CN106434497B - Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation - Google Patents
Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation Download PDFInfo
- Publication number
- CN106434497B CN106434497B CN201611138015.XA CN201611138015A CN106434497B CN 106434497 B CN106434497 B CN 106434497B CN 201611138015 A CN201611138015 A CN 201611138015A CN 106434497 B CN106434497 B CN 106434497B
- Authority
- CN
- China
- Prior art keywords
- siderophore
- high yield
- bacteria preparation
- bacterium
- yield bacteria
- 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
- 241000894006 Bacteria Species 0.000 title claims abstract description 76
- 239000000589 Siderophore Substances 0.000 title claims abstract description 56
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 41
- 239000002689 soil Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 244000025254 Cannabis sativa Species 0.000 claims abstract description 16
- 241000122971 Stenotrophomonas Species 0.000 claims abstract description 9
- 241001607911 Stenotrophomonas rhizophila Species 0.000 claims abstract description 9
- 241000589516 Pseudomonas Species 0.000 claims abstract description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 5
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 11
- 238000009655 industrial fermentation Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 8
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 7
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 7
- 235000013312 flour Nutrition 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 5
- 238000000855 fermentation Methods 0.000 claims description 4
- 230000004151 fermentation Effects 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims 2
- 241001024365 bacterium S17 Species 0.000 claims 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims 1
- 230000008439 repair process Effects 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 50
- 241000196324 Embryophyta Species 0.000 description 27
- 238000012360 testing method Methods 0.000 description 25
- 241000039970 Pseudomonas sp. S17 Species 0.000 description 18
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 17
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 14
- 241001042745 Bacillus sp. S86 Species 0.000 description 13
- 229910052751 metal Inorganic materials 0.000 description 12
- 239000002184 metal Substances 0.000 description 12
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 229910052698 phosphorus Inorganic materials 0.000 description 9
- 239000011574 phosphorus Substances 0.000 description 9
- 238000012545 processing Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000008901 benefit Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 229910052564 epsomite Inorganic materials 0.000 description 7
- 229910052742 iron Inorganic materials 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- 239000004098 Tetracycline Substances 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 238000005067 remediation Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 229960002180 tetracycline Drugs 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- 235000019364 tetracycline Nutrition 0.000 description 5
- 150000003522 tetracyclines Chemical class 0.000 description 5
- 241000222122 Candida albicans Species 0.000 description 4
- 241000223195 Fusarium graminearum Species 0.000 description 4
- 241000813090 Rhizoctonia solani Species 0.000 description 4
- 230000003042 antagnostic effect Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 229940095731 candida albicans Drugs 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 241000198596 Alternaria tomatophila Species 0.000 description 3
- 241000228245 Aspergillus niger Species 0.000 description 3
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- 244000241257 Cucumis melo Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 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 3
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
- 244000061458 Solanum melongena Species 0.000 description 3
- 235000002597 Solanum melongena Nutrition 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000008485 antagonism Effects 0.000 description 3
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 238000013329 compounding Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 235000002566 Capsicum Nutrition 0.000 description 2
- 240000008574 Capsicum frutescens Species 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 2
- 241000223221 Fusarium oxysporum Species 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 2
- 241001123668 Verticillium dahliae Species 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- WYWFMUBFNXLFJK-UHFFFAOYSA-N [Mo].[Sb] Chemical compound [Mo].[Sb] WYWFMUBFNXLFJK-UHFFFAOYSA-N 0.000 description 2
- 229910052793 cadmium Inorganic materials 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- 239000001390 capsicum minimum Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000536 complexating effect Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000009654 indole test Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 231100001231 less toxic Toxicity 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- -1 streptomysin Chemical compound 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 244000056139 Brassica cretica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 241000588700 Dickeya chrysanthemi Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 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
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 240000008488 Thlaspi arvense Species 0.000 description 1
- 235000008214 Thlaspi arvense Nutrition 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 238000002829 antibacterial sensitivity test Methods 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- NNTXFOAPABMVEG-MNOVXSKESA-N chrysobactin Chemical compound NCCCC[C@H](C(=O)N[C@@H](CO)C(O)=O)NC(=O)C1=CC=CC(O)=C1O NNTXFOAPABMVEG-MNOVXSKESA-N 0.000 description 1
- 108010066895 chrysobactin Proteins 0.000 description 1
- NNTXFOAPABMVEG-UHFFFAOYSA-N chrysobactrin Natural products [NH3+]CCCCC(C(=O)NC(CO)C([O-])=O)NC(=O)C1=CC=CC(O)=C1O NNTXFOAPABMVEG-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000003450 growing effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000011133 lead Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 230000004060 metabolic process Effects 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
- 239000002068 microbial inoculum Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the biological prosthetic field of environmental pollution, specially a kind of siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation.A kind of siderophore high yield bacteria preparation, in the siderophore high yield bacteria preparation containing bacillus (BacillusSp.) S86, pseudomonas bacterium (PseudomonasSp.) S17 and thermophilic Stenotrophomonas (Stenotrophomonas rhizophila) LD-6 combination.Above-mentioned siderophore high yield bacteria preparation can be used for farmland pollution heavy metal-polluted soil reparation, which mainly strengthens sugar grass and repair representative heavy metal contaminated soil.
Description
Technical field
The invention belongs to the biological prosthetic field of environmental pollution, specially a kind of siderophore high yield bacteria preparation and its in Polluted Soil
Earth heavy metal repairs the application of aspect.
Background technique
The heavy metal pollution of soil has become global environmental problem, pays close attention to by society and the common people, it would be highly desirable to solve.I
Many regional (especially East China) soil of state are in different degrees of heavy metal pollution, in many area grains, veterinary antibiotics
The content of beary metal such as cadmium, arsenic, copper, lead, zinc are exceeded or close to critical value, seriously endanger people's health.
Heavy metal pollution of soil improvement is extremely urgent but difficult, main reason is that heavy metal is unlike organic dirt
Dye object can be biodegradable like that, and migration is poor in the soil for heavy metal.Traditional physics, chemical method such as soil moved in improve the original, leaching
Wash, ion exchange etc. is often only suitable for the soil remediation of small-scale serious pollution, and because its is at high cost, destroys soil texture, with
And easily causes the disadvantages of secondary pollution and endure to the fullest extent and denounce.In contrast, biological prosthetic, because having, at low cost, effect is good, simple easy
The advantages that capable and without secondary pollution and by favor.Wherein, microorganism-is plant combined repairs as biological prosthetic one kind, because
Its toxicity that heavy metal in soil can be effectively reduced adsorbs heavy metal and changes rhizosphere environment, and then improves plant counterweight
The absorption of metal or fixed efficiency, and implementation cost relative moderate, environmental-friendly, and can extensive in-situ immobilization the advantages that and
It enjoys great popularity, is the new technology for being used to administer large area heavy metal pollution of soil greatly developed, carried out in recent years.On however,
The efficiency for stating combined remediation technology depends on the biomass of rehabilitation plant and the bioavailability of heavy metal in soil.It changes
Yan Zhi, phytomass is bigger and heavy metal can be as often as possible absorbed from soil, then remediation efficiency is higher, effect
Better.How to improve phytoremediation efficiency becomes the hot spot of research and concern.Hyperaccumulative plant (such as Btassica, front yard mustard category
With penny cress category) usually biomass is lower and be difficult to large-scale plantation, thus its in actual repair using extremely limited.And
The big and plant with heavy metal accumulation ability of some fast growings, biomass is just being increasingly being applied to heavy metal pollution field
Ground reparation.As a kind of energy crop that can be used for producing bio-ethanol, sugar grass is strong with voltage endurance capability, growth is rapid, raw
The advantages that object amount is big becomes heavy metal rehabilitation plant most competitive at present.
Siderophore (Siderophore) is the organic compound that a kind of pair of iron that microorganism generates has superpower complexing power,
Its function is to provide iron attainment point to microbial cell, especially under low iron hoop border.The combination of siderophore and heavy metal is main
It is embodied on the biological function of siderophore, i.e., in the iron being concentrated in environment and while promote it to be transported into cell, iron is carried
Body can also influence the bioavailability of other metals by complexing, to reduce its toxicity.In addition, Microbial Iron carrier
It can also be by inhibiting the growth of pathogenic microorganism to promote the disease resistance of root system of plant.Siderophore producing strains are because it is with upper
Advantage, speciality are stated, is waited so becoming the popular of microbial portion in the plant combined repairing heavy metal pollution system of microorganism-
Choosing.
Currently, having many siderophore producing strains by technologies such as manually enrichment, screenings and being isolated from environment, and is real
Pure culture is showed.However, siderophore superior strain is limited in these reported bacterial strains；It is (such as more to have both a variety of advantages, speciality
Heavy metal resistance and disease fungus knot resistance) bacterial strain it is deficient；Have practical fortification of plants and absorbs heavy metal in soil ability
Bacterial strain it is very few.Thus, finding and obtaining the excellent siderophore superior strain of character becomes the hot spot of the research field
And focus.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of siderophore high yield bacteria preparations.
It is a further object to provide above-mentioned siderophore high yield bacteria preparations in farmland pollution heavy metal-polluted soil reparation
The application of aspect, the application mainly strengthen sugar grass and repair representative heavy metal contaminated soil.
Above-mentioned technical problem of the invention is carried out by the following technical programs:
A kind of siderophore high yield bacteria preparation contains bacillus in the siderophore high yield bacteria preparation
(Bacillus sp.) S86, pseudomonas bacterium (Pseudomonas sp.) S17 and thermophilic Stenotrophomonas
The combination of (Stenotrophomonas rhizophila) LD-6.
High yield siderophore bacterium Pseudomonas sp.S17 and Bacillus sp.S86 according to the present invention is originated from Zhejiang
Experimental plot soil in the Scientific and Technological Institutes Of Zhejiang of province Hangzhou screens through artificial enrichment culture, pressure and isolates and purifies to obtain.The present invention
Another plant of bacterium --- the 5a,6,9,9a-hexahydro-6,9-methano-2,4 degradation bacteria being related to has bacterial strain by oneself for this experiment, which is thermophilic Stenotrophomonas
(Stenotrophomonas rhizophila) LD-6, on March 2nd, 2012 in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, number are CGMCC No.5839, which has the ability of degradation 5a,6,9,9a-hexahydro-6,9-methano-2,4.Depositary institution address:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica (similarly hereinafter).
S17 bacterial strain is pseudomonas bacterium, and Gram-negative contacts enzyme positive, oxidase negative, V.P. test
Feminine gender, indole test are positive, and thalli morphology is rod-short, and bacterium colony shows slightly burr, smooth wet in faint yellow, round, edge,
Its physiological and biochemical property is as shown in table 1, and the bacterial strain is on October 12nd, 2016 in China Committee for Culture Collection of Microorganisms
Common micro-organisms center preservation, number CGMCCNo.13104.
The bacterial strain S86 of bacillus (Bacillus sp.), the bacterial strain are micro- in China on October 12nd, 2016
The common micro-organisms center preservation of biological inoculum preservation administration committee, number are CGMCC No.13105.S86 bacterial strain is gemma bar
Campylobacter bacteria, Gram-positive contact enzyme positive, and oxidase negative, V.P. test is positive, and indole test is negative, thallus
Form be it is rod-shaped, bacterium colony is white, round, wax, and other physiological and biochemical properties are as shown in table 2.
The physiological and biochemical property of table 1, bacterial strain S17
Note: "+" indicates growth or positive reaction；"-" expression is not grown or negative reaction (similarly hereinafter).
The physiological and biochemical property of table 2, bacterial strain S86
The optimum growth temperature of S17 bacterial strain is 29 DEG C.With 1/5th LB (Luria-Bertani) or industrial fermentation culture
Base (corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·
3H2O 2g/L, MnSO40.05g/L, pH 7.0) after culture 24 hours, bacterium solution cell concentration is up to 6.5-9.2 × 109CFU/mL
(CFU, Colony Forming Unit).The bacterium (is all to seek to ampicillin, streptomysin, chloramphenicol, kanamycins and sensitive tetracycline
Normal antibiotic).It is considered that: when thallus is released in natural environment, will not be generated because of anti-(resistance to) pharmacological property problem super
Bacterium.In the test of pathogen antagonism, it is found that it is raw the bacterial strain can inhibit three kinds of mould, aspergillus niger and sclerotinia sclerotiorum disease fungus
It is long.The bacterium is to 12 kinds of test metal ion (Li+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+And Co4 +) different degrees of tolerance (or resistance) is presented.In view of China's heavy-metal contaminated soil status --- various heavy pollutes simultaneously
It deposits, it is believed that --- for the more other bacterial strains for not having various heavy tolerance or resistance of the bacterial strain, repaired implementing to strengthen
Repairing effect is influenced small by heavy metal inhibition during multiple.The bacterial strain siderophore active unit (Siderophore unit, SU)
It is 99.2%, it is highest up to siderophore bacterium is produced, it is that domestic reported produces in siderophore bacterium, produces the strongest bacterial strain of siderophore ability.
In addition, S17 is also equipped with the ability of certain Soluble phosphorus and production 3-indolyl acetic acid.Using 1.5% glucose as carbon source, 0.2% ammonium chloride
For nitrogen source, 30 DEG C, pH7.2, incubation time 66h (initial inoculum 2%), which is 120.35mg/L.To containing
The bacterium (inoculum concentration 1%), 28 DEG C, 160r/min culture 3 are accessed in the general LB liquid medium of L-Trp (200mg/L)
It, then measures the 3-indolyl acetic acid content in fermentation liquid, measured value 72.36mg/L.
The optimum growth temperature of S86 bacterial strain is 28 DEG C.With 1/5th LB or after the culture of industrial fermentation culture medium 24 hours,
Bacterium solution cell concentration is up to 6.1-8.6 × 109CFU/mL.The bacterium to ampicillin, streptomysin, chloramphenicol, kanamycins and
Sensitive tetracycline is highly sensitive.It is considered that: it, will not be because resisting (resistance to) pharmacological property problem when thallus is released in natural environment
And generate superbacteria.In the test of pathogen antagonism, it is found that the bacterial strain can inhibit graw mold of tomato, early blight of tomato, eggplant
Wilt disease, cotton wilt, sclerotinia sclerotiorum and the growth of Muskmelon Gummy Stem Blight Pathogen fungi.The bacterium is to 12 kinds of test metal ions
(Li+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+And Co4+) the different degrees of tolerance of presentation (or
Resistance).It is considered that --- for the more other bacterial strains for not having various heavy tolerance or resistance of the bacterial strain, implementing to strengthen
Repairing effect is influenced small by heavy metal inhibition in repair process.Bacterial strain SU value is 76.4%, belongs to strong siderophore producing strains.
Thermophilic Stenotrophomonas (Stenotrophomonas rhizophila) LD-6, bacterium can be under the conditions of supporting well, and 10 days
Interior realization is to the degradable of 100mg/L 5a,6,9,9a-hexahydro-6,9-methano-2,4, and catabolite is less toxic or nontoxic, environment degradable is quick；Bacterial strain LD-6 is to normal
It is sensitive with antibiotic tetracycline and kanamycins, tolerance is presented for test mass metal to 13 kinds；The bacterium is to Candida albicans
(Candida albicans), Rhizoctonia solani Kuhn (Rhizoctonia solani), sclerotinite (Sclerotinia
Sclerotiorum), verticillium wilt pathogen (Verticillium dahliae), fusarium graminearum (Fusarium
Graminearum) and obvious inhibiting effect is presented in capsicum wilt bacterium (Fusarium oxysporum)；In addition, the bacterial strain has
The ability of the standby efficient degradation 5a,6,9,9a-hexahydro-6,9-methano-2,4 under practical soil environment can significantly shorten 5a,6,9,9a-hexahydro-6,9-methano-2,4 degradation duration, have practical application meaning
Justice and value.The optimum growing condition of the bacterium are as follows: pH 7.2, temperature are 30 DEG C.With 1/5th LB (Luria-Bertani) or
Industrial fermentation culture medium (corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/
L, K2HPO4·3H2O2g/L, MnSO40.05g/L, pH 7.2) culture 24-36 hours after, bacterial concentration up to 5-8 ×
109CFU/mL.Thermophilic application of Stenotrophomonas (Stenotrophomonas rhizophila) LD-6 in terms of soil remediation
See ZL201210106224.1.
When S86 bacterial strain, S17 bacterial strain and LD-6 bacterial strain are 29 DEG C, pH7.3 in 1/5th LB or industrial fermentation culture medium,
After when 150r/min co-incubation 24 (one third when S86, S17, LD-6 initial inoculum are respective independent inoculated and cultured),
It was found that bacteria suspension cell density (5.6-10.2 × 109CFU/mL the bacteria suspension thallus that) be apparently higher than when respectively individually cultivating is close
Degree.
Preferably, the preparation is by 1/5th LB (Luria-Bertani) or industrial fermentation culture medium inoculated
Spawn incubation obtains after 22-24 hours, the formula of industrial fermentation culture medium are as follows: corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO40.05g/L.Further,
The temperature of the preparation fermentation culture is 29 ± 1 DEG C, pH7.2-7.4.
Application of the siderophore high yield bacteria preparation in terms of farmland pollution heavy metal-polluted soil reparation a kind of described in.
Preferably, the application is to strengthen sugar grass restoration of soil polluted by heavy metal, method is the sugar grass in growth period
Root sprinkling contain the siderophore high yield bacteria preparation, in siderophore high yield bacteria preparation the total concentration of thallus be 1-5 ×
108CFU/mL。
In conclusion the present invention compared with the existing technology has the advantages that
Pseudomonas sp.S17 and Bacillus sp.S86 bacterial strain of the invention is strong siderophore producing strains.Its
In, Pseudomonas sp.S17 bacterial strain, which has reached, produces the siderophore ability superlative degree, and producing siderophore ability is domestic bacterial strain of having registered
In it is most strong.S17 and S86 bacterial strain is sensitive to common antibiotics, different degrees of tolerance or resistance is presented to 12 heavy metal species, and simultaneous
Has disease fungus knot resistance.And thermophilic Stenotrophomonas (Stenotrophomonas rhizophila) LD-6 has efficiently drop
The ability of 5a,6,9,9a-hexahydro-6,9-methano-2,4 is solved, and catabolite is less toxic or nontoxic, environment degradable is quick；It is quick to common antibiotics tetracycline and kanamycins
Tolerance is presented for test mass metal to 13 kinds in sense；To Candida albicans (Candida albicans), Rhizoctonia solani Kuhn
(Rhizoctoniasolani), sclerotinite (Sclerotinia sclerotiorum), verticillium wilt pathogen (Verticillium
Dahliae), fusarium graminearum (Fusarium graminearum) and capsicum wilt bacterium (Fusarium oxysporum)
Obvious inhibiting effect is presented；In addition, the bacterial strain has the ability of the efficient degradation 5a,6,9,9a-hexahydro-6,9-methano-2,4 under practical soil environment, can significantly contract
Short 5a,6,9,9a-hexahydro-6,9-methano-2,4 degradation duration, has practical application meaning and value.And when by three's co-incubation, it is found that its cell density is significant
Higher than the cell density respectively singly cultivated under the same terms, there are complementary proliferative effects.In addition, S17 bacterial strain is also equipped with Soluble phosphorus energy
Power and production 3-indolyl acetic acid ability.Above-mentioned these advantages and feature are to the plant combined repairing heavy metal pollution soil of microorganism-
For, it is significant！Enable this reparation system it is more practical, more efficiently be applied to contaminated site reparation.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be appreciated that implementation of the invention not office
It is limited to the following examples, the accommodation in any form and/or change made to the present invention fall within present invention protection model
It encloses.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, used equipment and raw material etc.
It is commercially available or commonly used in the art.Method in following embodiments is unless otherwise instructed the normal of this field
Rule method.
The separation of embodiment 1:Pseudomonas sp.S17 and Bacillus sp.S86 and siderophore generate aptitude tests
With experimental plot soil 100g in Scientific and Technological Institutes Of Zhejiang, general five point samplings method collection in worksite Hangzhou, Zhejiang province city, with nothing
Bacterium envelope contains, and takes back laboratory rapidly.
Universal CAS detects liquid and MSA fluid nutrient medium and reports that (Chen Shaoxing, Zhao Xiang, Shen Ping wait Gao Ling according to Chen Shaoxing etc.
The plate detection method microbiology of quick pseudomonad siderophore is notified to, and 2006,33:122-127) it prepares.Solid detects plate
It is then that the CAS detection liquid of the addition 5% in MSA fluid nutrient medium and 2% agar powder solidify.
10 times of diminishing methods of pedotheque are diluted, being coated on 1/5th LB solid plates, (i.e. all nutritional ingredients are
The 1/5 of general LB culture medium) on.25 DEG C of cultures, after there is bacterium colony, the bacterium colony of different shape is chosen one by one with sterile toothpick,
It is transferred on 1/5th new LB solid plates, until purifying is at single colonie.All pure bacterial strain number consecutivelies, and carry out routine
Culture presevation is deposited in the form of inclined-plane among 4 DEG C of refrigerators.
By above-mentioned purifying bacterial strain, it is forwarded on solid detection plate one by one, 28 DEG C of inversions are cultivated 2 days.Observe periphery of bacterial colonies
Whether generation discoloration is enclosed, and does detailed record, produces siderophore ability power using the diameter for the circle that changes colour as siderophore bacterium is produced
Preliminary judgement.In addition, it is negative control that Escherichia coli DH5 α bacterial strain must be arranged simultaneously.
Siderophore quantitative determination reports that (Chen Shaoxing, Zhao Xiang, Shen Ping wait the highly sensitive pseudomonad iron of to carry according to Chen Shaoxing etc.
The plate detection method microbiology of body is notified to, and 2006,33:122-127) it carries out, by respective strains tested culture solution with 1%
Amount be linked into 30mL MSA fluid nutrient medium, 28 DEG C, 200r/min is cultivated for 24 hours, and 10000r/min is centrifuged 15min, supernatant
It is uniformly mixed with 0.22 micron of filtering with microporous membrane, and with isometric CAS detection liquid.Under room temperature, it is surveyed after placing sufficiently
Determine the OD of each sample680(As) value compares zeroing with distilled water.Same procedure measurement is made with each fluid nutrient medium for not connecing bacterium
For the light absorption value of supernatant, as reference value (Ar).The concentration of siderophore indicates that every processing is repeated with siderophore active unit
4 times.Then, according to the report of Persmark et al. (Persmark M, Expert D, Neilands JB.Isolation,
characterization,and synthesis of chrysobactin,a compound withsiderophore
activity from Erwinia chrysanthemi.The Journal of Biological Chemistry,1989,
264:3187-3193) siderophore ability is produced to each bacterial strain to be classified.
As a result, can be cultivated by amounting to obtain on 1/5th LB solid plates by 126 plants of bacterial strain, wherein 9 plants have siderophore
Generation ability (referring to table 3).In the bacterial strain for having siderophore generation ability, and, S86 bacterial strain most strong with the ability of S17 bacterial strain
Take second place.
Table 3, siderophore producing strains produce siderophore capability for registration table
The antibiotic susceptibility test and cause of disease of embodiment 2:Pseudomonas sp.S17 and Bacillus sp.S86 are true
The test of bacterium antagonistic ability
Antibiotic sensitivity test uses filter paper enzyme, selects ampicillin, tetracycline, chloramphenicol, kanamycins and chain
Five kinds of common antibiotics of mycin, with inhibition zone of each antibiotic filter paper on culture siderophore producing strains S17 and S86 plate
Diameter, the criterion as sensitive or resistance to (anti-) medicine.The test result that table 4 and table 5 provide show bacterial strain S17 and
S86 to it is above-mentioned for examination antibiotic be in different degrees of sensitivity response.
The antibiotic susceptibility test result of table 4, bacterial strain S17
Note: antibacterial circle diameter be greater than medium sensitivity range higher limit be it is highly sensitive, less than sensitive range lower limit value
For resistance (similarly hereinafter).
The antibiotic susceptibility test result of table 5, bacterial strain S86
This example demonstrates that when Pseudomonas sp.S17 and Bacillus sp.S86 thallus are released to natural environment
When middle, superbacteria will not be become because of anti-(resistance to) pharmacological property problem, this just provides safety assurance for its subsequent practical application.
With 10 kinds of pathogens --- graw mold of tomato germ, early blight of tomato germ, wilt of eggplant germ, muskmelon are climing withered
Germ, sclerotinia sclerotiorum germ, mould, cotton wilt germ, aspergillus niger, wheat sharp eyespot germ and wheat leaf blight disease
Bacterium is indicator bacteria, utilizes the germ fungi antagonistic ability of tablet face-off method measurement S17 and S86.It is inoculated in general PDA plate center
Indicator bacteria, surrounding are inoculated with S17 and S86 respectively, and every group of test is repeated 5 times, and pass through colony radius and the clear bacterial strain of antibacterial bandwidth
The antagonistic ability of S17 and S86.
The results are shown in Table 6, and Pseudomonas sp.S17 is in for the mould of examination, aspergillus niger and sclerotinia sclerotiorum germ
Obvious Antagonism, Bacillus sp.S86 is then to graw mold of tomato, early blight of tomato, wilt of eggplant, cotton wilt, oil
Dish sclerotiniose and gummy stem blight of melon germ are in knot resistance in various degree.
The antagonistic ability test of table 6, Pseudomonas sp.S17 and Bacillus sp.S86
Note: feminine gender is denoted as "-"；Antibacterial circle diameter 0-5mm is denoted as "+"；Antibacterial circle diameter 5-10mm is denoted as " ++ "；Inhibition zone
Diameter 10-15mm is denoted as " +++ "；Antibacterial circle diameter is greater than being denoted as of 15mm " ++++".
The heavy metal minimum inhibitory concentration (MIC) of embodiment 3:Pseudomonas sp.S17 and Bacillus sp.S86
Test
MIC experimental design carry out referring to the research of Filali et al. (Filali BK, Taoufik J, Zeroual Y,
Dzairi FZ, Talbi M, Blaghen M.Waste water bacterial isolates resistant to
Heavymetals and antibiotics.Current Microbiology, 2000,41:151-156), pick them separately
Greatly, S17 the and S86 single bacterium of activated processing is fallen in culture test tube, and 30 DEG C, 160r/min shaken cultivation 32 hours.According to thallus
Growing way determines MIC value, and every group sets 3 repetitions, respectively tolerance situation of the test strain to 12 metal ion species.As a result such as 7 institute of table
Show, strains tested has multiple heavy metal and is resistant to (anti-) property, and character is excellent.
Table 7, MIC test result
Note: data are the average value of 3 measured values in table.
The Soluble phosphorus and production 3-indolyl acetic acid ability of embodiment 4:Pseudomonas sp.S17 and Bacillus sp.S86 are surveyed
Examination
Take ring Pseudomonas sp.S17 and Bacillus a sp.S86 from test tube slant respectively with oese, point
It is not inoculated in the triangular flask for filling 50mL LB liquid medium, 160r/min shaken cultivation for 24 hours, then respectively asks for 1mL (about 1
×108CFU) culture is inoculated into respectively with Ca3(PO4)2For the 100mL Phos fermentation medium [glucose of unique phosphorus source
10g, (NH4)2SO41g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.002g, MnSO4·
H2O 0.002g, Ca3(PO4)25g, distilled water are settled to 1000mL, pH 7.5] in, 30 DEG C of shaken cultivations (160r/min)
3 repetitions are arranged in 48h.Phosphorus content is measured with general molybdenum antimony resistance colorimetric method --- 1mL culture solution is respectively asked in accordingly numbering
In centrifuge tube, 5000r/min is centrifuged 10min.It accurately takes 1mL supernatant in the 25mL volumetric flask accordingly numbered, adds 2.5mL
Then the anti-developer of molybdenum antimony uses distilled water constant volume.After reacting 30min at room temperature, spectrophotometric determination 700nm wavelength is used
Locate light absorption value, with mg P2O5/ L is indicated.Using the processing for not connecing bacterium as blank control, tests and set 3 repetitions.As a result, measuring
Pseudomonas sp.S17 amount of phosphorus dissolved is 88.6 ± 2.26mg/L, and Bacillus sp.S86 does not have phosphate solubilization.
To containing L-Trp (200mg/L) LB liquid medium in be respectively connected to Pseudomonas sp.S17 and
Bacillus sp.S86,28 DEG C, 160r/min cultivates 3 days.The OD of respective bacteria suspension is then measured with spectrophotometry600Value,
Then bacteria suspension 10000r/min is centrifuged 10min, takes supernatant that isometric Salkowski color solution (Libbert E is added
and Risch H.Interactions between plants andepiphytic bacteria regarding their
Auxin metabolism [J] .Physiologia Plantarum, 1969,22:51-58), being protected from light standing 30min keeps it aobvious
Color measures OD530Value.3 repetitions are arranged in the content of 3-indolyl acetic acid in unit of account volume bacterium solution, and not produce 3- indoles second
The E.coli DH5 α of acid is negative control.As a result, measuring S17 bacterial strain 3-indolyl acetic acid yield is 62.8 ± 0.86mg/L, S86
3-indolyl acetic acid is not detected in bacterial strain and negative control.
People have found during previous heavy metal pollution site remediation --- many reparation plant can be (special because of poor nutritional
Be not the absence of P elements), absorb limited, and the reasons such as growth is suppressed can not farthest play reparation absorption.
This example demonstrates that Pseudomonas sp.S17 has Soluble phosphorus and produces the ability of 3-indolyl acetic acid, and this is deposited to plant is strengthened
It is very practical and important for living, growth and its reparation effectiveness.
Embodiment 5:Pseudomonas sp.S17, Bacillus sp.S86 and Stenotrophomonas
RhizophilaLD-6 strengthens the application that sugar grass absorbs heavy metal jointly
A kind of siderophore high yield bacteria preparation, said preparation are obtained by tri- kinds of bacterium solution compoundings of A, B, C.
A bacterium solution: using Pseudomonas sp.S17 as initial inoculation object, 29 DEG C, industrial fermentation culture medium (corn flour 10g/
L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO4
0.05g/L, pH 7.2) it is made within fermented and cultured 24 hours.
B bacterium solution: using Bacillus sp.S86 as initial inoculation object, 28 DEG C, industrial fermentation culture medium (corn flour 10g/L,
Bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·3H2O 2g/L, MnSO4
0.05g/L, pH 7.5) it is made within fermented and cultured 24 hours.
C bacterium solution: using Stenotrophomonas rhizophila LD-6 as initial inoculation object, 30 DEG C, industrial fermentation training
Support base (corn flour 10g/L, bean cake powder 8g/L, (NH4)2SO46g/L, corn pulp 6g/L, MgSO4·7H2O 1g/L, K2HPO4·
3H2O 2g/L, MnSO40.05g/L, pH 7.2) it is made within fermented and cultured 24 hours.
Before compounding, tri- kinds of bacterium solutions of A, B, C respectively with sterile water or tap water be respectively self-regulated cell concentration to 1.0 ×
108CFU/mL.Then, according to VA:VB:VC=2:2:1 (volume ratio) ratio is compounded to get siderophore high yield bacteria preparation.
It is as follows that soil used in testing picks up from the not vegetable garden by heavy metal pollution, main physical and chemical: organic matter 4.84%,
Total nitrogen 832.6mg/kg, total phosphorus 292.1mg/kg, total potassium 220.3mg/kg, available nitrogen 68.2mg/kg, rapid available phosphorus 18.1mg/kg,
Available potassium 58.3mg/kg, pH6.2.Content of beary metal is cadmium 0.21mg/kg, copper 58mg/kg, zinc 101mg/kg.Air-dry sieving
Afterwards, it is separately added into solution state caddy, zinc chloride and the copper sulphate of different content, makes Cd in soil2+Concentration be 25mg/kg,
Zn2+Concentration be 800mg/kg, Cu2+Concentration be 800mg/kg.Contaminated soil is mixed well, stablizes 2 under 60% damp condition
It is tested after a month.
The seed of rehabilitation plant sugar grass is purchased from Linan City river bridge retail sales.The rudiment of seed elder generation, it is long to the left side 10cm to seedling
When right, select the consistent seedling replanting of growing way enter the good heavy-metal contaminated soil plastic tub of 1.5kg previous steady (high 13cm, directly
Diameter 17cm) in, 1, every basin.It is normal to plant strain growth after transplanting 7 days, the fresh configuration of 1mL is accessed in the root of respective plant, and
The siderophore high yield bacteria preparation shaken up.
To avoid nutritional ingredient in culture medium from influencing test result, above-mentioned access bacterium solution be after sterile water washing 3 times,
The bacterium solution to be suspended again with sterile water.Test sets 4 processing: not planting plant and does not add the blank control (C) of bacterium, only plants plant not
Bacterium (P) is connect, does not plant plant but adds bacterium (M), and had both planted plant or had added bacterium (PM), each processing sets 5 Duplicate Samples.Periodically pour
Water harvests after keeping the illumination of daily 8h, sugar grass to grow 82 days under greenhouse, and root system uses the EDTA solution of 10mmol after cleaning
30min is impregnated, then is rinsed 2 times with deionized water.Plant dries to constant weight for 80 DEG C after 105 DEG C of water-removings, weigh root, overground part it is dry
Weight.Plant tissue clears up (HClO4∶HNO3=1: 4) after, measuring its content of beary metal, it is dry to eliminate that Whole Process does blank control
It disturbs.
The results are shown in Table 8, access Pseudomonas sp.S17, Bacillus sp.S86 and
Stenotrophomonas rhizophila LD-6 can significantly improve the biomass and Metal uptake amount of sugar grass.Its
In, the dry weight of overground part and root has increased separately 31.2% and 36.0% (PM processing and P processing phase than not connecing the control of bacterium
Than), and sugar grass overground part Cd2+、Zn2+And Cu2+Total content be respectively increased 75.5%, 74.5% and 72.9%.It confirms
S17, S86 and LD-6 compounding microbial inoculum, which have, to be promoted sugar grass growth, strengthens sugar grass absorption for the ability of test mass metal.
The Metal uptake amount of table 8, sugar grass overground part and root
Note: content of beary metal=biomass × heavy metal concentration；Extraction efficiency is the total metals (root that plant is extracted
The sum of with overground part) the ratio between with heavy metal total content in initial soil；* indicating the value, there are significant differences with corresponding control value
(P<0.05)。
(Zhang Lu produces siderophore bacterium and strengthens sugar grass repairing heavy metal in soil pollution compared with the immediate prior art
Environmental science and technology, 2014,37:74-79), the overground part dry weight of " plant+microorganism " of the invention processing, overground part Cd2+、
Zn2+And Cu2+Total content, and respectively extraction efficiency compared with it improves 8.8%, 17.7%, 13.8% and 21.0% respectively, and
18.1%, 14.8% and 21.4%, better effect, advantage is significant.And (PM handles recovery rate/P processing in terms of recovery rate promotion
Recovery rate), three heavy metal species recovery rates of the invention promote effect and improve 39.5%, 95.6% and 26.0% respectively compared with it, excellent
Gesture is significant.Furthermore, it is necessary to be intended that, Pseudomonas sp.S17 is the same as the Pseudomonas in above-mentioned fine jade research
Sp.T07 is not same bacterium.Because obvious differences (referring to table 1), is in particular in terms of physiological and biochemical property
Catalase, oxidizing ferment, in four test items of glucose utilization ability and methyl red test.
Claims (5)
1. a kind of siderophore high yield bacteria preparation, it is characterised in that: contain bacillus in the siderophore high yield bacteria preparation
Bacterium (BacillusSp.) S86, pseudomonas bacterium (PseudomonasSp.) S17 and thermophilic Stenotrophomonas
(Stenotrophomonas rhizophila) LD-6 combination；
The bacillus S86 is commonly micro- in China Committee for Culture Collection of Microorganisms on October 12nd, 2016
Bio-Centers preservation, number are CGMCC No. 13105,
The pseudomonas bacterium S17 is commonly micro- in China Committee for Culture Collection of Microorganisms on October 12nd, 2016
Bio-Centers preservation, number are CGMCC No. 13104,
The thermophilic Stenotrophomonas LD-6 is commonly micro- in China Committee for Culture Collection of Microorganisms on March 2nd, 2012
Bio-Centers preservation, number are CGMCC No.5839.
2. siderophore high yield bacteria preparation described in claim 1, it is characterised in that: the preparation is by 1/5th LB
(Luria-Bertani) or industrial fermentation culture medium inoculated Spawn incubation obtains after 22-24 hours, and industrial fermentation culture medium is matched
Side are as follows: 10 g/L of corn flour, bean cake powder 8 g/L, (NH4)2SO46 g/L, corn pulp 6 g/L, MgSO4·7H21 g/L of O,
K2HPO4·3H2O 2 g/L, MnSO4 0.05 g/L。
3. siderophore high yield bacteria preparation as claimed in claim 2, it is characterised in that: the temperature of the preparation fermentation culture is 29
± 1 DEG C, pH7.2-7.4.
4. a kind of application of siderophore high yield bacteria preparation described in claim 1 in terms of farmland pollution heavy metal-polluted soil reparation.
5. application as claimed in claim 4, it is characterised in that: the application is to strengthen sugar grass restoration of soil polluted by heavy metal, side
Method is to contain the siderophore high yield bacteria preparation, bacterium in siderophore high yield bacteria preparation in the sprinkling of the root of the sugar grass in growth period
The total concentration of body is 1 × 108-5×108 CFU/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611138015.XA CN106434497B (en) | 2016-12-12 | 2016-12-12 | Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611138015.XA CN106434497B (en) | 2016-12-12 | 2016-12-12 | Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106434497A CN106434497A (en) | 2017-02-22 |
| CN106434497B true CN106434497B (en) | 2019-06-04 |
Family
ID=58216274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611138015.XA Expired - Fee Related CN106434497B (en) | 2016-12-12 | 2016-12-12 | Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106434497B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110252803B (en) * | 2019-07-23 | 2021-04-30 | 中国科学院成都生物研究所 | Cadmium-contaminated soil composite passivator and application thereof |
| CN110551652A (en) * | 2019-08-28 | 2019-12-10 | 中国科学院生态环境研究中心 | Compound microbial agent and preparation method and application thereof |
| CN111848734B (en) * | 2020-06-17 | 2022-04-05 | 自然资源部第三海洋研究所 | Ferro-carrier PVD with fluorescence characteristic, preparation method and application thereof |
| CN112725221B (en) * | 2020-12-15 | 2023-01-10 | 湘潭大学 | Pseudomonas fluorescens, method for preparing hydroxamic acid type siderophore by using pseudomonas fluorescens and application of pseudomonas fluorescens |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161976A (en) * | 2010-12-28 | 2011-08-24 | 湖南大学 | Endophyte for promoting growth of plants and remediation of heavy metal-contaminated soil and application |
| CN102757907A (en) * | 2012-03-30 | 2012-10-31 | 浙江农林大学 | Endosulfan degradation stain and application thereof in soil remediation |
| CN105985922A (en) * | 2016-01-20 | 2016-10-05 | 华南农业大学 | Bacillus aryabhattai J5 and application thereof |
-
2016
- 2016-12-12 CN CN201611138015.XA patent/CN106434497B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161976A (en) * | 2010-12-28 | 2011-08-24 | 湖南大学 | Endophyte for promoting growth of plants and remediation of heavy metal-contaminated soil and application |
| CN102757907A (en) * | 2012-03-30 | 2012-10-31 | 浙江农林大学 | Endosulfan degradation stain and application thereof in soil remediation |
| CN105985922A (en) * | 2016-01-20 | 2016-10-05 | 华南农业大学 | Bacillus aryabhattai J5 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani;Marco Kai et al.;《Arch Microbiol》;20061216;351-360 * |
| 产铁载体细菌强化甜高粱修复土壤重金属污染;张璐;《环境科学与技术》;20140430;第37卷(第4期);74-79 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106434497A (en) | 2017-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106544306B (en) | One plant of siderophore Producing Strain and its application in terms of farmland pollution heavy metal-polluted soil reparation | |
| Dey et al. | Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria | |
| CN106434497B (en) | Siderophore high yield bacteria preparation and its application in terms of Heavy Metals in Soil Contaminated reparation | |
| CN105586298B (en) | A kind of application of bacillus subtilis, microbial inoculum, the preparation method of microbial inoculum and microbial inoculum | |
| CN106119148A (en) | One pseudomonas and application thereof | |
| CN104877926B (en) | The bacterial strain of one plant of antagonism smoke tree wilt and its application | |
| CN108893419A (en) | Microbial strains and its screening technique and the application in processing heavy-metal contaminated soil | |
| CN105861351A (en) | Bacillus amyloliquefaciens with function of passivating heavy metals and application thereof | |
| CN109628437A (en) | Reduce the composite passivant and the preparation method and application thereof of soil cadmium content | |
| CN109868242B (en) | Salt-tolerant acetoin-producing bacillus subtilis and application thereof | |
| CN103146632A (en) | Method for screening multifunctional bacteria for degrading organophosphorus pesticides | |
| CN109112072B (en) | Application of trichoderma in promoting plant growth | |
| CN108753666B (en) | Salt-tolerant antagonistic bacterium M4-1 and preparation and application of microbial inoculum thereof | |
| CN110396485A (en) | Generate class Brevibacillus brevis and its application of auxin | |
| CN108097711A (en) | A kind of method for repairing Grown In Zinc Contaminated Soil using penicillium janthinellum joint Indian mustard | |
| CN106434490A (en) | Ginseng bacterium TY15-2 with effects of disease prevention and growth promotion and application thereof | |
| CN106754496B (en) | Siderophore high-yield bacterium preparation and application thereof in contaminated soil heavy metal remediation | |
| CN106244501B (en) | One plant of anti-antimony bacterium NXH1 and its application | |
| CN106434446B (en) | One plant of anti-antimony bacterium NXH3 and its application | |
| CN110117545A (en) | One kind having Applying Ectomycorrhizal Fungi and its application of Cr (VI) patience and reducing power | |
| CN102757907B (en) | Endosulfan degradation stain and application thereof in soil remediation | |
| CN114250177B (en) | Acinetobacter and application thereof in improving stress resistance of plants | |
| CN108130303B (en) | Acidovorax vorax TCP2011036 and application thereof | |
| CN110713942A (en) | Copper-zinc-resistant oxytetracycline hydrochloride degrading bacterium, and fermentation broth and application thereof | |
| CN109762766A (en) | One plant has absorption heavy metal, phosphorus decomposing and the prebiotic bacterium and its application of plant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into 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: 20190604 |