CN105505395A - Chitosan oligosaccharide heavy metal bioremediation agent, preparing method thereof and application - Google Patents
Chitosan oligosaccharide heavy metal bioremediation agent, preparing method thereof and application Download PDFInfo
- Publication number
- CN105505395A CN105505395A CN201510999534.4A CN201510999534A CN105505395A CN 105505395 A CN105505395 A CN 105505395A CN 201510999534 A CN201510999534 A CN 201510999534A CN 105505395 A CN105505395 A CN 105505395A
- Authority
- CN
- China
- Prior art keywords
- heavy metal
- chitin oligosaccharide
- soil
- renovation agent
- bacillusmusilaginosiengineering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 107
- 238000000034 method Methods 0.000 title abstract description 8
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 title abstract 7
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 92
- 239000002689 soil Substances 0.000 claims abstract description 78
- 238000000855 fermentation Methods 0.000 claims abstract description 28
- 230000004151 fermentation Effects 0.000 claims abstract description 28
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 26
- 238000002360 preparation method Methods 0.000 claims abstract description 25
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 24
- 244000005700 microbiome Species 0.000 claims abstract description 14
- 229920002101 Chitin Polymers 0.000 claims description 116
- 229920001542 oligosaccharide Polymers 0.000 claims description 112
- 150000002482 oligosaccharides Chemical class 0.000 claims description 112
- 238000009418 renovation Methods 0.000 claims description 71
- 239000007788 liquid Substances 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 51
- 239000002609 medium Substances 0.000 claims description 33
- 230000004048 modification Effects 0.000 claims description 33
- 238000012986 modification Methods 0.000 claims description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 30
- 239000000725 suspension Substances 0.000 claims description 30
- 241001465752 Purpureocillium lilacinum Species 0.000 claims description 27
- 239000001963 growth medium Substances 0.000 claims description 23
- 239000000203 mixture Substances 0.000 claims description 23
- 239000006916 nutrient agar Substances 0.000 claims description 21
- 238000011218 seed culture Methods 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 16
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 15
- 239000002054 inoculum Substances 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 11
- 238000002386 leaching Methods 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 229910052700 potassium Inorganic materials 0.000 claims description 9
- 241000238557 Decapoda Species 0.000 claims description 8
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 8
- 239000011591 potassium Substances 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- 241000881860 Paenibacillus mucilaginosus Species 0.000 claims description 7
- 239000007633 bacillus mucilaginosus Substances 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 7
- 239000010452 phosphate Substances 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 239000003945 anionic surfactant Substances 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 claims description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 4
- 239000013028 medium composition Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 235000011089 carbon dioxide Nutrition 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 235000012015 potatoes Nutrition 0.000 claims description 3
- 238000005204 segregation Methods 0.000 claims description 3
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 238000013022 venting Methods 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229960001763 zinc sulfate Drugs 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 150000004996 alkyl benzenes Chemical class 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 abstract description 18
- 241000196324 Embryophyta Species 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 11
- 238000010521 absorption reaction Methods 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 2
- 238000005067 remediation Methods 0.000 abstract description 2
- 239000000084 colloidal system Substances 0.000 abstract 2
- 230000004060 metabolic process Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 22
- 229920001661 Chitosan Polymers 0.000 description 15
- 241000208822 Lactuca Species 0.000 description 10
- 235000003228 Lactuca sativa Nutrition 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000003480 eluent Substances 0.000 description 8
- 239000013641 positive control Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000003321 atomic absorption spectrophotometry Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 229910052793 cadmium Inorganic materials 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000004913 activation Effects 0.000 description 3
- 230000000274 adsorptive effect Effects 0.000 description 3
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 3
- 239000003337 fertilizer Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- -1 amino, carboxyl Chemical group 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 244000075850 Avena orientalis Species 0.000 description 1
- 235000007319 Avena orientalis Nutrition 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
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000001455 metallic ions Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002688 soil aggregate Substances 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Soil Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a chitosan oligosaccharide heavy metal bioremediation agent, a preparing method thereof and application, and relates to heavy metal bioremediation. The chitosan oligosaccharide heavy metal bioremediation agent is prepared from 5-70 parts of a chitosan oligosaccharide solution, 10-30 parts of bacillus licheniformis fermentation liquor and 10-50 parts of colloid bacillus fermentation liquor. The preparing method includes the steps of preparing the modified chitosan oligosaccharide solution, preparing the bacillus licheniformis fermentation liquor, preparing the colloid bacillus fermentation liquor and preparing the chitosan oligosaccharide heavy metal bioremediation agent. The chitosan oligosaccharide heavy metal bioremediation agent can be applied to preparation of a soil eluting agent, a phytoremediation agent, a soil bioremediation agent and the like. Heavy metal in soil can be absorbed, the content of the heavy metal in the soil can be effectively reduced by using a soil washing technology, or the bioremediation agent is applied to the soil to enhance heavy metal absorption by plants to achieve the purpose of soil remediation, soil microorganism activities are facilitated, the soil structure is improved effectively, development of plant root systems is promoted, growth and metabolism of the plants are adjusted, and crop yield is increased.
Description
Technical field
The present invention relates to heavy metal biological reparation, especially relate to a kind of chitin oligosaccharide heavy metal biological renovation agent and preparation method thereof and application.
Background technology
Along with rapid industrial development and mankind's activity are strengthened gradually, the problem of heavy metal pollution of soil is also day by day serious, and wherein Cd Pollution in Soil is widest in area, pollution level is the most serious.Wherein Cd is the main source of causing Heavy Metal Pollution In Cultivated Land.In February, 2013, the article of " Hunan problem rice flows to Guangdong dining table " published by South Daily, and arable soil causes extensive concern by the pollution of heavy metal Cd.Foochow is except island district, strand, all there is Cd content overproof phenomenon in other a few class regions, in the vegetable field soil of suburb nearby, Cd content overproof rate is the highest, its exceeding standard rate is 40% (Ding Yujuan, Lin Changhu, He Tengbing, etc. Contamination of Heavy Metal in Vegetables present situation and progress [J]. Guizhou science, 2012,5:78-83).
China is serious by heavy metal contamination, microorganism remediation technology is by changing the biological effectiveness of heavy metal, reduce its toxicity or improve its hyperaccumulative plant enriching quantity, reduce heavy metal to the pollution of food chain, have low superiority and the science with promoting soil Sustainable Production of cost concurrently, Soil leaching technology is also remove the effective ways of heavy metal-polluted soil, the heavy metal that eluent dissolves in soil is utilized to make it flow out with leacheate, then subsequent disposal is carried out to leacheate, thus reach the object of rehabilitating soil pollution.Chinese patent CN201110334587.6 provides a kind of chemical leaching repairing method of heavy-metal contaminated soil, and the method uses Na
2eDTA solution drip washing heavy-metal contaminated soil, effectively removes the available Cd in soil and lead, but EDTA exists expensive and in physical environment system, is difficult to degraded.
Chitosan has stronger metallic ion coordination ability, and the amino in its molecule and the hydroxyl base adjacent with amino and many metal ions are (as Hg
2+, Ni
2+, Cu
2+, Pb
2+, Cd
2+deng) can be formed stable huge legendary turtle compound (Li Zengxin, Liang Qiang, Meng Yun, etc. chitosan is to the desorption [J] of ADSORPTION STATE Pb in contaminated soil (II). ecotope, 2008, (03): 1049-1052).Related researcher is according to containing a large amount of amino and hydroxyl in chitosan molecule chain; chitosan is carried out to the chemical modifications such as acidylate, esterification and etherificate; generate a series of chitosan derivatives; can improve that it is water-soluble, biological activity and mechanical property; expand chitosan to the repairing effect of water body, Mobility of Heavy Metals In Soil Environment (Wu Nana. the preparation of chitin modified sorbent material and the application [M] in the sewage and soil treatment of heavy metal contamination thereof. South China Science & Engineering University, 2014).Chitin oligosaccharide (chitooligosaccharides, be abbreviated as COS) be the class low polymerization degree, the water-soluble aminosaccharide compound that are produced after hydrolysis by chitin (chitin) and chitosan (chitosan), it is the general name of chitin oligopolymer (chitinoligosaccharides) and oligochitosan (chitosanoligosaccharides), there is comparatively highly water-soluble, microorganism system and the soil aggregate of soil can be improved.Because chitin oligosaccharide molecular weight is little, so after chitin oligosaccharide and heavy metal chelating, be easily absorbed by plants.Chinese patent CN201410078241.8 provides the preparation method of a kind of phosphite-oligochitosan composite biological medicinal fertilizer, this phosphite-oligochitosan composite biological medicinal fertilizer, being a kind of bio-feritlizer, is again a kind of biological pesticide, simultaneously still a kind of heavy-metal contaminated soil in-situ immobilization agent; Phosphorous acid can make heavy metal form the calcium phosphate precipitation of insoluble, but oligochitosan is easily absorbed by plants after being combined with heavy metal, causes plant heavy metals exceeding standard.Chinese patent CN201110413145.0 provides a kind of method of remediating heavy metal cadmium pollution soil, adopt chitosan, the heavy metal contamination of microbial association phytoremediation, but chitosan poorly water-soluble, needs to be dissolved in concentrated hydrochloric acid solution, limit its application to a certain extent.
Summary of the invention
The object of the invention is the problems referred to above existed for prior art, there is provided that not only preparation method is simple, environmental friendliness, and the increase of effective cadmium content of soil can be promoted, raising Soil leaching effect or fortification of plants are to a kind of chitin oligosaccharide heavy metal biological renovation agent of Cd uptake and preparation method thereof and application.
Described chitin oligosaccharide heavy metal biological renovation agent in mass ratio consist of chitin oligosaccharide solution 5 ~ 70, the lichen bacillus ferments liquid 10 ~ 30, bacillusmusilaginosiengineering fermented liquid 10 ~ 50.
Described chitin oligosaccharide heavy metal biological renovation agent composition is in mass ratio preferably chitin oligosaccharide solution 30 ~ 60, the lichen bacillus ferments liquid 10 ~ 20, bacillusmusilaginosiengineering fermented liquid 10 ~ 40.
Described chitin oligosaccharide utilizes Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind to be zymophyte, with shrimp and crab shells powder for raw material, produced by fermentable that the fermented supernatant fluid that obtains obtains through anion surfactant modification.Described Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind is preserved in China typical culture collection center on June 30th, 2010, address: China. Wuhan. Wuhan University, postcode: 430072, deposit number is CCTCCNO:M2010165.
Described Bacillus licheniformis is Bacillus licheniformis (Bacilluslicheniformis) three torch-10, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, register on the books numbering: CGMCCNO.10741 at preservation center.
Described bacillusmusilaginosiengineering is bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 2nd, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, register on the books numbering: CGMCCNO.3995 at preservation center.
The preparation method of described chitin oligosaccharide heavy metal biological renovation agent, comprises the following steps:
1) modification chitin oligosaccharide solution is prepared
Preparation PDA substratum, by Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind spore liquid, coat on nutrient agar plate and cultivate, by stroke-physiological saline solution, the spore of Paecilomyces lilacinus (Paecilomyceslilacinus) the three torch No. 03 kind spore liquid on nutrient agar plate is washed again, make spore suspension; Spore suspension is accessed in seed culture medium and cultivate to obtain seed liquor; Again seed liquor is seeded to fermentation culture 48 ~ 72h in fermention medium and obtains Paecilomyces lilacinus fermented liquid; By Paecilomyces lilacinus fermented liquid centrifugal segregation thalline, obtain supernatant liquor, concentrated, the chitin oligosaccharide solution of obtained content 10% ~ 15%; In obtained chitin oligosaccharide solution, the molar mass concentration such as to add is the anionic surfactant solution of 20 ~ 100mmol/L, vibration 2 ~ 48h, obtained modification chitin oligosaccharide solution;
2) the lichen bacillus ferments liquid is prepared
Preparation nutrient agar, Bacillus licheniformis (Bacilluslicheniformis) three torch-10 is seeded in nutrient agar plate carries out streak culture, from nutrient agar plate, the Bacillus licheniformis activated (Bacilluslicheniformis) three torch-10 stroke-physiological saline solution is washed, make bacteria suspension, cultivate in bacterial suspension inoculation to seed culture medium, obtain seed liquor, again seed liquor is seeded in fermention medium and ferments, obtained the lichen bacillus ferments liquid;
3) bacillusmusilaginosiengineering fermented liquid is prepared
Preparation is without nitrogen solid medium, be that bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 inoculation is streak culture without nitrogen solid medium flat board carries out by bacillusmusilaginosiengineering, from without the bacillusmusilaginosiengineering activated being by nitrogen solid medium flat board bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 bacterial strain is forwarded to and cultivates without in nitrogen solid medium, obtain seed liquor, again seed liquor is seeded in fermention medium and ferments, obtained bacillusmusilaginosiengineering fermented liquid;
4) chitin oligosaccharide heavy metal biological renovation agent is prepared
By step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) and gained bacillusmusilaginosiengineering fermented liquid in mass ratio (5 ~ 70): (10 ~ 30): (10 ~ 50) mix, and obtain chitin oligosaccharide heavy metal biological renovation agent.
In step 1) in, the composition of described PDA substratum can be: peeled potatoes 200g/L, glucose 20g/L, agar 15g/L, natural ph; The described condition that nutrient agar plate is cultivated of coating can cultivate 72 ~ 120h at 28 ~ 32 DEG C; The composition of described seed culture medium can be: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, zinc sulfate 0.1g/L, before sterilizing, pH value is 6.0; Described inoculum size spore suspension being accessed seed culture medium by volume per-cent can be 8% ~ 15%, is diluted to OD
600be 0.2; The described condition of cultivating in seed culture medium that accessed by spore suspension in 28 ~ 32 DEG C, can cultivate 72 ~ 96h under 150 ~ 200rpm; The composition of described fermention medium can be: shrimp and crab shells powder 25g/L, peptone 2g/L; Described seed liquor can by 5 ~ 10% inoculum size access fermention mediums; The condition of described fermentation culture can be: inoculation rear venting amount is 1.0vvm, during the fermentation, according to the generation of carbonic acid gas, progressively increases ventilation; During the fermentation intermediary and later stages, points three times progressively stream add the shrimp and crab shells powder mixing suspension of 10%; Described anion surfactant can be selected from least one in sodium laurylsulfonate (SDS), linear alkyl benzene sulphonate (LAS), rhamnolipid etc., preferred rhamnolipid.
In step 2) in, the composition of described nutrient agar can be extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, and before going out, pH value is 7.3; The composition of described seed culture medium can be extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, and before sterilizing, pH value is 7.0; The inoculum size of described bacterial suspension inoculation by volume per-cent can be 10% of bacteria suspension, bacteria suspension OD
600=0.2; The condition of cultivating in described seed culture medium in 28 DEG C, can cultivate 24 ~ 48h under 180rpm condition; The composition of described fermention medium can be glucose 20g/L, bean cake powder 9g/L, ammonium sulfate 15g/L, dipotassium hydrogen phosphate 2g/L, potassium primary phosphate 1g/L, sodium-chlor 5g/L, magnesium sulfate 0.5g/L, and before sterilizing, pH value is 7.0; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 8% ~ 15% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180 ~ 200rpm, and leavening temperature controls at 28 ~ 30 DEG C, and fermentation time is 48 ~ 72h.
In step 3) in, the described composition without nitrogen solid medium can be: sucrose 10g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.2g/L, sodium-chlor 0.2g/L, calcium carbonate 1.0g/L, agar 20g/L, and before sterilizing, pH is 7.5; Described fermention medium composition can be: yeast extract paste 1.579g/L, starch 5.5g/L, bean cake powder 5.76g/L, magnesium sulfate 1.4g/L, dipotassium hydrogen phosphate 2g/L, calcium carbonate 8.5g/L, sodium-chlor 0.2g/L, and before sterilizing, pH is 7.3; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 8% ~ 15% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180 ~ 200rpm, and leavening temperature controls at 28 ~ 30 DEG C, and fermentation time is 48 ~ 72h.
In step 4) in, described step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) mass ratio of bacillusmusilaginosiengineering fermented liquid of gained is preferably (30 ~ 60): (10 ~ 20): (10 ~ 40).
Described chitin oligosaccharide heavy metal biological renovation agent can prepare application in Soil leaching agent, phytoremediation agent, soil organisms renovation agent etc.
The present invention has following beneficial effect:
1, the present invention Bacillus licheniformis used, bacillusmusilaginosiengineering have stronger tolerance to various heavy, wide accommodation; Simultaneously can to the Cd in solution
2+there is very strong adsorptive power.
2, in the present invention's modification chitin oligosaccharide used structure with amino, carboxyl, hydroxyl, can complexation heavy metal ion.Bacillus licheniformis and bacillusmusilaginosiengineering, by mode enriching heavy metal ions such as ion-exchange, complexing action, precipitating action, active transport, change the existing forms of heavy metal in soil ion.
3, the present invention's bacillusmusilaginosiengineering used can secrete organic acid, has the solution effect of good ore deposit, can by Element releases such as K, Si, Mn, Mg, the Ca in soil out, these positively charged ions will with Cd
2+competitive adsorption point position, and due to K
+concentration increase makes soil ion intensity increase, and reduces soil to the absorption of cadmium, significantly improves soil available Cd content.
4, a kind of chitin oligosaccharide heavy metal biological renovation agent provided by the invention can promote that heavy metal-polluted soil activates, and can do Soil leaching agent, reduces heavy metal content in soil, or imposes on soil assistance plant absorption heavy metal, improves phytoremediation efficiency.
5, chitin oligosaccharide heavy metal biological renovation agent preparation of the present invention is simple, good to heavy metal-polluted soil repairing effect, and can not cause secondary pollution to environment, safety and environmental protection, has good promotional value.
Embodiment
Below by embodiment, the present invention is elaborated.Should be understood that these embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.
The preparation of embodiment 1 chitin oligosaccharide heavy metal biological renovation agent
1) modification chitin oligosaccharide solution is prepared
Preparation PDA substratum, by Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind spore liquid, coat on nutrient agar plate and cultivate, by stroke-physiological saline solution, the spore of Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind on nutrient agar plate is washed again, make spore suspension; Spore suspension is accessed in seed culture medium and cultivate to obtain seed liquor; Again seed liquor is seeded in fermentation tank culture medium the 48h that ferments; By Paecilomyces lilacinus fermented liquid centrifugal segregation thalline, obtain supernatant liquor, concentrated, obtained content 10% ~ 15%.Chitin oligosaccharide solution, the rhamnolipid solution that mass concentration is 100mmol/L such as to add, vibration 24h, obtained modification chitin oligosaccharide solution.
In step 1) in, the composition of described PDA substratum can be: peeled potatoes 200g/L, glucose 20g/L, agar 15g/L, natural ph; Described Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind, be preserved in China typical culture collection center on June 30th, 2010, address: China. Wuhan. Wuhan University, postcode: 430072, deposit number is CCTCCNO:M2010165; The described condition that nutrient agar plate is cultivated of coating can cultivate 72h under 28 DEG C of conditions; The composition of described seed culture medium can be: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, zinc sulfate 0.1g/L, and before sterilizing, pH value is 6.0; Described inoculum size spore suspension being accessed seed culture medium by volume per-cent can be 10%, is diluted to OD
600be 0.2; The described condition of cultivating in seed culture medium that accessed by spore suspension in 28 DEG C, can cultivate 72h under 180rpm;
Described fermention medium composition can be: shrimp and crab shells powder 25g/L, peptone 2g/L.Described seed liquor can by 5 ~ 10% inoculum size access fermention mediums.Described culture condition can be: inoculation rear venting amount is 1.0vvm, during the fermentation, according to the generation of carbonic acid gas, progressively increases ventilation; During the fermentation intermediary and later stages, points three times progressively stream add the shrimp and crab shells powder mixing suspension of 10%.
2) the lichen bacillus ferments liquid is prepared
Preparation nutrient agar, Bacillus licheniformis is seeded in nutrient agar plate carries out streak culture, from nutrient agar plate, the Bacillus licheniformis activated is washed by stroke-physiological saline solution, make bacteria suspension, cultivate in bacterial suspension inoculation to seed culture medium, seed liquor, then seed liquor be seeded in fermentation tank culture medium ferment, obtain the lichen bacillus ferments liquid;
In step 2) in, the composition of described nutrient agar plate can be extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, and before going out, pH value is 7.3; Described Bacillus licheniformis (Bacilluslicheniformis) three torch-10, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, register on the books numbering: CGMCCNO.10741 at preservation center;
The composition of described seed culture medium can be extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, and before sterilizing, pH value is 7.0; The inoculum size of described bacterial suspension inoculation by volume per-cent can be 10% of bacteria suspension, bacteria suspension OD
600=0.2; The condition of cultivating in described seed culture medium in 28 DEG C, can cultivate 48h under 180rpm condition;
The composition of described fermention medium can be glucose 20g/L, bean cake powder 9g/L, ammonium sulfate 15g/L, dipotassium hydrogen phosphate 2g/L, potassium primary phosphate 1g/L, sodium-chlor 5g/L, magnesium sulfate 0.5g/L, and before sterilizing, pH value is 7.0; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 12% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180rpm, and leavening temperature controls at 28 DEG C, and fermentation time is 48h.
3) bacillusmusilaginosiengineering fermented liquid is prepared
Preparation is without nitrogen solid medium, bacillusmusilaginosiengineering is seeded in without nitrogen solid medium flat board carries out streak culture, cultivate from without the bacillusmusilaginosiengineering activated is forwarded to by nitrogen solid medium flat board without in nitrogen liquid nutrient medium, obtain seed liquor, again seed liquor is seeded in fermention medium and ferments, obtain bacillusmusilaginosiengineering fermented liquid;
In step 3) in, the composition of described nitrogen-free agar can be: sucrose 10g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.2g/L, sodium-chlor 0.2g/L, calcium carbonate 1.0g/L, and before sterilizing, pH is 7.5, and solid medium adds 20g/L agar; Described bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch 01, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 2nd, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, register on the books numbering: CGMCCNO.3995 at preservation center;
Described fermention medium composition can be: yeast extract paste 1.579g/L, starch 5.5g/L, bean cake powder 5.76g/L, magnesium sulfate 1.4g/L, dipotassium hydrogen phosphate 2g/L, calcium carbonate 8.5g/L, sodium-chlor 0.2g/L, and before sterilizing, pH is 7.3; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 8 ~ 15% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180rpm, and leavening temperature controls at 28 DEG C, and fermentation time is 48h.
4) chitin oligosaccharide heavy metal biological renovation agent is prepared
Chitin oligosaccharide heavy metal biological renovation agent I: by step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) the bacillusmusilaginosiengineering fermented liquid in mass ratio 60: 20: 30 of gained, obtain chitin oligosaccharide heavy metal biological renovation agent I.
Chitin oligosaccharide heavy metal biological renovation agent II: by step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) the bacillusmusilaginosiengineering fermented liquid in mass ratio 45: 15: 20 of gained, obtain chitin oligosaccharide heavy metal biological renovation agent II.
Chitin oligosaccharide heavy metal biological renovation agent III: by step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) the bacillusmusilaginosiengineering fermented liquid in mass ratio 30: 15: 20 of gained, obtain chitin oligosaccharide heavy metal biological renovation agent III.
Chitin oligosaccharide heavy metal biological renovation agent IV: by step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) the bacillusmusilaginosiengineering fermented liquid in mass ratio 15: 15: 20 of gained, obtain chitin oligosaccharide heavy metal biological renovation agent IV.
Embodiment 2 chitin oligosaccharide heavy metal biological renovation agent is used for water body Cd
2+static Adsorption
Test design 7 test group, are respectively:
CK, negative control group: water;
A, positive controls 1: modification chitin oligosaccharide solution;
B, positive controls 2: microbiobacterial agent (the lichen bacillus ferments liquid and bacillusmusilaginosiengineering fermented liquid mixture);
C, experimental group 1: chitin oligosaccharide heavy metal biological renovation agent I;
D, experimental group 2: chitin oligosaccharide heavy metal biological renovation agent II;
E, experimental group 3: chitin oligosaccharide heavy metal biological renovation agent III;
F, experimental group 4: chitin oligosaccharide heavy metal biological renovation agent IV;
Get above-mentioned 7 test group heavy metal biological renovation agents (10g/L) respectively in 50mL Erlenmeyer flask, add heavy metal solution (first sterilising treatment), the final volume controlling solution is 10mL, Cd
2+final concentration be 100mg/L and 200mg/L, at 28 DEG C, 160rpm constant temperature oscillation 2h.Take out sample, vibrate centrifugal 10min under 6000rpm.Supernatant liquor is transferred in clean centrifuge tube, then through membrane filtration, detects remaining Cd in solution with atomic absorption spectrophotometry
2+concentration, and calculate Cd in solution
2+adsorption rate.
Gained different tests group is to Cd in water body
2+static Adsorption result as shown in table 1: 1) modification chitin oligosaccharide, microbiobacterial agent, chitin oligosaccharide heavy metal biological renovation agent are to Cd in solution
2+all there is certain adsorptive power, along with Cd
2+the rising of concentration, Cd
2+adsorption rate occur decline.2) chitin oligosaccharide heavy metal biological renovation agent is to Cd
2+adsorption rate all higher than two positive controls.3) along with the reduction of modification chitin oligosaccharide content in chitin oligosaccharide heavy metal biological renovation agent, heavy metal biological renovation agent is to Cd
2+adsorption rate also there is downtrending, when modification chitin oligosaccharide, the lichen bacillus ferments liquid, bacillusmusilaginosiengineering fermented liquid mass ratio are 60: 20: 30, Cd
2+adsorption rate reach 86.34%.As can be seen here, chitin oligosaccharide heavy metal biological renovation agent is to Cd
2+there is stronger adsorptive power.
Table 1
Embodiment 3 chitin oligosaccharide heavy metal biological renovation agent is used for laboratory Soil leaching
(1) test design 7 test group, are respectively:
CK, negative control group: water;
A, positive controls 1: modification chitin oligosaccharide solution;
B, positive controls 2: microbiobacterial agent (the lichen bacillus ferments liquid and bacillusmusilaginosiengineering fermented liquid mixture);
C, experimental group 1: chitin oligosaccharide heavy metal biological renovation agent I;
D, experimental group 2: chitin oligosaccharide heavy metal biological renovation agent II;
E, experimental group 3: chitin oligosaccharide heavy metal biological renovation agent III;
F, experimental group 4: chitin oligosaccharide heavy metal biological renovation agent IV;
By the modification chitin oligosaccharide solution in above-mentioned test group, microbiobacterial agent and chitin oligosaccharide heavy metal biological renovation agent dilute with water 300 times respectively.
Supply examination soil sample total Cd content to be 4.20mg/kg, available state Cd content is 1.27mg/kg.Take for examination pedotheque 2g in 50mL Erlenmeyer flask.Eluent selects the modification chitin oligosaccharide solution of above-mentioned dilution 300 times, microbiobacterial agent or chitin oligosaccharide heavy metal biological renovation agent.By 1mol/LHCl and NaOH adjust ph, make it to reach 5.Different eluents is added respectively to each Erlenmeyer flask by liquid-solid ratio 10: 1.At ambient temperature, 100rpm vibrates 2h, and each process repeats 3 times.After vibration, take out sample, vibrate centrifugal 10min under 6000rpm.Supernatant liquor is transferred in clean centrifuge tube, by Cd concentration in solution after atomic absorption spectrophotometry detection first time drip washing.Continue to add eluent by liquid-solid ratio 10: 1 in the soil that drip washing is crossed once, the drip washing time is 2h.After vibration, take out sample, vibrate centrifugal 10min under 6000rpm.Supernatant liquor is transferred in clean centrifuge tube, by Cd concentration in solution after the drip washing of atomic absorption spectrophotometry detection second time, calculates accumulative Cd and add up clearance.
Soil leaching heavy metal wash-out concentration and elution efficiency to affect result as shown in table 2, as can be seen from Table 2:
1) with modification chitin oligosaccharide solution, microbiobacterial agent, chitin oligosaccharide heavy metal biological renovation agent for Soil leaching agent time, the clearance of Cd far away higher than using water as eluent, especially chitin oligosaccharide heavy metal biological renovation agent I;
2) with microbiobacterial agent diluent for Soil leaching agent time, although centrifugal rear thalline is mixed in soil, but to heavy metal-polluted soil Cd, there is certain activation because of Bacillus licheniformis, bacillusmusilaginosiengineering, as can be seen from result also, in elute soln the concentration of Cd higher than control group.
3) along with the increase of drip washing number of times, in eluent solution, the content of Cd has decline in various degree than first time drip washing; After use chitin oligosaccharide heavy metal biological renovation agent I drip washing 2 times, in soil, the accumulative clearance of Cd is up to 54.52%, adds 44.00% than first time drip washing.A drip washing can not obtain the best effect removing heavy metal in soil, needs the raising realizing elution efficiency by increasing drip washing number of times.
Table 2
(2) on the basis of above-mentioned experiment, design another experiment, be Soil leaching agent with chitin oligosaccharide heavy metal biological renovation agent I, II, after diluting 300 times, regulates pH, make pH value reach 3,5,7,9,11 with 1mol/LHCl and NaOH.Supply examination soil sample total Cd content to be 4.20mg/kg, available state Cd content is 1.27mg/kg.Take for examination pedotheque 2g in 50mL Erlenmeyer flask.Different eluents is added respectively to each Erlenmeyer flask by liquid-solid ratio 10:1.At ambient temperature, 100rpm vibrates 2h, and each process repeats 3 times.After vibration, take out sample, vibrate centrifugal 10min under 6000rpm.Supernatant liquor is transferred in clean centrifuge tube, by Cd concentration in solution after atomic absorption spectrophotometry detection first time drip washing.Continue to add eluent by liquid-solid ratio 10: 1 in the soil that drip washing is crossed once, the drip washing time is 2h.After vibration, take out sample, vibrate centrifugal 10min under 6000rpm.Supernatant liquor is transferred in clean centrifuge tube, by Cd concentration in solution after the drip washing of atomic absorption spectrophotometry detection second time, calculates Cd and add up clearance.
PH value on heavy metal-polluted soil drip washing effect to affect result as shown in table 3:
1) chitin oligosaccharide heavy metal biological renovation agent is variant to heavy metal-polluted soil elutive power under different pH condition, is conducive to the wash-out of Cd in soil under pH=5 condition.
2) clearance of chitin oligosaccharide heavy metal biological renovation agent I and II couple of heavy metal-polluted soil Cd is similar, and the former slightly well.The modification chitin oligosaccharide content that chitin oligosaccharide heavy metal biological renovation agent II uses is less, sees economically, selects chitin oligosaccharide heavy metal biological renovation agent II for the more economical material benefit of actual heavy metal-polluted soil drip washing.
Table 3
Embodiment 4 chitin oligosaccharide heavy metal biological renovation agent is used for pot experiment
As shown in table 4 for examination pedotheque feature, for the air-dry ground 2mm sieve of examination soil, every basin will be tested and fills native 2kg, be mixed into CaCO
3with sprinkling CdSO
4solution, makes 2mg/kgCd Single Pollution soil, after mixing, balances 3 months.
Test design 7 test group, are respectively:
CK, negative control group: water;
A, positive controls 1: modification chitin oligosaccharide solution;
B, positive controls 2: microbiobacterial agent (the lichen bacillus ferments liquid and bacillusmusilaginosiengineering fermented liquid mixture);
C, experimental group 1: chitin oligosaccharide heavy metal biological renovation agent I;
D, experimental group 2: chitin oligosaccharide heavy metal biological renovation agent II;
E, experimental group 3: chitin oligosaccharide heavy metal biological renovation agent III;
F, experimental group 4: chitin oligosaccharide heavy metal biological renovation agent IV;
By the modification chitin oligosaccharide solution in above-mentioned test group, microbiobacterial agent and chitin oligosaccharide heavy metal biological renovation agent dilute with water 300 times respectively.Get during the above-mentioned diluted modification chitin oligosaccharide solution of 200mg/kg, microbiobacterial agent and chitin oligosaccharide heavy metal biological renovation agent be manured into soil.Conventional fertilizer application: urea 0.3g/kg, potassium primary phosphate 0.5g/kg, Repone K 0.085g/kg.Select the consistent Lettuce naked oats of growing way to carry out pot experiment, the strain of every basin 5, keep field capacity 60%, cultivate in the controlled environment chamber, gather after plantation 40d.
Result is as follows:
1) different tests group on soil physico-chemical property to affect result as shown in table 4, pollute test group A in (2mg/kg) soil at single Cd, B, C, D, E, F can make the soil organism, full nitrogen, available phosphorus, effectively potassium content have raising in various degree, illustrating that heavy metal-polluted soil renovation agent is used can improve soil nutrient content.And use Bacillus licheniformis and bacillusmusilaginosiengineering can make soil pH value to a certain degree reduce, may cause because Bacillus licheniformis and bacillusmusilaginosiengineering can secrete organic acid.The content of soil available Cd is detected according to GB/T23739-2009.Be in the soil of 2mg/kg in Cd pollution concentration, outside test group A, other test group all can make soil available Cd content increase.And test group A, namely use separately modification chitin oligosaccharide solution, because chitin oligosaccharide is very easily absorbed by plants, so modification chitin oligosaccharide is absorbed in a large number by plant after being combined with heavy metal, soil available Cd content is slightly declined.Experiment group B, namely uses microbiobacterial agent separately, can make soil available Cd content increase by 109.5%, illustrate Bacillus licheniformis and bacillusmusilaginosiengineering obvious to Cd activation effect in soil.Use after modification chitin oligosaccharide solution mixes with microbiobacterial agent, because the mixture of activation of microorganism Cd ability and modification chitin oligosaccharide absorption Cd is easily absorbed by plants, cause the reduction of total Cd content in soil.
Table 4
2) Lettuce plantation 40d gathers, and calculates plant plant height, root length, fresh weight.Different tests group on Lettuce grow to affect result as shown in table 5.Different tests group all can promote the growth of Lettuce, and its plant height, root length and fresh weight have raising in various degree.Wherein the growth-promoting effect of test group E is best, and Lettuce plant height improves 25.76%, and root is long improves 77.65%, and fresh weight improves 65.40%.
Table 5
| Test group | Plant height (cm) | Root long (cm) | Fresh weight (g) |
| CK | 13.2 | 8.5 | 4.22 |
| A | 15.4 | 14.3 | 5.61 |
| B | 14.5 | 13.2 | 5.32 |
| C | 15.2 | 13.7 | 6.12 |
| D | 15.9 | 14.5 | 6.03 |
| E | 16.6 | 15.1 | 6.98 |
| F | 16.3 | 15.0 | 6.54 |
3) Lettuce plantation 40d gathers, and detects the Cd content of Lettuce according to GB5009.15-2014.Different tests group on Cd content in Lettuce to affect result as shown in table 6, the impact of different tests group on Absorption of Rape heavy metal Cd is variant, but compared with blank, the Cd content in Lettuce cauline leaf, root has had larger increase.Wherein, the effect of test group E is the most obvious, Cd content in Lettuce cauline leaf can be made to improve 96.88%, make Cd content in root improve 61.80%.As can be seen here, chitin oligosaccharide heavy metal biological renovation agent can significantly improve Cd content in plant, thus reduces total Cd content in soil.
Table 6
Consider, the present invention's preferred chitin oligosaccharide heavy metal biological renovation agent III is used as heavy metal-polluted soil bioremediation agents.
In addition, can arbitrary combination in the middle of the various different embodiment of the present invention, as long as without prejudice to thought of the present invention, content disclosed in this invention should be considered as.
Claims (10)
1. a chitin oligosaccharide heavy metal biological renovation agent, it is characterized in that its in mass ratio consist of chitin oligosaccharide solution 5 ~ 70, the lichen bacillus ferments liquid 10 ~ 30, bacillusmusilaginosiengineering fermented liquid 10 ~ 50.
2. as claimed in claim 1 chitin oligosaccharide heavy metal biological renovation agent in mass ratio consist of chitin oligosaccharide solution 30 ~ 60, the lichen bacillus ferments liquid 10 ~ 20, bacillusmusilaginosiengineering fermented liquid 10 ~ 40.
3. a kind of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 1 or 2, it is characterized in that described chitin oligosaccharide utilizes Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind to be zymophyte, with shrimp and crab shells powder for raw material, produced by fermentable that the fermented supernatant fluid that obtains obtains through anion surfactant modification; Described Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind is preserved in China typical culture collection center on June 30th, 2010, and deposit number is CCTCCNO:M2010165;
Described Bacillus licheniformis is Bacillus licheniformis (Bacilluslicheniformis) three torch-10, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2015, register on the books numbering: CGMCCNO.10741 at preservation center;
Described bacillusmusilaginosiengineering is bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 2nd, 2010, register on the books numbering: CGMCCNO.3995 at preservation center.
4. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 1, is characterized in that comprising the following steps:
1) modification chitin oligosaccharide solution is prepared
Preparation PDA substratum, by Paecilomyces lilacinus (Paecilomyceslilacinus) three torch No. 03 kind spore liquid, coat on nutrient agar plate and cultivate, by stroke-physiological saline solution, the spore of Paecilomyces lilacinus (Paecilomyceslilacinus) the three torch No. 03 kind spore liquid on nutrient agar plate is washed again, make spore suspension; Spore suspension is accessed in seed culture medium and cultivate to obtain seed liquor; Again seed liquor is seeded to fermentation culture 48 ~ 72h in fermention medium and obtains Paecilomyces lilacinus fermented liquid; By Paecilomyces lilacinus fermented liquid centrifugal segregation thalline, obtain supernatant liquor, concentrated, the chitin oligosaccharide solution of obtained content 10% ~ 15%; In obtained chitin oligosaccharide solution, the molar mass concentration such as to add is the anionic surfactant solution of 20 ~ 100mmol/L, vibration 2 ~ 48h, obtained modification chitin oligosaccharide solution;
2) the lichen bacillus ferments liquid is prepared
Preparation nutrient agar, Bacillus licheniformis (Bacilluslicheniformis) three torch-10 is seeded in nutrient agar plate carries out streak culture, from nutrient agar plate, the Bacillus licheniformis activated (Bacilluslicheniformis) three torch-10 stroke-physiological saline solution is washed, make bacteria suspension, cultivate in bacterial suspension inoculation to seed culture medium, obtain seed liquor, again seed liquor is seeded in fermention medium and ferments, obtained the lichen bacillus ferments liquid;
3) bacillusmusilaginosiengineering fermented liquid is prepared
Preparation is without nitrogen solid medium, be that bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 inoculation is streak culture without nitrogen solid medium flat board carries out by bacillusmusilaginosiengineering, from without the bacillusmusilaginosiengineering activated being by nitrogen solid medium flat board bacillusmusilaginosiengineering (Bacillusmucilaginosus) three torch No. 01 bacterial strain is forwarded to and cultivates without in nitrogen solid medium, obtain seed liquor, again seed liquor is seeded in fermention medium and ferments, obtained bacillusmusilaginosiengineering fermented liquid;
4) chitin oligosaccharide heavy metal biological renovation agent is prepared
By step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) and gained bacillusmusilaginosiengineering fermented liquid in mass ratio (5 ~ 70): (10 ~ 30): (10 ~ 50) mix, and obtain chitin oligosaccharide heavy metal biological renovation agent.
5. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 4, is characterized in that in step 1) in, consisting of of described PDA substratum: peeled potatoes 200g/L, glucose 20g/L, agar 15g/L, natural ph; The described condition that nutrient agar plate is cultivated of coating can cultivate 72 ~ 120h at 28 ~ 32 DEG C; The composition of described seed culture medium can be: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, zinc sulfate 0.1g/L, before sterilizing, pH value is 6.0; Described inoculum size spore suspension being accessed seed culture medium by volume per-cent can be 8% ~ 15%, is diluted to OD
600be 0.2; The described condition of cultivating in seed culture medium that accessed by spore suspension in 28 ~ 32 DEG C, can cultivate 72 ~ 96h under 150 ~ 200rpm.
6. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 4, is characterized in that in step 1) in, consisting of of described fermention medium: shrimp and crab shells powder 25g/L, peptone 2g/L; Described seed liquor can by 5 ~ 10% inoculum size access fermention mediums; The condition of described fermentation culture can be: inoculation rear venting amount is 1.0vvm, during the fermentation, according to the generation of carbonic acid gas, progressively increases ventilation; During the fermentation intermediary and later stages, points three times progressively stream add the shrimp and crab shells powder mixing suspension of 10%; Described anion surfactant can be selected from least one in sodium laurylsulfonate, linear alkyl benzene sulphonate, rhamnolipid etc., preferred rhamnolipid.
7. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 4, it is characterized in that in step 2) in, described nutrient agar consist of extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, before going out, pH value is 7.3;
The composition of described seed culture medium can be extractum carnis 3g/L, peptone 10g/L, sodium-chlor 5g/L, and before sterilizing, pH value is 7.0; The inoculum size of described bacterial suspension inoculation by volume per-cent can be 10% of bacteria suspension, bacteria suspension OD
600=0.2; The condition of cultivating in described seed culture medium in 28 DEG C, can cultivate 24 ~ 48h under 180rpm condition;
The composition of described fermention medium can be glucose 20g/L, bean cake powder 9g/L, ammonium sulfate 15g/L, dipotassium hydrogen phosphate 2g/L, potassium primary phosphate 1g/L, sodium-chlor 5g/L, magnesium sulfate 0.5g/L, and before sterilizing, pH value is 7.0; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 8% ~ 15% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180 ~ 200rpm, and leavening temperature controls at 28 ~ 30 DEG C, and fermentation time is 48 ~ 72h.
8. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 4, it is characterized in that in step 3) in, described consisting of without nitrogen solid medium: sucrose 10g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.2g/L, sodium-chlor 0.2g/L, calcium carbonate 1.0g/L, agar 20g/L, before sterilizing, pH is 7.5;
Described fermention medium composition can be: yeast extract paste 1.579g/L, starch 5.5g/L, bean cake powder 5.76g/L, magnesium sulfate 1.4g/L, dipotassium hydrogen phosphate 2g/L, calcium carbonate 8.5g/L, sodium-chlor 0.2g/L, and before sterilizing, pH is 7.3; Described seed liquor be seeded to the inoculum size of fermenting in fermention medium by volume per-cent can be 8% ~ 15% of seed liquor; The condition of described fermentation can be: rotating speed controls at 180 ~ 200rpm, and leavening temperature controls at 28 ~ 30 DEG C, and fermentation time is 48 ~ 72h.
9. the preparation method of chitin oligosaccharide heavy metal biological renovation agent as claimed in claim 4, it is characterized in that in step 4) in, described step 1) modification chitin oligosaccharide solution, the step 2 of gained) the lichen bacillus ferments liquid, the step 3 of gained) mass ratio of bacillusmusilaginosiengineering fermented liquid of gained is (30 ~ 60): (10 ~ 20): (10 ~ 40).
10. chitin oligosaccharide heavy metal biological renovation agent is applied preparing in Soil leaching agent, phytoremediation agent, soil organisms renovation agent as claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510999534.4A CN105505395B (en) | 2015-12-28 | 2015-12-28 | A kind of chitin oligosaccharide heavy metal biological renovation agent and the preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510999534.4A CN105505395B (en) | 2015-12-28 | 2015-12-28 | A kind of chitin oligosaccharide heavy metal biological renovation agent and the preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105505395A true CN105505395A (en) | 2016-04-20 |
| CN105505395B CN105505395B (en) | 2018-08-10 |
Family
ID=55713705
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510999534.4A Expired - Fee Related CN105505395B (en) | 2015-12-28 | 2015-12-28 | A kind of chitin oligosaccharide heavy metal biological renovation agent and the preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105505395B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106180175A (en) * | 2016-07-11 | 2016-12-07 | 董晓 | A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal |
| CN107087641A (en) * | 2017-02-08 | 2017-08-25 | 福建三炬生物科技股份有限公司 | A kind of marine oligosaccharide biological agent for improving crop salt-resistance and preparation method thereof |
| CN110218679A (en) * | 2019-06-21 | 2019-09-10 | 漳州三炬生物技术有限公司 | A kind of secondary salinization soil remediation microbial inoculum and its application |
| CN112522155A (en) * | 2020-11-17 | 2021-03-19 | 上海圣珑环境修复材料有限公司 | Bacillus licheniformis and application thereof |
| CN115502195A (en) * | 2022-09-16 | 2022-12-23 | 浙江乾精新材料科技有限责任公司 | Method for quickly restoring saline-alkali soil |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695315A (en) * | 2013-10-12 | 2014-04-02 | 福建三炬生物科技股份有限公司 | Method for producing chitooligosaccharide by microbial fermentation |
| CN104744181A (en) * | 2015-03-06 | 2015-07-01 | 贵州地宝生物科技有限公司 | Bio-organic fertilizer for promoting growth of tea plant and preparation method of bio-organic fertilizer |
| CN105112057A (en) * | 2015-07-29 | 2015-12-02 | 福建三炬生物科技股份有限公司 | Soil heavy metal repairing agent, preparation thereof and application |
-
2015
- 2015-12-28 CN CN201510999534.4A patent/CN105505395B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695315A (en) * | 2013-10-12 | 2014-04-02 | 福建三炬生物科技股份有限公司 | Method for producing chitooligosaccharide by microbial fermentation |
| CN104744181A (en) * | 2015-03-06 | 2015-07-01 | 贵州地宝生物科技有限公司 | Bio-organic fertilizer for promoting growth of tea plant and preparation method of bio-organic fertilizer |
| CN105112057A (en) * | 2015-07-29 | 2015-12-02 | 福建三炬生物科技股份有限公司 | Soil heavy metal repairing agent, preparation thereof and application |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106180175A (en) * | 2016-07-11 | 2016-12-07 | 董晓 | A kind of method planting Brassica campestris L restoration of soil polluted by heavy metal |
| CN106180175B (en) * | 2016-07-11 | 2019-04-16 | 绿艺园林建设有限公司 | A method of plantation rape restoration of soil polluted by heavy metal |
| CN107087641A (en) * | 2017-02-08 | 2017-08-25 | 福建三炬生物科技股份有限公司 | A kind of marine oligosaccharide biological agent for improving crop salt-resistance and preparation method thereof |
| CN107087641B (en) * | 2017-02-08 | 2021-04-13 | 福建三炬生物科技股份有限公司 | Marine oligosaccharide biological preparation for improving salt resistance of crops and preparation method thereof |
| CN110218679A (en) * | 2019-06-21 | 2019-09-10 | 漳州三炬生物技术有限公司 | A kind of secondary salinization soil remediation microbial inoculum and its application |
| CN110218679B (en) * | 2019-06-21 | 2020-09-08 | 漳州三炬生物技术有限公司 | Secondary salinization soil remediation microbial inoculum and application thereof |
| CN112522155A (en) * | 2020-11-17 | 2021-03-19 | 上海圣珑环境修复材料有限公司 | Bacillus licheniformis and application thereof |
| CN112522155B (en) * | 2020-11-17 | 2021-09-21 | 上海圣珑环境修复材料有限公司 | Bacillus licheniformis and application thereof |
| CN115502195A (en) * | 2022-09-16 | 2022-12-23 | 浙江乾精新材料科技有限责任公司 | Method for quickly restoring saline-alkali soil |
| CN115502195B (en) * | 2022-09-16 | 2024-01-09 | 浙江乾精新材料科技有限责任公司 | Quick restoration method for saline-alkali soil |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105505395B (en) | 2018-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105505395B (en) | A kind of chitin oligosaccharide heavy metal biological renovation agent and the preparation method and application thereof | |
| CN106493167B (en) | Bacillus licheniformis and microbial inoculum and their application and heavy metal-passivated method | |
| CN107774704A (en) | A kind of method of heavy metal copper in plant animal microbial association rehabilitating soil | |
| CN105234167B (en) | A kind of renovation agent of heavy metal biological containing alga oligosaccharides and preparation method thereof | |
| CN106734159A (en) | A kind of restorative procedure of heavy-metal contaminated soil | |
| CN103614302B (en) | One strain has the efficient phosphate-solubilizing penicillium oxalicum of heavy metal tolerance characteristic | |
| CN105112057A (en) | Soil heavy metal repairing agent, preparation thereof and application | |
| CN105132300A (en) | Method for preparing natural water ecological purification microbial inoculum | |
| CN105884415A (en) | Compound alga oligosaccharide microbial fertilizer and preparation method thereof | |
| CN106734188A (en) | A kind of nitrate nitrogen contamination method of heavy metals in farmland pollution | |
| CN104195070B (en) | A kind of reduce the bacterial strain of cadmium content, microbial inoculum and preparation and application thereof | |
| CN108575994A (en) | The preparation and application of growth-promoting type trichoderma and bacillus composite wettable powder | |
| CN104531585A (en) | Delftia tsuruhatensis and application thereof | |
| CN113980685B (en) | Bioactive soil conditioner for repairing chromium pollution of soil and preparation method and application thereof | |
| CN105149343A (en) | Remediation method of heavy metal contaminated soil | |
| CN105964680B (en) | A kind of beach saline land continuous cropping cotton soil ecology renovation agent and the preparation method and application thereof | |
| CN105013815A (en) | Biological remediation method for polycyclic aromatic hydrocarbon and heavy metal compound contaminated soil | |
| CN103451105B (en) | A kind of filamentous fungus Penicllium chrysogenum J-5 of high absorption cadmium and preparation method and application | |
| CN107815428A (en) | One plant of cadmium removes rhizobium KG2, microbial inoculum containing the rhizobium and application thereof | |
| CN104707864A (en) | Compound photosynthetic bacteria preparation for enhancing phytoremediation for heavy metal pollution of soil and preparation method thereof | |
| CN103911331A (en) | Composite microbial agent used for restoring wheat-field heavy metal chromium-contaminated soil | |
| CN104496725B (en) | Organic bacterial manure for lead zinc ore contaminated soil remediation and preparation method of organic bacterial manure | |
| CN104152432A (en) | Petroleum hydrocarbon degrading bacteria carrier material and preparation method and purpose thereof | |
| CN109576181A (en) | A kind of modified illite powder microbial bacterial agent and the preparation method and application thereof | |
| CN110257293A (en) | P. amylolyticus KY15, microbial inoculum, application and the product using it |
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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190418 Address after: Unit B1, Room 501, No. 16 Xinfeng Third Road, Torch Garden, Xiamen Torch High-tech Zone, Xiamen City, Fujian Province Co-patentee after: ZHANGZHOU SUNJU BIOLOGY TECHNOLOGY Co.,Ltd. Patentee after: FUJIAN SANJU BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. Address before: 361006 Unit B1, Room 501, Rihua International Building, 16 Xinfeng Third Road, Torch Garden, Xiamen City, Fujian Province Patentee before: FUJIAN SANJU BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180810 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |