CN105505830B - The acinetobacter calcoaceticus strain and its application of one plant of biosurfactant production - Google Patents
The acinetobacter calcoaceticus strain and its application of one plant of biosurfactant production Download PDFInfo
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- CN105505830B CN105505830B CN201610038100.2A CN201610038100A CN105505830B CN 105505830 B CN105505830 B CN 105505830B CN 201610038100 A CN201610038100 A CN 201610038100A CN 105505830 B CN105505830 B CN 105505830B
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- Prior art keywords
- biosurfactant
- strain
- acinetobacter calcoaceticus
- acinetobacter
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- 239000003876 biosurfactant Substances 0.000 title claims abstract description 38
- 241000588624 Acinetobacter calcoaceticus Species 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000005862 Whey Substances 0.000 claims abstract description 8
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 8
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 8
- 239000004094 surface-active agent Substances 0.000 claims description 22
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 230000003213 activating effect Effects 0.000 claims description 8
- 229910052751 metal Inorganic materials 0.000 claims description 7
- 239000002184 metal Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 235000010344 sodium nitrate Nutrition 0.000 claims description 5
- 239000004317 sodium nitrate Substances 0.000 claims description 5
- 241000588625 Acinetobacter sp. Species 0.000 claims description 4
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 4
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- ZMQBAPPSYGILGT-UHFFFAOYSA-N sodium;2,3-bis(hydroxymethyl)butanedioic acid Chemical compound [Na+].OCC(C(O)=O)C(CO)C(O)=O ZMQBAPPSYGILGT-UHFFFAOYSA-N 0.000 claims description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 239000002609 medium Substances 0.000 abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 9
- 230000001580 bacterial effect Effects 0.000 abstract description 9
- 239000000126 substance Substances 0.000 abstract description 9
- 229910052799 carbon Inorganic materials 0.000 abstract description 8
- 239000013527 degreasing agent Substances 0.000 abstract description 5
- 238000005237 degreasing agent Methods 0.000 abstract description 4
- 238000005265 energy consumption Methods 0.000 abstract description 4
- 241000589291 Acinetobacter Species 0.000 abstract description 3
- 235000013351 cheese Nutrition 0.000 abstract description 3
- 238000010790 dilution Methods 0.000 abstract description 3
- 239000012895 dilution Substances 0.000 abstract description 3
- 230000004060 metabolic process Effects 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 239000003513 alkali Substances 0.000 abstract 1
- 239000003921 oil Substances 0.000 description 39
- 235000019198 oils Nutrition 0.000 description 39
- 239000000243 solution Substances 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 229910000831 Steel Inorganic materials 0.000 description 6
- 238000007747 plating Methods 0.000 description 6
- 239000010959 steel Substances 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 229930186217 Glycolipid Natural products 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 230000002421 anti-septic effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000000693 micelle Substances 0.000 description 3
- -1 phosphoric acid hydrogen Chemical class 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 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 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 229960002337 magnesium chloride Drugs 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- JNRLEMMIVRBKJE-UHFFFAOYSA-N 4,4'-Methylenebis(N,N-dimethylaniline) Chemical compound C1=CC(N(C)C)=CC=C1CC1=CC=C(N(C)C)C=C1 JNRLEMMIVRBKJE-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 150000007942 carboxylates Chemical group 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000009671 shengli Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000012138 yeast extract Substances 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
- C25D5/34—Pretreatment of metallic surfaces to be electroplated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The present invention provides the acinetobacter calcoaceticus strain and its application of one plant of biosurfactant production, which is obtained from oil pollution using enrichment culture, dilution spread, screening, purifying, is identified as acinetobacter.The invention discloses the enriched medium, the fermentation mediums that screen the bacterial strain, and are analyzed the characteristic for the biosurfactant that the bacterial strain generates.Carbon source used in culture medium is to extract remaining leftover bits and pieces whey powder after cheese in the present invention, cheap, at low cost；Bacterial strain breeding is fast, metabolism is fast, the biosurfactant generated is metabolized as biological degreasing agent, it plays a role at normal temperature, product is environmentally friendly, nontoxic and pollution-free, biological degradability is good, high temperature strong alkali environment, reduces energy consumption and operation risk factor needed for avoiding conventional chemical methods oil removing, avoids secondary environmental pollution；Performance is stablized, and has good industrial application value.
Description
Technical field
The present invention relates to the applications of the acinetobacter calcoaceticus of field of biotechnology, and in particular to one plant of biosurfactant production
Acinetobacter calcoaceticus strain and its application.
Background technique
Biosurfactant be one kind that microorganism secrets out of in the metabolic process contain hydrophilic radical (such as amino acid or
Polypeptide, disaccharides, oligosaccharides or polysaccharide etc.) and lipophilic group (such as saturated or unsaturated fatty alcohol or fatty acid) amphiphilic chemical combination
Object.Biosurfactant also has a characteristic that (1) in oil-water other than having the characteristics that chemical surfactant
There is higher surface-active at interface；(2) nontoxic, safety, easily biological-degradable, environmental-friendly；(3) there is very strong grease emulsifying energy
Power；(4) reaction product can introduce the chemical group of new type, and some of them group is that chemical method is difficult to synthesize, molecule knot
Structure type multiplicity；(5) biosurfactant can be applied under room temperature, condition of normal pressure；(6) fermentation strain produces biological table
The raw material of face activating agent is widely present and inexpensive in nature, is typical " green " technique.
Biosurfactant mainly includes glycolipid, lipopeptid, phosphatide, fatty acid, polysaccharide-composite of lipid and neutral lipid
Derivative etc., it is known that biosurfactant based on glycolipid.Biosurfactant due to its it is good solubilising, emulsification,
The features such as surfactant and environment friendly are reduced, in the improvement of oily waste water, the environment remediation on oil pollution ground, oil recovery
It has been widely used in the fields such as industry, cleaning, and is penetrated into other field.Studies have shown that biosurfactant
Oil displacement efficiency is 3.5-8 times higher than the oil displacement efficiency of chemical surfactant, and price is only the 30% of chemical surfactant.It is raw
Object surfactant substituted chemistry surfactant gradually becomes a kind of new research tendency.
The oil removing and derusting of the normal nulling part of metal treatment before plating are the important procedure of plating, electroplated component treatment before plating matter
The quality of amount directly affects electroplated layer quality.According to statistics, the main reason for 90% quality accident of plating piece be due to treatment before plating not
Caused by.At present before metal-plated oil removing traditional handicraft, mainly have thermokalite high temeperature chemistry ablution, ultrasonic oil removal, chemistry
Reagent oil removing etc.；Wherein high temperature alkaline process energy consumption is high, it is seriously polluted met with eliminate；Ultrasonic oil removal limits it because equipment investment is big
Application in practice；Chemical reagent oil removing is at present using most deoiling methods, however, traditional chemical reagent oil removing is deposited
In following disadvantage: deoiling effect is poor under room temperature, and when use must heat (mostly at 70 DEG C or more), and deoiling effect is with tank liquor
Aging and gradually die down, not only waste of resource but also caused the secondary pollution of greasy dirt, body refuse, there is also high spumescence, high alkalinity and corruption
Lose the problems such as serious.Therefore it is badly in need of a kind of low energy consumption, safety and environmental protection, degradable, efficient degreaser in industrial production.
Summary of the invention
The purpose of the present invention is the bacterial strain by filtering out one plant of biosurfactant production, the life generated using the bacterial strain
Object surfactant reaches room temperature oil removing, and biodegradable, free of contamination effect, is applied to metal plating pre-treatment.
The technical scheme is that obtaining strain X H- from oil pollution using enrichment culture, dilution spread, purifying
2, the identified bacterium is acinetobacter (Acinetobacter sp.).The acinetobacter calcoaceticus (Acinetobacter sp.)
XH-2, oneself is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 6th, 2016 (referred to as
CGMCC, address are as follows: city, BeiJing, China, North Star West Road 1, Chaoyang District institute 3), deposit number is CGMCC No. 11960.
Enriched medium used is (w/v, %) are as follows: magnesium chloride 2, dipotassium hydrogen phosphate 6, potassium dihydrogen phosphate 3.8, tri-chlorination
Iron 0.5, ammonium chloride 10 add antiseptic oil as sole carbon source and carry out enrichment culture.
Fermentation medium used is (w/v, %): whey powder 0.1, sodium nitrate 0.1, potassium dihydrogen phosphate 0.2, phosphoric acid hydrogen two
Sodium 0.6, magnesium chloride hexahydrate 0.02, sodium chloride 2, microelement 1mL/L, Tween80 1, pH 6, distilled water are prepared.
Strain X H-2 fermented and cultured 48h in the fermentation medium, 4 DEG C of 10000rpm are centrifuged 20min, take
Supernatant adjusts pH to 2.0 with 6mol/L HCl, after being extracted 2 times with isometric ethyl acetate solution, merges organic phase in rotation
Turn to be concentrated on evaporimeter, concentrate is poured into beaker, up to the crude product of surfactant after volatilizing naturally.
In order to further study the characteristic of the biosurfactant, constituent analysis, critical glue have been carried out using the crude product
The measurement and stability analysis of beam concentration.
The biosurfactant main component that the present invention announces, is tentatively judged as through qualitative analysis and the results of FT-IR
Glycolipid class.
After measured, the critical micelle concentration of the biosurfactant is 200mg/L, is substantially lower than common chemical surface
Activating agent such as dodecyl sodium sulfate (2120mg/L), cetyl trimethylammonium bromide (1300mg/L).
It is respectively the removal of 4cm, 2cm and 0.3cm steel disc surface anticorrosion oil applied to length, deoiling effect is good, when
Between it is short, it is only necessary to 16min.
The advantages and positive effects of the present invention are:
Applicant from oil pollution screens the bacterial strain of one plant of biosurfactant production, is named as acinetobacter calcoaceticus XH-
2, using the method for biofermentation, to extract the byproduct whey powder after cheese, sodium nitrate, inorganic salts as raw material, utilization is motionless
Bacillus XH-2 produces Glycolipids Biosurfactantss, which under normal temperature conditions, can remove metal surface
Antiseptic oil.
Carbon source used in the present invention is to extract remaining leftover bits and pieces whey powder after cheese, cheap, at low cost；Micro- life
Object breeding is fast, and metabolism is fast, and the biosurfactant generated using microbial metabolism is sent out at normal temperature as biological degreasing agent
The effect of waving, product is environmentally friendly, nontoxic and pollution-free, biological degradability is good, high temperature highly basic ring needed for avoiding conventional chemical methods oil removing
Border reduces energy consumption and operation risk factor, avoids secondary environmental pollution；Biosurfactant made by the present invention is through difference
After high Inversion phenomenon, pH processing, surface tension variations are little, and the biosurfactant removes after 100 DEG C are boiled 10min
Oily efficiency does not reduce, and has good industrial application value.
Detailed description of the invention
Fig. 1 is the Determination of Critical Micelle Concentration figure of object surfactant produced by strain X H-2 of the invention
Fig. 2 is the infrared absorpting light spectra of biosurfactant of the invention
Fig. 3 is the effect picture of biosurfactant of the invention for oil removing before the coat of metal
Specific embodiment
The screening of acinetobacter calcoaceticus (Acinetobacter sp.XH-2) bacterial strain of 1 biosurfactant production of embodiment.
It is sampled from the soil by oil pollution, the sample that the present embodiment uses is Shandong region Shengli Oil Field oil recovery scene
By the soil sample of crude oil pollution, 5g soil sample is weighed in 50mL enriched medium (w/v, %: magnesium chloride 2, dipotassium hydrogen phosphate 6, di(2-ethylhexyl)phosphate
Hydrogen potassium 3.8, ferric trichloride 0.5, ammonium chloride 10, add antiseptic oil as sole carbon source) in cultivated, taking-up 1mL culture solution
It is forwarded in enriched medium again, after switching 3 times；Dilution spread on blood plate, to have the bacterial strain of transparent circle LB solid train
It supports and carries out scribing line purifying on base.
Purified single colonie is inoculated in the LB liquid medium of 5mL, is transferred to the fermentation being not optimised after activating 12h
Culture medium (w/v, %: glucose 1, peptone 1, potassium dihydrogen phosphate 0.2, disodium hydrogen phosphate 0.6, magnesium chloride hexahydrate 0.02, micro
Element 1mL/L) in, after fermented and cultured 2d, centrifugation fermentation liquid abandons thallus, does the experiment of oil extraction circle with supernatant and utilizes JK9913 type
Full-automatic tension instrument measures surface tension using hanging ring method, and carries out oil removing experiment.
Its oil extraction loop diameter of the strain X H-2 screened is 4.5cm, surface tension 32.58mN/m, has certain remove
Oily effect.
Select strain X H-2 as the original strain of biological degreasing agent, the identified Pseudomonas is in acinetobacter.
The fermentation technology optimization of 2 strain X H-2 of embodiment.
Using surface tension and oil removing time as index, the fermentation medium and fermentation condition of strain X H-2 are optimized.
From the LB liquid medium that picking strain X H-2 single colonie is inoculated in 5mL on LB solid plate, after activating 12h,
It is transferred in fermentation medium, the revolving speed of shaking table is 160rpm, and temperature is set as 30 DEG C.
Using glucose, sucrose, lactose, whey powder, glycerol, olive oil, deep-fried twisted dough sticks waste oil as carbon source, by peptone, nitre
Sour sodium, ammonium sulfate, yeast extract (ferment mentions), dregs of beans after the 2d that ferments, carry out the survey of surface tension and oil removing time as nitrogen source
It is fixed, it the results are shown in Table 1 and 2, most suitable carbon nitrogen source mainly determined according to the oil removing time；Then concentration optimization is carried out again, and carbon source concentration is set
It is set to (w/v, %) 0.1,0.5,1.5,3, nitrogen concentration (w/v, %) is set as and is set as 0.1,0.2,1,3；Determine that carbon source is
0.1% whey powder, nitrogen source are 0.1% sodium nitrate.
Initial pH is set as 5,6,7,8,9,10, adds 1%Tween80 and SDS surfactant.
Fermentation medium (w/v, %) after optimization are as follows: whey powder 0.1, sodium nitrate 0.1, potassium dihydrogen phosphate 0.2, phosphoric acid hydrogen
Disodium 0.6, magnesium chloride hexahydrate 0.02, sodium chloride 2, microelement 1mL/L, Tween80 1, pH 6, distilled water are prepared.
Fermentation condition includes the optimization of inoculum concentration and kind age, and inoculum concentration (v/v, %) is set as 1,2,3,4,8；It will be in life
Long initial stage (6h), exponential phase (12h), stationary phase (20h), decline phase (30h) bacterial strain be transferred to the fermentation medium optimized
In, it determines optimum inoculation amount 4% and kind age is 12h.
Surface tension list of the 1 different carbon source type of table to metal surface oil removing time and produced surfactant
Surface tension list of the 2 different nitrogen sources type of table to metal surface oil removing time and produced surfactant
Embodiment 3 prepares biological degreasing agent using the produced biosurfactant of strain X H-2 fermentation.
Strain X H-2 single colonie on picking LB solid plate is after 5mL LB liquid medium activates 12h, by 4% inoculation
Amount is transferred in the fermentation medium of the optimization of embodiment 2, after 30 DEG C, 160rpm shaking table top fermentation culture 2d, fermentation liquid centrifugation
It takes in supernatant culture dish holding, by long 4cm, wide 2cm, high 0.3cm steel disc is placed in one, when observing and recording needed for oil removing
Between.The results are shown in attached figure 3, and Fig. 3 A show steel disc surface covering oil film, and the oil film that Fig. 3 B show steel disc surface is cleared,
It is 16min that the oil removing time is most short, and Fig. 3 C is shown after steel disc places a period of time after oil removing, iron rust occurs, shows steel disc oil film
It is divided.
Embodiment 4 prepares biosurfactant using strain X H-2 fermented and cultured, and carries out the survey of critical micelle concentration
Fixed, ingredient Preliminary Identification and stability research.
The fermentation liquid that strain X H-2 fermented and cultured 2d is obtained according to the method for embodiment 3, in 4 DEG C, the revolving speed of 10000rpm
It is centrifuged 20min, supernatant 6mol/L HCl is taken to adjust pH to 2.0, after being extracted 2 times with isometric ethyl acetate solution, is closed
And organic phase pours into concentrate in beaker in being concentrated on Rotary Evaporators, up to the thick of surfactant after volatilizing naturally
Product, yield 3.1g/L.
Crude product obtained is made into the aqueous solution of 10,20,50,100,200,300,400,500,1000mg/L, is surveyed respectively
Its fixed surface tension is simultaneously depicted as curve, as shown in attached drawing 1.When biosurfactant solution is from 0-50mg/L, surface tension
38.58mN/m is dropped to quickly from 75mN/m；Decline is slow later；When surfactant concentration reaches 200mg/L, surface
Power is reduced to 33.12mN/m；After sample concentration is higher than 200mg/L, the surface tension of solution almost no longer declines；Therefore, the surface
The CMC value of activating agent is 200mg/L, hence it is evident that is lower than common chemical surfactant dodecyl sodium sulfate (SDS, 2120mg/
L), cetyl trimethylammonium bromide (CTAB, 1300mg/L), good surface-active are more conducive to practical application.
The charging property of the biosurfactant is identified using methylene blue-chloroform test.In methylene blue-chloroform test
In, above-mentioned surfactant crude product is made into the solution of 0.3g/L, takes 5mL surfactant solution, sequentially adds 10mL methylene
Base indigo plant and 5mL chloroform after mixing well, are observed after standing a few minutes.Experimental result darkens for water layer, shows the biology table
Face activating agent is cationic surface active agent.
Surfactant crude product is subjected to infrared spectroscopy (FT-IR) analysis with KBr pressed disc method.The results are shown in attached figure 2, Cong Tuzhong
It can be seen that 3390cm-1Nearby there is the absorption peak of Qiang Erkuan, shows there are a large amount of-OH in the molecule；2924 cm-1With
2854cm-1Absorption vibration belong to the absorption peak of aliphatic C-H, and 1460cm-1Neighbouring absorption peak can belong to molecule carbon chain
Upper continuous C-H vibration causes；1671cm-1Stretching vibration of the absorption peak at place from carbonyl, 1728 cm-1Nearby have it is relatively strong and
The absorption peak of point, shows the presence for having ester group；1074cm-1With 1025cm-1Stretching vibration of the absorption peak at place from C-O-C key,
Show that there are carboxylate groups in the molecule.With the glycolipid class infrared spectrogram comparison delivered, the biological surface is primarily determined
Activating agent is glycolipid compound.
Above-mentioned surfactant crude product is made into the solution of 0.3g/L, carry out the biosurfactant different temperatures,
Stability analysis under pH and salinity processing.
(1) temperature stability: the biosurfactant solution is at different temperature (4 DEG C, room temperature, 60 DEG C, 100 DEG C)
After placing 30min, surface tension is measured；
(2) pH stability: the biosurfactant crude product different pH (2,4,6,8,10,12) buffer solution, in
After being placed at room temperature for 12h, surface tension is measured；
(3) salinity stability: the biosurfactant crude product is made into concentration with the solution of different salinity (0-9%) and is
The solution of 0.3g/L measures surface tension after being placed at room temperature for 12h.
As a result 3 be see the table below, under the processing of different temperatures, pH and salinity, surface tension variations are little, the Bio-surface active
Agent has preferable stability and tolerance.After boiling water bath handles 10min, oil removal efficiency influences not the supernatant of fermentation liquid
Greatly.
3 temperature of table, the influence of pH and salinity to biosurfactant stability
One embodiment of the present invention has been described in detail above, but the content is only preferable implementation of the invention
Example, should not be considered as limiting the scope of the invention.It is all according to all the changes and improvements made by the present patent application range
Deng should still be within the scope of the patent of the present invention.
Claims (4)
1. the acinetobacter calcoaceticus strain of one plant of biosurfactant production, it is characterised in that: the acinetobacter calcoaceticus strain is acinetobacter calcoaceticus
(Acinetobacter sp.)XH-2CGMCC No.11960。
2. the acinetobacter calcoaceticus strain using one plant of biosurfactant production described in claim 1 produces biosurfactant
Method, it is characterised in that: from the LB liquid medium that picking single colonie is inoculated in 5mL on LB solid plate, after activating 12h,
It is transferred in fermentation medium, inoculum concentration 4%, fermentation medium (w/v, %) are as follows: whey powder 0.1, sodium nitrate 0.1, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.2, disodium hydrogen phosphate 0.6, magnesium chloride hexahydrate 0.02, sodium chloride 2, microelement 1mL/L, Tween80 1, pH 6 steam
Distilled water is prepared, shaking speed 160rpm, and temperature is set as 30 DEG C, cultivates 2d, is centrifuged 20min in 4 DEG C, 10000rpm, is taken supernatant
Liquid adjusts pH to 2.0 with 6mol/L HCl, after being extracted 2 times with isometric ethyl acetate solution, merges organic phase and steams in rotation
It is concentrated on hair instrument, concentrate is poured into beaker, up to the crude product of surfactant after volatilizing naturally.
3. the biosurfactant that method obtains according to claim 2.
4. application of the biosurfactant according to claim 3 before the coat of metal in oil removing.
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