showing the effect of C/N on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 in accordance with the present invention.
FIG. 11 is a graph showing the effect of pH on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 12 is a graph showing the effect of temperature on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 13 is a graph showing the effect of inoculum size on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 14 is a graph showing the influence of rotational speed on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 15 is a graph showing the effect of different ammoniacal nitrogen concentrations on the heterotrophic nitrification denitrification performance of Pseudomonas YG8 in accordance with the present invention.
FIG. 16 is a graph showing the effect of C/N on aerobic denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 17 is a graph showing the effect of C/N on aerobic nitrosification denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 18 is a graph showing the effect of different nitrate nitrogen concentrations on the aerobic denitrification performance of Pseudomonas YG8 according to the present invention.
FIG. 19 is a graph showing the effect of different nitrite nitrogen concentrations on the aerobic denitrification performance of Pseudomonas YG8 according to the present invention.
Detailed Description
The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.
The media formulations used in the examples are as follows:
LB Medium (g.L) -1 ): 10 parts of peptone, 5 parts of NaCl, 5 parts of yeast extract and 7.0-7.5 parts of pH.
Enrichment medium (g.L) -1 )：(NH 4 ) 2 SO 4 0.5, 2.17 percent of sodium succinate and 50ml/L of Vickers salt solution.
Vickers salt solution: KHPO 4 ·3H 2 O 6.05，MgSO 4 ·7H 2 O 2.50，NaCl 2.50，FeSO 4 ·7H 2 O 0.05，MnSO 4 ·H 2 O 0.04。
Heterotrophic nitrification medium (g.L) -1 )：(NH 4 ) 2 SO 4 0.24, other components and contents are the same as those of the enrichment medium.
Aerobic Denitrification Medium (DM): KNO 3 0.36, sodium succinate 2.81, Na 2 HPO 4 ·7H 2 O7.9, trace element solution 2 ml. L -1 pH7.0-7.5; solution of trace elements (g.L) -1 )：EDTA 50，CuSO 4 ·5H 2 O 1.57，ZnSO 4 2.20， CaCl 2 5.50，FeSO 4 ·7H 2 O 5.00，MnCl 2 ·4H 2 O 5.06，CoCl·6H 2 O 1.61，pH7.0。
Broomthymol blue Medium (BTB) (g.L) -1 )：KNO 3 1.00, L-asparagine 1.00, 0.1% bromothymol blue 5ml (diluted with alcohol), sodium citrate 8.50, KH 2 PO 4 1.00，MgSO 4 ·7H 2 O 1.00，CaCl 2 0.2，FeCl 3 ·6H 2 O0.05, and the pH is adjusted to 7.0-7.3.
Beef extract peptone medium (g.L) -1 ): 3.00 of beef extract, 10.00 of peptone and 5.00 of NaCl, and adjusting the pH value to 7.0-7.2.
If solid culture medium is prepared, agar 15.00-20.00 g.L is added -1 . The above culture medium is sterilized at 121 deg.C for 20 min.
Reagent: greens reagent: solution A: 0.50g of sulfanilic acid and 150ml of dilute acetic acid (10 percent), and can be used within one month after being stored away from light; and B, liquid B: 0.10g of 1-naphthylamine, 20ml of distilled water and 150ml of diluted acetic acid (10%). The two solutions AB are mixed evenly in equal amount before use.
The experimental instruments used in the examples are shown in table 1:
TABLE 1 Experimental apparatus
The water quality testing and analyzing items and methods in the examples are shown in table 2:
TABLE 2 items and methods of Water quality testing analysis
Example (b):
the Pseudomonas strain YG8 of the present invention is heterotrophic nitrification-aerobic denitrification bacterium YG8(Pseudomonas mendocina YG8), which is deposited in Guangdong province microorganism strain collection center with the deposition time of 2018, 3-21 days, and the deposition number of GDMCC NO: 60340.
1) separating and screening strains
The pseudomonas YG8 of the above example was obtained by the following method:
1.1) preliminary screening
Activated sludge collected from Longjin sewage treatment plant of Longyan city, Fujian province is added into a conical flask filled with glass beads in an enrichment medium for enrichment culture. After the enrichment is finished, the bacterial liquid is diluted and coated on a beef extract peptone solid plate. After a single colony grows out, further streaking and purifying. And (3) selecting the purified strain, inoculating the purified strain into a sterilized heterotrophic nitrification culture medium, and taking the bacterial liquid every 2 days and dropwise adding a Grignard reagent to confirm whether the strain has nitrification activity. And inoculating the primarily screened heterotrophic nitrifying strains to a BTB primary screening culture medium for coating culture, wherein each strain is at least 2 parallel samples, and culturing in a constant-temperature incubator at 30 ℃. The denitrification process can increase the pH value of the BTB culture medium, so that blue halos can be generated around colonies, single colonies with the blue halos are selected, streaking separation is carried out on the DM solid culture medium, the separation operation is repeated for 3 times, and the single colonies are selected for storage after growth. The screened strain has heterotrophic nitrification and aerobic denitrification activities.
1.2) double sifting
And (3) selecting the strains obtained by primary screening, respectively inoculating the strains into sterilized heterotrophic nitrification culture medium and aerobic denitrification culture medium, carrying out shake culture on a shaker at the temperature of 30 ℃ and the rotating speed of 150rpm, detecting the content of ammonia nitrogen and nitrate nitrogen in the culture medium after 48 hours, screening out the strains with high degradation rate of ammonia nitrogen and nitrate nitrogen, and selecting the strains for subsequent research on denitrification performance.
2) Strain identification
The identification process and the results of the pseudomonas YG8 strain of the above example are as follows:
2.1) morphological identification
And (3) culturing, selecting strains to be identified, streaking the strains on a beef extract peptone flat culture medium, and observing the colony morphology after culturing for 48 hours.
The colony morphology results of YG8 plate are shown in FIG. 1: the colony surface is yellow, opaque, clear and round, and has a sticky texture.
2.2) gram stain visualization
Colony smears in the logarithmic growth phase (18-24h) were picked, air-dried, fixed on a flame and then gram-stained. The color and size of the cells were observed by an optical microscope.
Gram staining results show that: YG8 is a gram-negative bacterium, and is rod-shaped.
2.3) Transmission Electron microscopy morphology
The examination was carried out by the institute for microorganisms of Guangdong province.
The transmission electron microscope morphology results are shown in fig. 2: the single bacterium is in the shape of a short rod, and the size of the bacterial body is 362X 253 nm.
2.4) physiological and biochemical identification
The procedure was carried out according to Bergey Manual of bacteriology, and other references were made in part.
The physiological and biochemical reactions of bacteria are one of the important bases for the classification and identification of bacteria. The YG8 strain was identified according to Bergey Manual of bacteriology and Manual of systematic identification of common bacteria. The results of the identification are shown in table 3: YG8 can grow at 4 deg.C and 41 deg.C, and can react in the experiment of catalase, starch hydrolysis, urea hydrolysis, pectin hydrolysis, acetic acid oxidation, etc., and the experimental result is positive. And (3) identification through physiological and biochemical experiments: YG8 is similar to Pseudomonas mendocina (Pseudomonas sp.).
TABLE 3 physiological and biochemical experimental results of YG8
| Detecting the index | Results | Detecting the index | Results |
| Contact enzyme | + | Hydrolysis of urea | + |
| Oxidase enzyme | - | Pectin hydrolysis | + |
| V.P | - | Oxidation of acetic acid | + |
| Methyl Red | - | Production of hydrogen sulfide | - |
| Fermentation of glucose | -- | Citric acid salt | + |
| Lactose fermentation | -- | Milk with litmus | Acidity |
| Pyocyancin | + | Nitrate reduction | + |
| Liquefaction of gelatin | + | Indole experiments | + |
| Starch hydrolysis | + | Oxidative fermentation | ++ |
| Hydrolysis of fats and oils | - | Growth temperature | Growth at both 4 ℃ and 41 ℃ |
Note: + indicates a positive result, a reaction occurs; a negative result was indicated, and the reaction was absent. In the glucose fermentation, the first +/-indicates acid production/non-acid production and the second +/-indicates gas production/non-gas production. In the oxidative fermentation, the first +/-indicates reaction/non-reaction under aerobic conditions, and the second +/-indicates reaction/non-reaction under anaerobic conditions.
2.5) identification of the bacterial species in molecular biology
Extracting strain DNA as PCR template for amplification. The primers are universal primers, 27F: 5 'AGAGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. PCR reaction system (50 ul): 41.5ul of sterile water, 5ul of Buffer, 1492R 1ul, 27F 1ul, 0.5ul of taq enzyme and 1ul of dNTP. The PCR reaction condition is that firstly, the mixture is pre-denatured for 3min at 95 ℃; ② denaturation at 95 ℃ for 45s, annealing at 55 ℃ for 45s and extension at 72 ℃ for 45 s; thirdly, the step 2 is circulated for 30 times; fourthly, final extension is carried out for 10min at 72 ℃ and storage is carried out at 4 ℃. And purifying the obtained PCR amplification product by agarose gel electrophoresis, sending the product to Shanghai Huamao gene company to complete the determination of the 16S rDNA sequence of the strain, and performing strain homology analysis according to the result to determine the phylogenetic position of the strain.
Amplification of NapA functional gene. Nitrate reductases are classified into membrane-bound nitrate reductases (Nar) and periplasmic nitrate reductases (Nap), in which periplasmic nitrate reductase genes are preferentially expressed under aerobic conditions and function under both anaerobic and aerobic conditions. Using primer nap 1: 5 '-TCTGGACCATGG-GCTTCAACCA-3', nap 2: 5 '-ACGACGACCGGCCAGCGCAG-3' and carrying out amplification on a PCR instrument. Reaction system (25 ul): 10 Xbuffer 2.5ul, dNTP 0.5ul, NAP10.5ul, NAP20.5ul, template DNA 1ul, Taq enzyme 0.25ul, sterile water 19.75 ul. Setting PCR conditions: firstly, pre-denaturation is carried out for 10min at 94 ℃; ② denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1.5 min; thirdly, the step 2 is circulated for 30 times; fourthly, final extension is carried out for 10min at 72 ℃ and storage is carried out at 4 ℃. And (3) carrying out agarose gel electrophoresis purification and imaging and photographing by a gel imaging system on the obtained PCR amplification product, and then sending the PCR amplification product to Shanghai Huada Gene company to complete sequence determination.
YG8 strain 16S rDNA amplification is shown in FIG. 3: the PCR product band was brighter and the channel had no other bands, which were between 1000-2000bp, indicating that the 16S rDNA target band of YG8 was obtained. The 16S rDNAPCR product of strain YG8 was purified and sequenced to a sequence length of 1294 bp. Phylogenetic analysis of the strains was performed based on the sequencing results, which are shown in FIG. 4: YG8 has 100% similarity to Pseudomonas mendocina, and YG8 is named as Pseudomonas mendocina YG8 (hereinafter referred to as YG 8).
Amplification of periplasmic nitrate reductase subunit genes:
the DNA sample of YG8 strain was amplified by primer nap1/nap2 and subjected to agarose gel electrophoresis, and the amplification results are shown in FIG. 5: about 750-1000bp has clear specific band, the periplasmic nitrate reductase subunit gene napA separated from the poplar seed is 870bp, and the bacteria contains the napA gene by preliminary judgment, which shows that the bacterial strain YG8 has aerobic denitrification function.
And (2) taking the YG8 strain to be subjected to activated culture in an LB culture medium until the strain liquid reaches the logarithmic phase, culturing for 48 hours in a constant-temperature shaking table at the temperature of 30 ℃, pouring the strain liquid in the LB culture medium into a centrifuge tube, centrifuging for 10min at 4000rpm, collecting thalli, washing the thalli with sterile water, centrifuging, and repeating the operation for 2-3 times. Then, the bacterial cells were resuspended in sterile water, and the concentration of the bacterial cells was adjusted to an OD600 value within the range of 0.65-0.80, to obtain a seed solution.
The application example is as follows:
(1) aerobic denitrification performance
Inoculating the bacterial suspension into the initial nitrate with the nitrogen concentration of 50 mg.L by using the inoculation amount of 3 percent -1 Culturing in 500mL conical flask containing 200mL aerobic denitrification culture medium with C/N of 8 and pH of 7.5 at 30 deg.C and 150rpm shaking table, sampling every 6h, and determining ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, COD and TN (total nitrogen) content, pH and OD 600 。
The aerobic denitrification performance with potassium nitrate as the only nitrogen source is shown in figure 6: OD for 0-6 h 600 The curve increases more gradually, OD 600 The growth is only increased from 0.07 to 0.11, which is the adaptation process of the strain to the culture environment and is the strain growth adaptation period; 6-18h OD 600 The slope of the straight line is increased, the strain is multiplied and reaches the maximum value of 0.31 at 18h, and the strain is in the logarithmic growth phase; thereafter OD 600 The slope of the straight line is negative, and the strain enters the decline stage. The nitrate nitrogen removal rate is increased from 16.51% to 96.77% during the logarithmic growth period of the strain, the TN removal rate is increased from 12.95% to 67.13%, and the COD removal rate is increased from 17.35% to 96.61%, which indicates that the nitrate reduction process mainly occurs in the logarithmic phase; nitrite nitrogen is rapidly accumulated in logarithmic growth phase and reaches a maximum value of 4.35 mg.L at 24h -1 . And the TN removal rate is reduced from 67.13% to 53.90%. The pH of the medium was increasing throughout the cultivation, indicating that the denitrification process was accompanied by alkali production.
In the whole denitrification process, a small amount of nitrite accumulation is detected and then reduced by a small amount, which indicates that the thalli YG8 should firstly reduce nitrate nitrogen into nitrite, induce the generation of nitrite reductase and further reduce nitrite into gas to overflow the system. Initial nitrogen concentration was 40 mg.L as compared with the same species of strain -1 In the denitrification system of (1), the nitrate nitrogen removal rate of Pseudomonas mendocina HAD-2 at 24h is less than 86.40%, and YG8 at the initial nitrogen concentration is 50 mg.L -1 The nitrate nitrogen removal rate reaches 96.77% after 24h, compared with YG8, which has higher nitrate nitrogen removal rate and fast reaction rate; under aerobic conditions, Zooglose sp.ZN2, ZN1 and ZN3 (iron reducing bacteria) take sodium nitrate as the only nitrogen source,the results of the determination of the denitrification capacity showed that 3 strains of bacteria entered the logarithmic growth phase at 6h as with YG8, but the OD of the 3 strains after 50h of culture 600 Only reaches the maximum value of 0.06, and the removal rate of nitrate nitrogen is 0.06mg (L.h) when the culture is carried out for 47h -1 About, and the removal rate of YG8 to nitrate nitrogen at 24h reaches 2.02mg (L.h) -1 . Compared with the thallus growth speed of ZN1, ZN2 and ZN3, the YG8 growth speed is higher, and the YG8 has higher removal rate of nitrate nitrogen, so that the YG8 strain can remove the nitrate nitrogen in the water body with higher efficiency.
(2) Aerobic nitrosation performance
Inoculating the bacterial suspension into the initial nitrite nitrogen concentration of 50 mg.L with the inoculation amount of 3 percent -1 Culturing in 500mL conical flask containing 200mL aerobic nitrosation culture medium with C/N of 8 and pH of 7.5 at 30 deg.C and 150rpm shaking table, sampling every 6h, and determining ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, COD and TN content, pH and OD 600 。
The aerobic nitrosation denitrification performance with sodium nitrite as the only nitrogen source is shown in figure 7: OD is between 0 and 6h 600 The growth is slow, indicating that the strain is in a lag phase during this time; OD during 6-12h 600 The growth is multiplied and reaches a maximum value of 0.41 at 12h, and the stage is the logarithmic growth phase of the strain; after 12h, the thallus growth begins to enter the decline stage, and the OD is 12-24h 600 Decline, nutrient reduction and accumulation of metabolites inhibit growth of bacteria. The removal rates of nitrite nitrogen, TN and COD are respectively increased to 91.11%, 53.85% and 92.51% in 18h, and the removal rates of nitrite nitrogen, TN and COD tend to be stable after 18h and respectively reach 91.74%, 56.23% and 93.29%. During the whole culture process, the pH value of the culture medium is increased, which indicates that the aerobic nitrosation process is accompanied with alkali production. Nitrate nitrogen is little, and the accumulated amount reaches 1.58 mg.L at 6h -1 And tends to be stable.
YG8 is capable of using nitrate nitrogen and nitrite nitrogen as nitrogen sources like most heterotrophic nitrification-aerobic denitrification bacteria, for example, Acinetobacter calco aceticus N7 strain, with initial nitrate nitrogen and nitrite nitrogen concentrations of 50 mg.L -1 Removal rate of nitrate nitrogen and nitrite nitrogen product at 24hThe cumulative amounts were 65.4% and 0.7 mg.L, respectively -1 The removal rate of nitrite nitrogen and the accumulated amount of nitrate nitrogen are respectively 80.8 percent and 4.3 mg.L -1 YG8 showed nitrite nitrogen removal rate and nitrate nitrogen accumulation amount of 91.74% and 1.58 mg.L, respectively -1 Therefore, YG8 has better aerobic denitrification performance than N7 strain. It was reported that Alcaligenes faecalis No.4 could not use nitrite nitrogen as a nitrogen source for growth nor for denitrification, unlike the use of nitrate and nitrite nitrogen by strain YG 8.
(3) Heterotrophic nitrification performance
Inoculating the bacterial suspension into the initial ammonia nitrogen concentration of 50 mg.L with the inoculation amount of 3 percent -1 Culturing in 500mL conical flask containing 200mL heterotrophic nitrification medium with C/N of 8 and pH of 7.5 at 30 deg.C and 150rpm shaking table, sampling every 6h, and determining ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, COD and TN content, pH and OD 600 。
The results of the heterotrophic nitrification denitrification performance of YG8 using ammonium sulfate as the nitrogen source are shown in FIG. 8: OD for 0-12h 600 The growth is geometric multiple, and is logarithmic growth phase; OD after 12h 600 The growth rate is slow, and the OD is at 24h 600 A maximum of 0.35 is reached. The removal rate of the ammonia nitrogen is rapidly increased within 0-12h, and the removal rate of the ammonia nitrogen reaches 100% within 12 h. The nitrifying bacteria produce acid in the heterotrophic denitrification process to reduce the pH, and the aerobic denitrification process produces alkali. The pH of the strain YG8 was increased from 7.34 to 8.61 during the heterotrophic nitrification denitrification process, indicating that YG8 was accompanied by aerobic denitrification during the heterotrophic nitrification process. During the nitration reaction, the accumulation amount of nitrate nitrogen in 18h reaches 2.83 mg.L -1 And subsequently degraded completely, also indicating that the heterotrophic nitrification process at YG8 is accompanied by aerobic denitrification.
The removal of ammonia nitrogen and COD in the heterotrophic nitrification process of many strains occurs in the logarithmic growth phase like that of the YG8 strain, for example, the removal rates of ammonia nitrogen and COD in the logarithmic growth phase of the Klebsiella sp.y6 strain reach 99.67 percent and 82.17 percent respectively, and almost no nitrate nitrogen and nitrite nitrogen accumulate in the whole process. Compared with Klebsiella sp.HLNR02 cultured for 24h, the ammonia nitrogen removal rate reaches 87.7%, the ammonia nitrogen removal rate of YG8 reaches 100% in 12h, and the ammonia nitrogen removal efficiency of the YG8 strain is higher.
(4) Factor influencing heterotrophic nitrification denitrification performance
(4.1) Effect of carbon Source on the heterotrophic nitrification denitrification Performance
Respectively inoculating the bacterial suspension into different heterotrophic nitrification culture media (a 250mL conical flask with the loading of 100 mL) which take sodium succinate, sodium citrate, glucose, sodium acetate, sodium bicarbonate, sodium oxalate and sucrose as carbon sources and do not contain the carbon sources at the inoculation amount of 3 percent, wherein the initial ammonia nitrogen concentration is 50 mg.L -1 C/N8, pH 7.5, incubated at 30 ℃ on a shaker at 150rpm, sampled for 24h, and assayed for ammonia nitrogen and TN content as well as pH and OD 600 。
The effect of carbon source on the denitrification performance of strain YG8 is shown in FIG. 9: when sodium citrate, sodium acetate and sodium succinate are used as carbon sources, the YG8 bacterial biomass reaches more than 0.2, the ammonia nitrogen removal rate is higher than 89%, and the TN removal rate is higher than 65%, which shows that the growth condition and the denitrification effect of the strain YG8 are better when the three organic acids are used as carbon sources. When glucose and sucrose were used as carbon sources, the ammonia nitrogen degradation rates of YG8 were 32.75% and 28.58%, respectively, and the ammonia nitrogen removal rates were poorer when glucose and sucrose were used as carbon sources than those of sodium acetate with a lower molecular weight, indicating that the carbon source has a simple structure and a lower molecular weight, and is more easily utilized by the bacteria. When sodium oxalate was used as the carbon source, the strain did not grow substantially, indicating that sodium oxalate, an organic carbon source, was not suitable for the growth of YG 8. When sodium bicarbonate is used as a carbon source, YG8 basically does not grow, the ammonia nitrogen degradation rate is 65%, and the TN removal rate is 0, which indicates that the strain YG8 can utilize an inorganic carbon source and has autotrophic nitrification capability, but the inorganic carbon is not suitable for the growth of the strain and the degradation of total nitrogen. In addition, the medium without carbon source had the lowest ammonia nitrogen removal rate and no degradation of TN compared to other medium with carbon source, indicating that YG8 requires carbon source for growth and denitrification. In conclusion, when sodium acetate is used as a carbon source, YG8 has high biomass and high ammonia nitrogen and TN removal rate, so sodium acetate is selected as the carbon source for heterotrophic nitrification denitrification.
(4.2) influence of C/N on the heterotrophic nitrification denitrification Performance
Inoculating the bacterial suspension with 3% inoculum size respectively with C/N of 6, 8, 10, 12, 16, 20, carbon source of sodium acetate, nitrogen source of ammonium sulfate, and initial ammoniaThe nitrogen concentration is 50 mg.L -1 100mL of heterotrophic nitrification medium with pH of 7.5 is cultured in a shaker at 30 ℃ and 150rpm, and after 24 hours, sampling is carried out, and the content of ammonia nitrogen and TN as well as pH and OD are measured 600 。
The effect of C/N on the heterotrophic nitrification-denitrification performance of the strain YG8 is shown in FIG. 10: when C/N is 6, the ammonia nitrogen degradation rate of YG8 can reach 71.74%, C/N is in the range of 6-12, the biomass and ammonia nitrogen removal rate of the strain YG8 increase with the increase of C/N, and when C/N is 12, the ammonia nitrogen removal rate of YG8 reaches the maximum value 95.49%, and the total nitrogen removal rate is 89.73%; when C/N is increased to 30, ammonia nitrogen removal rate is basically unchanged, but OD 600 And the total nitrogen removal rate is obviously reduced, and the total nitrogen removal rate is reduced to 41.74 percent. This indicates that sufficient amount of carbon source is helpful to promote the heterotrophic nitrification denitrification performance of YG8 strain, but when the amount of carbon source is larger than the amount required by YG8 strain, the ammonia nitrogen removal rate is not affected by C/N basically, but the growth of cells is easily inhibited by too high carbon source, and the proper C/N enables the strain to grow rapidly and exert better denitrification capability. Therefore, C/N-12 was selected as the C/N for the subsequent experiments.
Compared with the C/N of heterotrophic nitrification-aerobic denitrification bacteria reported at present, such as Acinetobacter calcoeceticus N7, the initial ammonia nitrogen concentration is 50 mg.L -1 C/N is 35, and the removal rates of ammonia nitrogen and total nitrogen within 24 hours reach maximum values of 88.6 percent and 90.0 percent respectively. In contrast, the best C/N for the growth of the strain YG8 and the heterotrophic nitrification denitrification is smaller, and is particularly suitable for the low C/N sewage treatment.
(4.3) influence of pH on the heterotrophic nitrification denitrification Performance
Respectively inoculating the bacterial suspension with the inoculation amount of 3 percent into the bacteria with the pH values of 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, wherein the carbon source is sodium acetate, and the initial ammonia nitrogen concentration is 50 mg.L -1 100mL of heterotrophic nitrification culture medium with C/N of 12 is cultured in a shaking table at 30 ℃ and 150rpm, sampling is carried out after 24 hours, and the content of ammonia nitrogen and TN as well as pH and OD are measured 600 。
The microorganisms can grow over a wide pH range, but all have an optimum pH. The pH in the environment is closely related to the growth metabolism of the bacteria: the activity of the microbial enzyme is influenced by pH, and the stability of the enzyme is influenced by higher or lower pH, so that the activity of the enzyme is inhibited; secondly, the pH can change the potential of the microbial cell membrane, thereby influencing the absorption of the microbes on nutrient substances; in addition, too low a pH may result in hydrolytic deterioration of nucleic acids and microbial surface proteins, inhibiting the growth and metabolism of microorganisms. Therefore, pH is an important influence factor on the heterotrophic nitrification denitrification performance.
The effect of pH on the heterotrophic nitrification denitrification performance of YG8 is shown in FIG. 11: the YG8 strain can grow well in the pH range of 6.5-9 and has an ammonia nitrogen removal rate of over 90%, but the total nitrogen removal rate is reduced with the increase of pH. When the pH value is equal to 6.5, the ammonia nitrogen degradation rate and the total nitrogen removal rate of the strain are both maximum and respectively reach 98.23% and 83.02%, which indicates that YG8 is more suitable for a weakly acidic environment. The pH value of the current practical sewage treatment biochemical pond is generally 6-7, which indicates that YG8 has potential application value in the practical sewage treatment. Therefore, pH6.5 was selected as the initial pH of the medium for the subsequent experiments.
(4.4) influence of temperature on the heterotrophic nitrification denitrification Performance
Inoculating the bacterial suspension with 3% inoculum size, sodium acetate as carbon source, ammonium sulfate as nitrogen source, and initial ammonia nitrogen concentration of 50 mg.L -1 C/N12, pH6.5, shaking table culturing at 25, 30, 35 and 40 deg.C and 150rpm, sampling after 24 hr, measuring ammonia nitrogen and TN content and pH and OD 600 。
The temperature has obvious influence on the survival and metabolism of the microorganisms, and the bacteria stop growing when the temperature is too low; when the temperature is higher, proteins, nucleic acids and the like in cells are destroyed, and the cells are inactivated; suitable temperatures enable faster growth of the bacteria. The effect of temperature on the nitrification denitrification performance of the strain YG8 is shown in FIG. 12: when the temperature is increased to 30 ℃ at 25 ℃, the ammonia nitrogen removal rate is respectively 74.34 percent and 75.10 percent; when the temperature is 35 ℃, the removal rate of ammonia nitrogen is reduced to 65.74%; the ammonia nitrogen removal rate at 40 ℃ is only 35.54 percent. The ammonia nitrogen removal rate of strain YG8 decreased by nearly 40% during the temperature increase from 25 ℃ to 40 ℃, indicating that YG8 did not tolerate high temperatures and the denitrification efficiency decreased with increasing temperature, probably due to the decreased activity of the enzyme involved in denitrification. Therefore, 30 ℃ was selected as the incubation temperature for the subsequent experiments.
(4.5) influence of inoculum size on heterotrophic nitrification denitrification Performance
Inoculating the bacterial suspension with 3%, 5%, 7%, 10% and 15% of inoculum size to obtain a carbon source of sodium acetate, wherein the initial ammonia nitrogen concentration is 50 mg.L -1 Culturing 100mL heterotrophic nitrification culture medium with C/N of 12 and pH of 6.5 in a shaker at 30 ℃ and 150rpm for 24h, sampling, measuring ammonia nitrogen and TN content and pH and OD 600 。
The effect of the inoculum size on the heterotrophic nitrification denitrification performance of YG8 is shown in FIG. 13: when the inoculation amount is 3% -10%, the biomass of the strain YG8 is above 0.38 after 24h of culture, and the removal rates of ammonia nitrogen and TN are both above 92% and above 85%; under the inoculation amount of 10%, the YG8 has the maximum removal rate of ammonia nitrogen and TN, which respectively reach 93.36% and 85.53%. And when the inoculation amount is 15%, the OD 600 Greatly increased, reduced ammonia nitrogen and total nitrogen removal rate, and reduced OD 600 The reason for the great increase is that the inoculation amount is large, the basic number of the bacterial biomass is large, and the denitrification rate is reduced because the inoculation amount is too high to meet the nutrient substances required by the growth amount of microorganisms under certain nutritional conditions, so that the bacteria grow and even die, the dead bacterial strains can generate autolysis, and then intracellular organic nitrogen is released, and the denitrification rate is reduced. Therefore, the inoculation amount of 10 percent is selected as the inoculation amount of the subsequent heterotrophic nitrification denitrification performance influencing factors. Compared with Raoultella sp.sari01, the ammonia nitrogen removal rate of 30h is 99.8% with the inoculation amount of 10%, and the ammonia nitrogen degradation rate of the strain YG824h is 93.36%, the reaction rate of YG8 for removing ammonia nitrogen is faster.
(4.6) influence of rotational speed on heterotrophic nitrification denitrification Performance
Inoculating 10% inoculum size of bacterial suspension into carbon source sodium acetate with initial ammonia nitrogen concentration of 50 mg.L -1 Culturing 100mL heterotrophic nitrification medium with C/N of 12 and pH of 6.5 in a shaking table at 30 ℃ and rotating speeds of 0, 75, 150 and 180rpm respectively for 24h, sampling and determining ammonia nitrogen and TN contents and pH and OD 600 。
The dissolved oxygen content has a significant influence on nitrification, and the dissolved oxygen is positively correlated with the rotation speed, so that the dissolved oxygen in the culture medium can be adjusted by changing the rotation speed of the shaking table. The effect of rotational speed on the heterotrophic nitrification denitrification performance of the YG8 strain is shown in FIG. 14: the rotating speed is increased from 0rpm to 75rpm, the biomass of YG8 thalli is increased from 0.116 to 0.26, the ammonia nitrogen removal rate and the TN removal rate are respectively increased from 0 percent and 0 percent to 55.77 percent and 47.06 percent, which indicates that the static culture is insufficient in dissolved oxygen and is unfavorable for the growth of YG 8; when the rotating speed is increased to 150rpm, the ammonia nitrogen removal rate is increased to 99.84%, the total nitrogen removal rate reaches 85.42%, which indicates that along with the increase of the rotating speed of the shaking table to 150rpm, the liquid in the culture medium forms vortex, the air retention time is prolonged, the air contact area is enlarged, the dissolved oxygen of the culture medium is increased, the growth requirement of the bacterial strain YG8 is met, and therefore, the bacterial strain denitrification rate is high; when the rotating speed is continuously increased to 180rpm, the rotating speed of the shaking table is higher, the denitrification rate of the strain YG8 is reduced, the reason may be that the dissolved oxygen in the culture medium is too high, and simultaneously, the bacterial liquid and the triangular flask collide violently under high-speed operation, so that the bacterial cell damage is caused, and the synthesis capacity of the enzyme related to the denitrification process is inhibited. In summary, the rotation speed of 150rpm was selected as the subsequent experimental condition.
The ammonia nitrogen removal rate and the rotation speed change rules of different strains are different. For example, the ammonia nitrogen removal rate of the strain TAD1 increases with the increase of the rotating speed in the rotating speed range of 0-180rpm, and the ammonia nitrogen removal rate decreases with the increase of the rotating speed of 180rpm and 210rpm, which is approximately the same as the ammonia nitrogen removal change rule of YG8 with the change of the rotating speed. And the heterotrophic nitrifying bacteria Alcaligenes sp.HN-S bred by the Zangqingmei and the like are in the range of 0-250rpm, and the higher the rotating speed is, the more beneficial to the heterotrophic nitrification and denitrification of the Alcaligenes.
(4.7) influence of Ammonia Nitrogen concentration on heterotrophic nitrification denitrification Performance
Inoculating the bacterial suspension with the initial ammonia nitrogen concentration of 50, 100, 150, 200, 300, 400, 500 and 800 mg.L at the inoculation amount of 10 percent -1 The carbon source is sodium acetate, 100ml heterotrophic nitrification culture medium with the content of sodium acetate adjusted to make C/N12 and pH6.5 is cultured in a shaking table at 30 ℃ and 150rpm, samples are taken every 24h, and the content of ammonia nitrogen, TN, COD, nitrate nitrogen and nitrite nitrogen, pH and OD are measured 600 。
The effect of ammonia nitrogen concentration on the performance of YG8 heterotrophic nitrification denitrification is shown in FIG. 15: 50-500 mg.L on the seventh day of culture -1 Ammonia nitrogen concentration range, OD 600 With ammoniaThe nitrogen concentration increases and is in positive correlation. When the ammonia nitrogen concentration is 500 mg.L -1 Then, OD 600 The maximum value is 1.17, which indicates that the strain YG8 can grow in a higher ammonia nitrogen environment; the ammonia nitrogen concentration is 800 mg.L -1 Then, OD 600 The decrease to 0.100 indicates 800 mg.L -1 The ammonia nitrogen concentration of the strain exceeds the tolerance degree of the strain. 50-200 mg.L -1 The ammonia nitrogen concentration range, the ammonia nitrogen removal rate is increased along with the increase of the ammonia nitrogen concentration; at 200 mg.L -1 When the method is used, the ammonia nitrogen removal rate reaches 98.05 percent to the maximum; the ammonia nitrogen concentration is 300-500 mg.L -1 The YG8 removal rate of ammonia nitrogen and TN is reduced; the ammonia nitrogen concentration is 500 mg.L -1 The ammonia nitrogen removal rate is 56.02%, and the TN removal rate is 18.77%. The experimental results show that: under the condition of low ammonia nitrogen concentration, the improvement of nutrient substances is beneficial to the growth and the propagation of bacteria; and when the mass concentration of the ammonia nitrogen is too high, the inhibition can be generated on the bacterial strain, and the growth metabolism and the effective reaction of the bacterial strain are reduced. The strain YG8 can tolerate the ammonia nitrogen concentration of 500-800 mg.L -1 And on the other hand, the method proves that the method has potential application value in denitrification of high ammonia nitrogen wastewater.
The strain Pseudomonas putida LY1 has an initial ammonia nitrogen concentration of 50 mg.L -1 The highest ammonia nitrogen removal efficiency is 64%, and the ammonia nitrogen degradation rate is gradually reduced along with the increase of the ammonia nitrogen concentration; sp YN3 with the increase of the initial ammonia nitrogen concentration, the ammonia nitrogen removal effect is gradually reduced, and the initial ammonia nitrogen concentration is 150 mg.L -1 The removal rate of the ammonia nitrogen is only 18.7 percent in 24 hours, and when the initial ammonia nitrogen concentration is improved to 200 mg.L -1 In this case, denitrification reaction hardly occurs. LY1 and YN3 have the same law with YG8, but YG8 has better tolerance and removal effect on ammonia nitrogen concentration. Some species of aerobic denitrifying strains have larger ammonia nitrogen tolerance range and higher concentration, but the ammonia nitrogen removal rate is not high, such as R aoultella sp.sari01 which increases from 200 to 2000 mg.L with the ammonia nitrogen concentration -1 The ammonia nitrogen concentration is reduced from 99.7 percent to 12.3 percent; alcaligenes farecalis No.4 is increased from 100 to 1200 mg.L with ammonia nitrogen concentration -1 But the ammonia nitrogen removal rate is reduced from 100% to 8.33%.
(5) Influence factor of aerobic denitrification performance
(5.1) influence of C/N on aerobic denitrification and nitrosation denitrification Performance
Inoculating 10% of the bacterial suspension into sodium acetate as a carbon source, wherein the initial nitrate nitrogen and nitrite nitrogen concentrations are both 50 mg.L -1 At pH6.5, culturing in a nitrate nitrogen culture medium with C/N of 4, 8, 12, 16, 20, 25 and 30 and a nitrite nitrogen culture medium with C/N of 4, 8, 12, 16 and 20 at 30 ℃ and 150rpm in a shaking table, sampling every 24h, and measuring the contents of nitrate nitrogen, nitrite nitrogen and TN, and the pH and OD 600 。
C/N is one of the important factors influencing the denitrification effect. The effect of different C/N on the aerobic denitrification performance of YG8 with potassium nitrate as the only nitrogen source is shown in FIG. 16: when C/N is 4, the nitrate nitrogen removal rate in 24h of the strain reaches 41.51%, and the TN removal rate is 15.13%. The reason is that the carbon source is insufficient under low C/N, and the growth of thalli is limited due to the lack of sufficient energy, the synthesis of a denitrification enzyme system is incomplete, and the reduction of nitrate nitrogen is a process requiring energy; when the C/N is increased to 12, the removal rate of YG8 on nitrate nitrogen reaches 100%, and the removal rate of TN is over 75%; the biomass basically shows a reduction trend along with the increase of C/N, the nitrate nitrogen removal rate is basically unchanged, the TN removal rate is reduced, and when the C/N is equal to 30, the nitrate nitrogen removal rate and the TN removal rate are the lowest and are respectively 52.53 percent and 49.54 percent, which shows that the carbon source amount is excessive, and the excessive carbon source amount has an inhibiting effect on the removal of the nitrate nitrogen of the thalli. As mentioned above, C/N12 is the experimental condition suitable for YG8 aerobic denitrification process.
The effect of different C/N on the aerobic nitrosation denitrification performance of YG8 with sodium nitrite as the only nitrogen source is shown in FIG. 17: when C/N is 4, the removal rate of nitrite nitrogen reaches 22.82%, the removal rate of total nitrogen is 0, which indicates that the low C/N carbon source is insufficient, and the energy is insufficient, so that the growth of thalli is limited, the synthesis of a nitrosase system is incomplete, and the reduction of nitrite nitrogen is a process requiring energy; when C/N is 12, the nitrite nitrogen removal rate reaches 84.13%, and the total nitrogen removal rate is 47.25%; as C/N is increased, the removal rates of nitrite nitrogen and TN are decreased. This indicates that the amount of carbon source is excessive and that the excessive amount of carbon source inhibits the denitrification effect of the cells. In conclusion, C/N-12 is a suitable experimental condition for the aerobic nitrosation process. The nitrate nitrogen removal is facilitated under the condition of low carbon nitrogen ratio.
(5.2) influence of nitrate nitrogen concentration on aerobic Denitrification Performance
Inoculating the bacterial suspension with 10% inoculum size, sodium acetate as carbon source, initial nitrate nitrogen concentration of 50, 100, 150 and 200 mg.L -1 The nitrate nitrogen medium with C/N of 12 and pH of 6.5 is cultured in a shaker at 30 ℃ and 150rpm, and the nitrate nitrogen and TN contents and the pH and OD are measured by sampling every 24 hours 600 。
The effect of nitrate nitrogen concentration on the aerobic denitrification performance of YG8 with potassium nitrate as the only nitrogen source is shown in FIG. 18: the nitrogen concentration of nitrate is 50 mg.L -1 In time, the highest removal rate of YG8 on nitrate nitrogen is 97.76%, and the removal rate of TN is 57.91%; YG8 had a decreasing effect on nitrate nitrogen removal as the nitrate nitrogen concentration increased. When the nitrate nitrogen concentration rises to 200 mg.L -1 In this case, the nitrate nitrogen removal rate was reduced to 56.91%, and the TN removal rate was 27.63%. Strain YG8 at a nitrate nitrogen concentration of 200 mg.L -1 In the 24h process, the removal rate of nitrate nitrogen can reach 4.74mg (L.h) -1 It is shown that YG8 has a higher removal rate for nitrate nitrogen concentration.
(5.3) influence of nitrite Nitrogen concentration on aerobic Denitrification Performance
Inoculating 10% of the bacterial suspension into sodium acetate as a carbon source, wherein the initial nitrite nitrogen concentrations are respectively 50, 100, 150 and 200 mg.L -1 Culturing in nitrite nitrogen culture medium with C/N of 12 and pH of 6.5 at 30 deg.C and 150rpm, sampling every 24 hr, and determining nitrite nitrogen and TN content and pH and OD 600 。
The effect of nitrite nitrogen concentration on the aerobic denitrification performance of YG8 with sodium nitrite as the only nitrogen source is shown in FIG. 19: nitrite nitrogen concentration is 50 mg.L -1 In the process, the highest removal rate of nitrite nitrogen is 90.43%, and the removal rate of TN is 53.14%; the effect of YG8 on removing nitrite nitrogen gradually decreased with the increase of nitrite nitrogen concentration, and when the nitrite nitrogen concentration increased to 200 mg.L -1 When the temperature of the water is higher than the set temperature,the nitrite nitrogen removal rate is only 12.85%, and the TN removal rate is 0.
Different species of bacteria are not equally tolerant to nitrite nitrogen, for example: strain Bacillus hwajinpoensis. slwx 2 When the nitrite nitrogen concentration is 100 mg.L -1 In time, the nitrite removal rate of 48h is 1.06mg (h.L) -1 . Bacillus coaguluns YX-6 with initial nitrite nitrogen concentration of 20 mg.L -1 Meanwhile, the removal rate of nitrite nitrogen is close to 100%; when the initial concentration is increased to 100 mg.L -1 The removal rate of nitrite nitrogen was only 20%. Compare SLWX 2 And YX-6, nitrite nitrogen 100 mg.L -1 In time, the removal rate of the 24h bacterial strain YG8 nitrite nitrogen can reach 69.87%, and the removal rate is 2.91 mg (h.L) -1 It is shown that YG8 is more effective in removing nitrite nitrogen.
In conclusion, the YG8 strain has high removal rate on ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, has excellent comprehensive performance, can provide inoculum resources for biological treatment of complex nitrogen-containing wastewater, and has great application value.
The above description is only for the preferred embodiment of the present application and should not be taken as limiting the present application in any way, and although the present application has been disclosed in the preferred embodiment, it is not intended to limit the present application, and those skilled in the art should understand that they can make various changes and modifications within the technical scope of the present application without departing from the scope of the present application, and therefore all the changes and modifications can be made within the technical scope of the present application.
Claims (10)
1. The Pseudomonas strain is Pseudomonas mendocina YG8(Pseudomonas indoxydans YG8), which is heterotrophic nitrification-aerobic denitrification bacterium and is preserved in Guangdong province microorganism strain preservation center, wherein the preservation time is 3 and 21 days in 2018, and the preservation number is GDMCC NO: 60340.
2. a seed liquid of Pseudomonas mendocina strain YG8 according to claim 1, which is activated and cultured.
3. The method of preparing a seed liquid of a pseudomonas strain according to claim 2, comprising the steps of:
inoculating the pseudomonas mendocina strain YG8 into an LB culture medium to be cultured to a logarithmic phase, centrifuging the obtained bacterial suspension to remove supernatant, washing, and adding sterile water to obtain a seed solution of the pseudomonas strain.
4. The method according to claim 3, wherein the culturing is carried out by: performing shaking culture at 25-35 ℃ and 100-180 rpm for 12-18 h.
5. The production method according to claim 3, wherein the LB medium is composed of: peptone 10 g.L -1 5 g.L yeast extract -1 ，NaCl 10 g·L -1 ，pH 7.0～7.5。
6. The method according to claim 3, wherein sterile water is added to the OD of the bacterial liquid 600 0.65 to 0.80.
7. Use of a pseudomonas strain according to claim 1 for the treatment of nitrogen-containing wastewater.
8. Use according to claim 7, wherein the nitrogen-containing waste water contains NH 4 + 、NO 3 - And NO 2 - One or more of (a).
9. The use of claim 8, wherein the nitrogen-containing wastewater further comprises a carbon source.
10. The use of claim 9, wherein the carbon source is one or more of sodium citrate, sodium succinate and sodium acetate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810695307.6A CN110656059B (en) | 2018-06-29 | 2018-06-29 | Pseudomonas strain YG8, seed liquid and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810695307.6A CN110656059B (en) | 2018-06-29 | 2018-06-29 | Pseudomonas strain YG8, seed liquid and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110656059A CN110656059A (en) | 2020-01-07 |
| CN110656059B true CN110656059B (en) | 2022-08-09 |
Family
ID=69027539
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810695307.6A Expired - Fee Related CN110656059B (en) | 2018-06-29 | 2018-06-29 | Pseudomonas strain YG8, seed liquid and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110656059B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951658B (en) * | 2020-01-15 | 2022-11-18 | 广州市微生物研究所有限公司 | Pseudomonas and application thereof |
| CN113998832A (en) * | 2020-11-02 | 2022-02-01 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for advanced treatment of total nitrogen in amino acid wastewater |
| CN112852658B (en) * | 2020-12-14 | 2022-10-25 | 暨南大学 | Pseudomonas DNF-23 and method for improving denitrification efficiency of pseudomonas |
| CN113005063B (en) * | 2021-03-25 | 2022-08-23 | 中国科学院微生物研究所 | Pseudomonas putida GY13 and application thereof in sewage treatment |
| CN113087142A (en) * | 2021-04-16 | 2021-07-09 | 北京涞澈科技发展有限公司 | Rapid start accelerator for sulfur autotrophic microorganisms in high-nitrogen domestic sewage |
| CN113717877B (en) * | 2021-07-05 | 2023-09-01 | 中国水产科学研究院珠江水产研究所 | Heterotrophic nitrification-aerobic denitrification bacteria DS2 from seawater and application thereof |
| CN113604379B (en) * | 2021-07-06 | 2023-01-20 | 广州大学 | Pseudomonas holothurians with heterotrophic nitrification-aerobic denitrification function and application thereof |
| CN113968629B (en) * | 2021-11-09 | 2023-09-08 | 上海水源地建设发展有限公司 | Application of pseudomonas TY1 in water treatment and microbial inoculum for water treatment |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812416A (en) * | 2009-11-20 | 2010-08-25 | 东华大学 | Method for extracting pseudomonas mendocina strain |
| CN103484398A (en) * | 2013-08-28 | 2014-01-01 | 温州大学 | Heterotrophic nitrification-aerobic denitrification pseudomonas mendocina as well as culture and application thereof |
| CN105861359A (en) * | 2016-05-17 | 2016-08-17 | 中国石油大学(华东) | Heterotrophic nitrification-aerobic denitrification high temperature resisting strain for producing floc, and application thereof |
| CN106520624A (en) * | 2016-12-07 | 2017-03-22 | 暨南大学 | Pseudomonas mendocina MKC-02 strain and application of pseudomonas mendocina MKC-02 strain to waste water denitrification |
-
2018
- 2018-06-29 CN CN201810695307.6A patent/CN110656059B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812416A (en) * | 2009-11-20 | 2010-08-25 | 东华大学 | Method for extracting pseudomonas mendocina strain |
| CN103484398A (en) * | 2013-08-28 | 2014-01-01 | 温州大学 | Heterotrophic nitrification-aerobic denitrification pseudomonas mendocina as well as culture and application thereof |
| CN105861359A (en) * | 2016-05-17 | 2016-08-17 | 中国石油大学(华东) | Heterotrophic nitrification-aerobic denitrification high temperature resisting strain for producing floc, and application thereof |
| CN106520624A (en) * | 2016-12-07 | 2017-03-22 | 暨南大学 | Pseudomonas mendocina MKC-02 strain and application of pseudomonas mendocina MKC-02 strain to waste water denitrification |
Non-Patent Citations (3)
| Title |
|---|
| Xu et al..Nitrogen Removal Characteristics of Pseudomonas putida Y-9 Capable of Heterotrophic Nitrification and Aerobic Denitrification at Low Temperature.《BioMed Research Internationa》.2017,第1-7页. * |
| 范铮等.冬季微污染河流脱氮菌的筛选及鉴定.《安全与环境工程》.2015,第22卷(第6期),第17-22页. * |
| 郑建龙等.一株好氧反硝化细菌的分离与鉴定.《中国畜牧兽医》.2015,第1877-1882页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110656059A (en) | 2020-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110656059B (en) | Pseudomonas strain YG8, seed liquid and preparation method and application thereof | |
| CN110655199B (en) | Method for treating ammonia nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain | |
| CN110655198B (en) | Method for treating nitrogen-containing wastewater by using heterotrophic nitrification-aerobic denitrification paracoccus strain | |
| CN110656058B (en) | Heterotrophic nitrification-aerobic denitrification pseudomonas strain, seed liquid, and preparation method and application thereof | |
| CN109517770B (en) | Aerobic facultative autotrophic denitrifying bacterium and application thereof | |
| CN111172061B (en) | Aerobic denitrification composite microbial inoculant and application thereof | |
| CN111534449B (en) | Aerobic denitrifying pseudomonas and culture method and application thereof | |
| CN110655200B (en) | Method for treating nitrogen-containing wastewater by using pseudomonas strain YG8 | |
| CN110656057B (en) | Heterotrophic nitrification-aerobic denitrification paracoccus strain, seed liquid, preparation method and application thereof | |
| CN110964663A (en) | Heterotrophic nitrifying bacteria for low-temperature sewage denitrification and application thereof | |
| CN114703095B (en) | Pseudomonas adulthood and application thereof in field of sewage and wastewater purification | |
| CN112551692B (en) | Halomonas with aerobic denitrification and heterotrophic sulfur oxidation functions and application thereof | |
| CN111961609A (en) | Heterotrophic nitrification-aerobic denitrification bacterium agent and application thereof | |
| CN104726366A (en) | Denitrifying phosphorus accumulation organism (DPAO) with function of efficiently removing nitrogen and phosphorus and application of DPAO | |
| CN112266885B (en) | Heterotrophic nitrification aerobic denitrifying bacteria Y16 and application thereof | |
| CN111139198A (en) | Lactobacillus parvum GBW-HB1903 and application thereof | |
| CN113736700B (en) | Heterotrophic nitrification-aerobic denitrification bacteria and application thereof | |
| CN110951647B (en) | Bacillus psychrophilus GBW-HB1901 and application thereof | |
| CN116376756B (en) | Aerobic denitrifying bacteria and application thereof in sewage treatment | |
| CN111269861A (en) | Providencia rettgeri with aniline degradation and denitrification capabilities and application thereof | |
| CN115820466B (en) | Sulfur autotrophic denitrification strain, bacterial preparation and application thereof | |
| CN117821330B (en) | Marine heterotrophic nitrification-aerobic denitrification bacterium with biological denitrification characteristic and biological denitrification application thereof | |
| CN117866826B (en) | Marine heterotrophic nitrification-aerobic denitrification bacterium with high dissolved oxygen characteristic and denitrification application thereof | |
| CN111100811B (en) | Heterotrophic nitrification composite microbial inoculum and application thereof | |
| CN115975845B (en) | Application of salt-resistant/acid-resistant heterotrophic nitrification-aerobic denitrification bacteria in environmental wastewater treatment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220809 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |