CN110257268A - One plant of degrading polyethylene generates the Pichia guilliermondii of alkane simultaneously - Google Patents
One plant of degrading polyethylene generates the Pichia guilliermondii of alkane simultaneously Download PDFInfo
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- CN110257268A CN110257268A CN201910630385.2A CN201910630385A CN110257268A CN 110257268 A CN110257268 A CN 110257268A CN 201910630385 A CN201910630385 A CN 201910630385A CN 110257268 A CN110257268 A CN 110257268A
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- Prior art keywords
- polyethylene
- guilliermondii
- alkane
- pichia guilliermondii
- meyerozyma
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- -1 polyethylene Polymers 0.000 title claims abstract description 57
- 229920000573 polyethylene Polymers 0.000 title claims abstract description 57
- 239000004698 Polyethylene Substances 0.000 title claims abstract description 55
- 241000235048 Meyerozyma guilliermondii Species 0.000 title claims abstract description 39
- 150000001335 aliphatic alkanes Chemical class 0.000 title claims abstract description 32
- 230000000593 degrading effect Effects 0.000 title claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 241000255896 Galleria mellonella Species 0.000 claims 2
- 230000000968 intestinal effect Effects 0.000 claims 1
- 230000001360 synchronised effect Effects 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 11
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 239000013618 particulate matter Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000311506 Meyerozyma Species 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 208000016261 weight loss Diseases 0.000 description 5
- 230000004580 weight loss Effects 0.000 description 5
- 241000235648 Pichia Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical group C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000000446 fuel Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241001351995 Aphomia sociella Species 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
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000001035 drying 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
- 239000007789 gas Substances 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- BJQWYEJQWHSSCJ-UHFFFAOYSA-N heptacosane Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCCC BJQWYEJQWHSSCJ-UHFFFAOYSA-N 0.000 description 2
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- YKNWIILGEFFOPE-UHFFFAOYSA-N pentacosane Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC YKNWIILGEFFOPE-UHFFFAOYSA-N 0.000 description 2
- YCOZIPAWZNQLMR-UHFFFAOYSA-N pentadecane Chemical compound CCCCCCCCCCCCCCC YCOZIPAWZNQLMR-UHFFFAOYSA-N 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- BGHCVCJVXZWKCC-UHFFFAOYSA-N tetradecane Chemical compound CCCCCCCCCCCCCC BGHCVCJVXZWKCC-UHFFFAOYSA-N 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- HGUFODBRKLSHSI-UHFFFAOYSA-N 2,3,7,8-tetrachloro-dibenzo-p-dioxin Chemical compound O1C2=CC(Cl)=C(Cl)C=C2OC2=C1C=C(Cl)C(Cl)=C2 HGUFODBRKLSHSI-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 108020000949 Fungal DNA Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 229920001617 Vinyon Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000005649 metathesis reaction Methods 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000033772 system development Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000002341 toxic gas Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
- C12R2001/16—Corynebacterium diphtheriae
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- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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Abstract
One plant of degrading polyethylene generates the Pichia guilliermondii of alkane simultaneously, is related to one plant of Pichia guilliermondii.The present invention provides one plant of Pichia guilliermondii, provides new bacterium source for the biodegrade of polyethylene and the production of alkane.The Pichia guilliermondii is Pichia guilliermondii Meyerozyma guilliermondii ZJC-1, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is the institute 3 of Chaoyang District, Beijing City Beichen Lu 1, the deposit date is on December 17th, 2018, deposit number was CGMCC No:16956.Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 degradable polyethylene of the present invention generates alkane substance simultaneously, provides new bacterium source for the biodegrade of polyethylene and the biosynthesis of alkane.For degrading polyethylene, it is also used for preparing alkane.
Description
Technical field
The present invention relates to one plant of Pichia guilliermondiis.
Background technique
Currently, most waste polyethylene plastics are mainly handled by the way of burning and filling, wherein largely
The landfill of vinyon waste results in serious environmental hazard, such as destroys soil physico-chemical property, in turn results in soil hardening,
And CO, HCl, NOx, SO can be generated by burning2And a large amount of toxic gases such as dioxin pollute atmospheric environment in turn, both processing sides
Formula causes serious pollution to environment, thus, a promising solution of waste polyethylene plastics is to turn them
Turn to valuable liquid fuel or chemical raw material.
The main source of alkane is natural gas and petroleum at this stage.Although the gas component of various regions is different, nearly all
Containing 75% methane, 15% ethane and 5% propane, remaining be higher alkane.Alkane is not only the weight of fuel
Source is wanted, and is also the raw material of modern chemical industry.The alkane of microbial method production at present is in microbial body by a system
Column metabolic pathway synthesizing alkanes, the route of synthesis there is also metabolic by-product excessively and alkane carbon chain lengths be difficult to control etc. ask
Topic, and the route of synthesis of fatty acid is all strictly regulated and controled on transcriptional level and protein level in cell, therefore in natural item
It cannot achieve the mesostate for largely accumulating its route of synthesis under part in microbial cell.So Biodegradable polyethylene
Generating alkane has investment small, low without adding other a large amount of chemical reagent, operating cost, without being transformed to cell interior
The advantages that, there are huge potentiality.In short, microorganism exists due to being limited to the route of synthesis of fatty acid in cell under natural conditions
Strictly regulated and controled on transcriptional level and protein level, cannot achieve the mesostate for largely accumulating its route of synthesis.Cause
This, currently with micro-organisms alkane still in laboratory stage, and the intracorporal alkane separation of cell faces huge challenge.
It is mostly that physico-chemical process, such as Jia X etc. are ground at present about the research for being converted into alkanes using polyethylene
Studying carefully report makes different molecular weight, different types of polyethylene using sequential catalyst intersection alkane metathesis method in a mild condition
It is converted into useful liquid fuel and wax, but rarely therefore research report is visited by microbial degradation polyethylene generation alkane
Ask can degrading polyethylene and also generate simultaneously alkane bacterial strain have good researching value, be Biodegradable polyethylene and
The synthesis of mixed alkanes substance provides new research direction.
Summary of the invention
The present invention provides one plant of Pichia guilliermondii, can be polyethylene to generate alkane while degrading polyethylene
Degradation and the generation of alkane provide new bacterium source.
Degradable polyethylene of the present invention generates the Pichia guilliermondii Meyerozyma of alkane substance simultaneously
Guilliermondii ZJC-1 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC),
Preservation address is the institute 3 of Chaoyang District, Beijing City Beichen Lu 1, and the deposit date is on December 17th, 2018, deposit number CGMCC
No:16956.
Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 separation screening of the present invention is from bee moth larvae
In enteron aisle liquid.
Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 of the present invention is on YPD solid medium
After cultivating 2d, single colonie diameter is 2~3mm, is creamy white, and round, surface is smooth glossy, and quality is more sticky, opaque.
It can be observed that the bacterium is elliposoidal under oil mirror after the dyeing of methylene blue decoration method.
Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 of the present invention can give birth on YPD culture medium
It is long, 30 DEG C of optimum growth temperature.
Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 degradable polyethylene of the present invention, for degradation
Polyethylene provides new bacterium source.
Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 of the present invention is using polyethylene as sole carbon source
It can produce alkanes substance.
Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 of the present invention belongs to pichia
(Pichia), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is north
The institute 3 of the Chaoyang District Jing Shi Beichen Lu 1, the deposit date is on December 17th, 2018, deposit number was CGMCC No:16956.
Detailed description of the invention
Fig. 1 is the PCR amplification electropherogram of bacterial strain Meyerozyma guilliermondiiZJC-1 of the present invention；Fig. 2 is this
The close bacterial strain included in invention bacterial strain MeyerozymaguilliermondiiZJC-1 and GenBank carries out sequence analysis
Constructed systematic evolution tree；Fig. 3 is the polyethylene table of bacterial strain Meyerozyma guilliermondiiZJC-1 degradation front and back
Face water contact angle figure.；Fig. 4 is the table of polyethylene before and after bacterial strain Meyerozyma guilliermondiiZJC-1 degrading polyethylene
The infrared spectrum of face chemical structure；Fig. 5 is that bacterial strain Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 exists
The weight-loss ratio of polyethylene during 60 days before and after degrading polyethylene.Fig. 6 is Meyerozyma of the present invention
The stereomicroscope figure for the alkane that guilliermondiiZJC-1 degrading polyethylene generates；Fig. 7 is bacterial strain of the present invention
The infrared spectrogram for the alkane that Meyerozyma guilliermondiiZJC-1 degrading polyethylene generates；Fig. 8 is bacterial strain
The gas chromatography mass spectrometry spectrogram for the alkane that Meyerozyma guilliermondiiZJC-1 degrading polyethylene generates.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the Pichia guilliermondii of present embodiment degradable polyethylene
MeyerozymaguilliermondiiZJC-1 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart (CGMCC), preservation address are the institutes 3 of Chaoyang District, Beijing City Beichen Lu 1, and the deposit date is on December 17th, 2018, preservations
Number is CGMCC No:16956.
Present embodiment Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 is by bee moth larvae enteron aisle
Screening obtains in liquid, and is identified by the following method.
Molecular biology identification is carried out to the bacterial strain screened: Meyerozyma guilliermondii ZJC-1 is connect
Kind in YPD fluid nutrient medium, in 30 DEG C of constant-temperature shaking incubator 120r/min cultures 16~for 24 hours, 2mL bacterium solution is taken
Thalline were collected by centrifugation by 8000rpm, extracts strain gene group DNA according to OMEGA HP Fungal DNA Kit kit.With bacterial strain
Total DNA is template, uses -3 ' and NL4:5 ' of universal primer NL1:5 '-GCATATCAATAAGCGGAGGAAAAG -
GGTCCGTGTTTCAAGACGG-3 ' and Jin Weizhi PCR kit carry out PCR amplification, PCR to the bacterial strain area 26SrDNA D1/D2
Amplification condition: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 60s, 32 recycle；Last 72
DEG C extend 10min, 4 DEG C preservation.Pcr amplification product is detected using 1% agarose gel electrophoresis, in ultraviolet gel imaging system
Middle comparison Marker analyzes pcr amplification product size.
PCR amplification system: 50 μ L reaction systems are as follows: 5 μ L DNA profilings, 5 μ 10 × PCR of L Buffer, 4 μ L dNTP
(2.5 mmol-1), 1 μ L primer NL1,1 μ L primer NL4,0.5 μ L Taq DNA polymerase, 34.5 μ L ddH2O。
As shown in Figure 1, the amplified band of Meyerozyma guilliermondii ZJC-1 clearly becomes clear, and no miscellaneous band, nothing
Hangover, no non-specific amplification.Sheng Gong bioengineering Co., Ltd is sent to the PCR product of the 26SrDNA of bacterial strain ZJC-1 to carry out
Sequencing is obtained sequence and is compared with blast program and the sequence in GenBank, and chooses the higher sequence of similarity by sequencing
Column, use MEGA7.0 software building bacterial strain phylogenetic tree.
Molecular biology identification result: the 26SrDNA sequence of bacterial strain Meyerozyma guilliermondii ZJC-1 is long
Degree is 593bp, and all sequences obtained in sequence blast program and GenBank are carried out nucleotide homology comparison, are looked into
See base sequence similitude.It chooses the ITS sequence of the high known bacterial strain of tetraploid rice and is used with surveyed bacterial strain ITS sequence
MEGA7.0 software building phylogenetic tree bootstraps 1000 with the reliability of method of bootstrapping (Bootstrap) checking system development tree
It is secondary.
As shown in Fig. 2, Meyerozyma guilliermondii ZJC-1 and Pichia guilliermondii bacterium form a race
Group, and homology is up to 98%.According to the morphological feature, sequence homology and phylogenetic tree of bacterial strain as a result, determining the bacterium
Belong to pichia (Pichia), is Pichia guilliermondii (Meyerozyma guilliermondii).
Present embodiment Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 degrading polyethylene simultaneously produces
The method of raw alkane is as follows.
Carbon-free culture medium (LCFBM): 0.7g KH is weighed2PO4, 0.7g K2HPO4, 0.7g MgSO4·7H2O, 1.0g
NH4NO3, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O and 0.001g MnSO4·H2O is mended and is steamed
Distilled water is to 1000ml (carbon-free solid medium need to add 15~20g of agar), 121 DEG C of high pressure sterilization 15min.
Yeast extract powder peptone dextrose culture-medium (YPD): tryptone 20g, yeast extract 10g, glucose 20g,
(solid YPD culture medium adds 15~20g of agar, melts), distilled water is mended to 1000ml, 115 DEG C of high pressure sterilization 20min.
Activated strains Pichia guilliermondii MeyerozymaguilliermondiiZJC-1, prepares cell suspending liquid.It will
The ZJC-1 bacterium suspension of 10ml is added in the 500mL conical flask of 90mL LCFBM, is then placed in the pretreated polyethylene of 0.2g
Film (3 × 10cm) is used as sole carbon source.In 30 DEG C, the constant-temperature shaking incubator culture 60 or so of 120r/min.It is taken out after 60 days
Experimental group and control group polyethylene film remove biomembrane, using video optics contact angle measurement (OCA20) measurement experiment group and
The variation of control group polyethylene film surface hydrophobic.The minimizing technology of biomembrane is as follows:
Remaining polyethylene film is collected, 2min is cleaned with the phosphate buffer (p H7.2) of 0.01M, in 2% SDS
2h is embathed, impregnates 2h after being cleaned with warm distilled water in 2% glutaraldehyde, is cleaned twice using 50% EtOH Sonicate ripple later
(30min is primary), in 75% ethyl alcohol overnight, with washes of absolute alcohol 3 times (30min is primary) of 100%, finally with sterile
Distilled water cleaning.
The pretreatment of polyethylene: polyethylene film used in testing is cut to the strip diaphragm of 10 × 3cm, in superclean bench
Middle ultraviolet irradiation 2h, impregnates 2h in 75% ethyl alcohol later, with sterile water wash 3 times
As shown in figure 3, the polyethylene contact angle after bacterial strain Meyerozyma guilliermondii ZJC-1 degradation is divided into
78.6 ± 1.2 °, the contact angle for compareing polyethylene is 99.9 ± 0.8 °, the polyethylene contact angle control group after degradation, contact angle
Become smaller and indicate that polyethylene obtains that hydrophobicity is lower, hydrophily is got higher, and can tentatively illustrate polyethylene surface by bacterial strain Meyerozyma
Guilliermondii ZJC-1 oxidation generates hydrophilic radical.
Polyethylene film pieces before and after taking bacterial strain Meyerozyma guilliermondii ZJC-1 to degrade 60 days, removal life
Object film.Using FTIR to the surface chemistry knot of the polyethylene film of Meyerozyma guilliermondii ZJC-1 degradation front and back
Structure carries out analysis detection.Processing is fitted to its detection data using Origin 8.5.
As shown in figure 4, the polyethylene film after Meyerozyma guilliermondii ZJC-1 degradation is in 1735cm-1Place
There is peak, and control group does not have then, 1735cm-1Caused by the appearance of place's peak value is the stretching vibration as carbonyl (- C=O-), carbonyl
Appearance prove polyethylene surface functional group be oxidized.
90mL LCFBM is added in 10mL bacterium suspension by activated strains Meyerozyma guilliermondii ZJC-1
500 mL conical flasks in, be then placed in the pretreated polyethylene film of 0.2g as sole carbon source.Control group is the pre- place of 0.2g
Manage the sterile saline of polythene strip and 10mL.3 repetitions are prepared respectively during culture in 10,30,50,60 days, 30
DEG C, the constant-temperature shaking incubator culture of 120r/min.10,30,50,60 days when take out experimental group and control group polyethylene film, go
It is except biomembrane, the polythene strip for removing biomembrane is dry in electric drying oven with forced convection.Poly- second is observed after drying process
Alkene film has lossless, weighs weight in superior balance, calculates weight-loss ratio.Weight-loss ratio calculation formula is as follows:
Weight-loss ratio=bodies lost weight/original weight
As shown in figure 5, polyethylene is during Meyerozyma guilliermondiiZJC-1 degrades 10,30,50,60 days
Weight-loss ratio be respectively 1.24%, 3.87%, 9.39%, 13.97%, control group does not change significantly.
Conical flask bottom is observed during the culture of Meyerozyma guilliermondii ZJC-1 degrading polyethylene,
After having the generation of particulate matter, residual polyethylene film and culture solution are removed, particulate matter is taken out using 75% alcohol and impregnates 3 points
Clock uses sterile water wash 3 times.Particulate matter is taken to be observed under stereomicroscope.As shown in Figure 6, it can be seen that the substance
Be white for translucent, color, length 0 between 1mm, it is mostly rodlike.
Particulate matter is ground into powder, its chemical structure is detected using infrared spectrometer, using Origin 8.5 to it
Ir data is handled, and result such as Fig. 7, spectrogram shows the substance in 571.16cm-1、759.21cm-1、
1004.56cm-1、 1435.54cm-1、1682.04cm-1、2924.54cm-1、3236cm-1There is peak value at place.571cm-1Peak value
For C-X vibration cause, 759.21cm-1For C-H out-of-plane bending vibration absorption peak, 1004.56cm-1It is inhaled for the stretching vibration of C-O
Receive peak, 1435.54cm-1For the stretching vibration absworption peak of CH3,1682.04cm-1For the stretching vibration absworption peak of C=O,
2924.54cm-1For the characteristic peak of alkane C-H, 3236cm-1For the stretching vibration absworption peak of-OH.1004.56cm-1、
1682.04cm-1、 3236cm-1The appearance of peak value indicates that the solid catabolite contains organic acid.759.21cm-1、
1435.54cm-1、2924.54cm-1Peak value implies that the particulate matter is alkane organic acid mixture.
Powder after particulate matter is ground is dissolved by heating using ethyl acetate at 70 DEG C.Lysate uses gas chromatograph-mass spectrometer
To (Agilent 7890A-7000B), it carries out qualitative analysis detection.Testing conditions are shown in as follows:
Gc-mss detection carrier gas be helium, chromatographic column HP-5MS, 30mx250 μm x0.25 μm of size, initially
45 DEG C of column temperature, heating rate is 3 DEG C/min, in 200 DEG C of holding 5min, 220 DEG C of injector temperature, and 280 DEG C of transmission line temperature, from
230 DEG C of source temperature, level four bars temperature be 150 DEG C, ion source be the source EI, scan pattern mass range be 45amu~
550amu。
As shown in figure 8, particulate matter gas chromatography mass spectrometry spectrogram shows that the unknown particulate matter peak value is more, miscellaneous peak is less, can be preliminary
Judge the substance for organic mixture, according in spectrogram characteristic peak and standard spectrum picture library be compared, confirmation characteristic peak institute it is right
The chemical combination matter answered, the correspondence compound and its retention time of matching degree high peaks are shown in Table 1, retention time 12.778,
19.766,22.715,27.871,35.826,39.862 compounds corresponding with the peak value of 43.025min are 4,5- bis- respectively
Methyl-nonane, pentadecane, the tetradecane, hexadecane, pentacosane and heptacosane, it can be seen that the unknown particulate matter is mainly
Mixed alkanes substance, for the carbon chain lengths of alkanes substance between 10~30, the generation of the result may be due to bacterium
Degradation so that the long-chain of polyethylene has been broken to form the alkanes substance of short chain.
Table 1
Claims (2)
1. the Pichia guilliermondii that one plant of degrading polyethylene generates alkane simultaneously, it is characterised in that the Pichia guilliermondii
Meyerozyma guilliermondiiZJC-1 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, preservation address are the institutes 3 of Chaoyang District, Beijing City Beichen Lu 1, and the deposit date is on December 17th, 2018, deposit number was
CGMCC No:16956.
2. the separation method of the guilliermondii of Pichia guilliermondii Meyerozyma described in claim 1 ZJC-1
Are as follows: using polyethylene as sole carbon source, enrichment culture is carried out to bee moth larvae (Galleria mellonella) intestinal flora, in
30 DEG C, screening obtains the degradable polyethylene and synchronized compound alkane after constant-temperature shaking culture 60 days under the conditions of 120r/min
The bacterial strain of substance.
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| CN102242071B (en) * | 2011-03-28 | 2013-08-14 | 国家海洋局第三海洋研究所 | Application of Pichia guilliermondii 510-6jm in treatment of petroleum hydrocarbon contaminations |
| WO2018040372A1 (en) * | 2016-09-05 | 2018-03-08 | 华南理工大学 | Yeast and use thereof in catalytical synthesis of 2,5-dihydroxymethylfuran |
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| CN114456958B (en) * | 2021-12-20 | 2023-08-22 | 辽宁师范大学 | Salt-tolerant pichia pastoris strain with azo dye degradation function, culture method and application thereof |
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