CN110257267A - The Pichia guilliermondii of one plant of degradable polyethylene - Google Patents

The Pichia guilliermondii of one plant of degradable polyethylene Download PDF

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CN110257267A
CN110257267A CN201910629725.XA CN201910629725A CN110257267A CN 110257267 A CN110257267 A CN 110257267A CN 201910629725 A CN201910629725 A CN 201910629725A CN 110257267 A CN110257267 A CN 110257267A
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polyethylene
guilliermondii
meyerozyma
pichia guilliermondii
pichia
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张�杰
刘长莉
王滨松
池明眼
张国财
王珊珊
郝孔利
董欣欣
姬彦飞
高超
刘燕
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Northeast Forestry University
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

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Abstract

The Pichia guilliermondii of one plant of degradable polyethylene is related to one plant of Pichia guilliermondii.The present invention provides one plant of Pichia guilliermondii, can provide new bacterium source with degrading polyethylene for the biodegrade of polyethylene.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, new bacterium source is provided for Biodegradable polyethylene.For degrading polyethylene.

Description

The Pichia guilliermondii of one plant of degradable polyethylene
Technical field
The present invention relates to one plant of Pichia guilliermondiis.
Background technique
The production of polymer plastic is global industry one of field with fastest developing speed, and usage history is 100 years existing.From 20 Since the fifties in century, plastic products start by large-scale production and consumption, and global plastics-production amount, which is presented, significantly to be referred to Number trend, 50 years since 1964, plastics-production scale increased 20 times, and the annual plastics-production in the whole world is more than 300,000,000 Ton, global plastics yield in 2014 are 3.11 hundred million tons, and 2015 are 3.35 hundred million tons, it is contemplated that are up to about 1,800,000,000 to the year two thousand fifty Ton.Polyethylene is one of main raw material(s) of plastic products, and relative molecular weight is big, hydrophobicity is relatively strong and surface can be very low.It is poly- Ethylene usually has 3 kinds, respectively high density polyethylene (HDPE), low density polyethylene (LDPE) and linear low density polyethylene.With industrial development Speed is accelerated, the increase of plastics manufacture, increases waste plastic processing load.Since polyethylene has durability and good Material property is generally difficult to degrade, so largely, widely being caused using ecological environment of the polyethylene plastic products to the earth Serious pollution.
Currently, most waste polyethylene plastics are mainly handled by the way of burning and filling, waste plastic The 5-25wt% of the total amount of waste of refuse landfill is accounted for, wherein the landfill of a large amount of plastic wastes results in problem of management and environment danger Evil such as destroys soil physico-chemical property, causes soil hardening, and CO, HCl, NOx, SO can be generated by burning2And dioxin etc. largely has Poisonous gas pollutes air in turn, both processing modes cause serious pollution to environment.
Therefore, the fungi for filtering out energy degrading polyethylene, for further being solved using environmentally protective processing method The pollution problem of waste polyethylene plastics is of great significance.
Summary of the invention
The present invention provides one plant of Pichia guilliermondii, can with degrading polyethylene, for polyethylene degradation provide it is new Bacterium source.
The Pichia guilliermondii Meyerozyma guilliermondii ZJC-1 of degradable polyethylene of the present invention is protected It ensconces China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is Chaoyang District, Beijing City The institute 3 of Beichen Lu 1, the deposit date is on December 17th, 2018, deposit number was 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 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 Meyerozyma guilliermondiiZJC-1 and GenBank carries out sequence analysis Constructed systematic evolution tree；Fig. 3 is poly- second before and after bacterial strain Meyerozyma guilliermondiiZJC-1 degrading polyethylene The infrared spectrum of the surface chemical structure of alkene；Fig. 4 is bacterial strain Meyerozyma guilliermondiiZJC-1 degradation front and back Polyethylene surface water contact angle figure.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.
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 Meyerozyma of present embodiment degradable polyethylene GuilliermondiiZJC-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.
Present embodiment Pichia guilliermondii Meyerozyma guilliermondiiZJC-1 is by bee moth larvae enteron aisle Screening obtains in liquid.Screening sequentially includes the following steps:
Prepare 5 age bee moth larvae 100, using wheat bran as processing on an empty stomach is done after diatery supplement feeding polyethylene film 2~3 days, on an empty stomach Bee moth larvae is immersed into 75% ethyl alcohol the 1min that sterilizes afterwards for 24 hours, is then rinsed 2 times with Sterile Saline, is carried out in superclean bench Dissection obtains enteron aisle liquid.Enteron aisle liquid is put into the centrifuge tube containing 40mL physiological saline, 5min is shaken in vortex instrument, it The impurity such as extra intestinal tissue are removed with pipettor afterwards.The bee moth larvae enteron aisle liquid that will acquire will be used as microbial inoculant object suspension It is inoculated into the carbon-free liquid medium (LCFBM) of 80mL in superclean bench, and polyethylene film pieces 0.2g is added, at 30 DEG C Constant-temperature shaking incubator 120r/min shaken cultivation 60 days.After 60 days, 1mL culture solution is taken to be inoculated in containing final concentration of 30 μ In the potato glucose solid medium (PDA) of the streptomysin of g/mL, in 30 DEG C, 120 turns of constant-temperature shaking incubator culture. For 24 hours afterwards by the different colony inoculations of growth in carrying out scribing line separation on PDA agar medium, until dividing for pure bacterial strain can be obtained From object.
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.
PDA culture medium (1000mL) is by 200g potato, 20g glucose, 1.5g MgSO4·7H2O、3g KH2PO4With it is remaining The distilled water of amount forms；PDA solid medium (1000mL) is by 200g potato, 20g glucose, 1.5g MgSO4·7H2O、 3g KH2PO4, 15g agar powder and surplus distilled water composition.
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.
The bacterial strain Meyerozyma guilliermondiiZJC-1 filtered out is according to " fungal taxonomy ", " Fungal identification Handbook " and " saccharomycete feature and identification handbook " progress Morphological Identification.The bacterium single colonie is creamy white, round, and surface is smooth Glossy, quality is more sticky, opaque, and thallus is elliposoidal.
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 universal primer NL 1:5 '-G C A T A T C A A T A A G C G G A G G A A A A G -3 ' and NL4:5 '-G G T C C G T G T T T C A A G A C G G-3 ' and Jin Weizhi PCR kit PCR amplification is carried out to the bacterial strain area 26SrDNA D1/D2, PCR amplification condition: 94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 30s, 59 DEG C Anneal 30s, 72 DEG C of extension 60s, 32 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.Using 1% agarose gel electrophoresis Pcr amplification product is detected, Marker is compared in ultraviolet gel imaging system and 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。
PCR product by 1% Agar Gel sugar detected through gel electrophoresis, electrophoresis result as shown in Figure 1, the bacterium amplification item Band is clear bright, no miscellaneous band, no hangover, no non-specific amplification.Raw work is sent to the PCR product of the 26SrDNA of bacterial strain ZJC-1 Bioengineering Co., Ltd is sequenced, and sequencing is obtained sequence and is compared with blast program and the sequence in GenBank, And the higher sequence of similarity is chosen, 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 Secondary, phylogenetic tree is as shown in Fig. 2, bacterial strain Meyerozyma guilliermondii ZJC-1 and Pichia guilliermondii Form a group, and homology is up to 98%.According to the morphological feature of bacterial strain, sequence homology and phylogenetic tree knot Fruit determines that the Pseudomonas is Pichia guilliermondii (Meyerozyma in pichia (Pichia) guilliermondii)。
The Pichia guilliermondii Meyerozyma of YPD slant medium is stored in using transfer needle scraping GuilliermondiiZJC-1 is inoculated in new YPD solid medium, and 28 DEG C are cultivated 3-5 days, carries out bacterial strain activation, preparation Bacterium suspension.The ZJC-1 bacterium suspension of 10mL is added in the 500mL conical flask of 90mL LCFBM, is then placed in 0.2g and locates in advance The polyethylene film (3 × 10cm) of reason makes it as sole carbon source.The nothing of pretreatment polyethylene film and 10mL that control group is 0.2g Bacterium physiological saline.At 30 DEG C, constant-temperature shaking incubator culture 60 days of 120r/min.Experimental group is taken out after 60 days and control group is poly- Vinyl film removes biomembrane, uses video optics contact angle measurement (OCA20) measurement experiment group and control group polyethylene film table The hydrophobic variation in face.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 guilliermondiiZJC-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 GuilliermondiiZJC-1 oxidation generates hydrophilic radical.
Polyethylene film pieces before and after taking bacterial strain Meyerozyma guilliermondiiZJC-1 to degrade 60 days, removal biology Film.Using FTIR to the surface chemical structure of the polyethylene film of Meyerozyma guilliermondiiZJC-1 degradation front and back Carry out analysis detection.Processing is fitted to its detection data using Origin 8.5, as a result sees Fig. 4.Meyerozyma Polyethylene film after guilliermondiiZJC-1 degradation is in 1735cm-1There is peak at place, and control group does not have then, 1735cm-1Place Caused by the appearance of peak value is the stretching vibration as carbonyl (- C=O-), the appearance of carbonyl proves the functional group of polyethylene surface It is oxidized.
10mL bacterium suspension is added 90mL LCFBM's by activated strains Meyerozyma guilliermondiiZJC-1 In 500 mL conical flasks, the pretreated polyethylene film of 0.2g is then placed in as sole carbon source.Control group is the pretreatment of 0.2g The sterile saline of polythene strip and 10mL.Prepare 3 repetitions 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, removal is given birth to Object film, the PE piece for removing biomembrane is dry in electric drying oven with forced convection.There is lossless observation PE film after drying process, Weight is weighed 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.

Claims (2)

1. the Pichia guilliermondii of one plant of degradable polyethylene, it is characterised in that Pichia guilliermondii Meyerozyma GuilliermondiiZJC-1 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address It 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。
2. the separation method of the guilliermondii of Pichia guilliermondii Meyerozyma described in claim 1 ZJC-1 Are as follows: using polyethylene as carbon source, enrichment isolation is carried out to bee moth larvae (Galleria mellonella) intestinal flora, obtains institute State degradable polyethylene bacterial strain.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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
CN107937284A (en) * 2017-11-23 2018-04-20 青岛农业大学 A kind of Pichia guilliermondii and its application
CN110257268B (en) * 2019-07-12 2022-05-03 东北林业大学 Pichia guilliermondii strain for degrading polyethylene and producing alkane simultaneously

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
KR101218275B1 (en) * 2011-11-22 2013-01-03 이경태 Methods for preparing fermented ginseng or fermented red ginseng using yeast
CN107937284A (en) * 2017-11-23 2018-04-20 青岛农业大学 A kind of Pichia guilliermondii and its application
CN110257268B (en) * 2019-07-12 2022-05-03 东北林业大学 Pichia guilliermondii strain for degrading polyethylene and producing alkane simultaneously

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