<i>Microbacterium oleivorans </i>capable of degrading polyethylene terephthalate and intermediate thereof
11654317 · 2023-05-23
Assignee
Inventors
Cpc classification
Y02W30/62
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A62D2101/28
HUMAN NECESSITIES
A62D3/02
HUMAN NECESSITIES
C12R2001/01
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure discloses Microbacterium oleivorans capable of degrading polyethylene terephthalate and an intermediate thereof, and belongs to the technical field of microorganisms. The present disclosure provides Microbacterium oleivorans JWG-G2 capable of degrading the polyethylene terephthalate. After Microbacterium oleivorans JWG-G2 is inoculated into an inorganic salt liquid medium containing 2 g/L polyethylene terephthalate plastic particles with an inoculation quantity of 1×10.sup.8 CFU/mL to be cultivated for 5 d, the polyethylene terephthalate plastic particles can be partially degraded into monohydroxyethyl terephthalate and terephthalic acid capable of being directly recycled, ester bond functional groups on surfaces of the polyethylene terephthalate plastic particles can be reduced, and a weight loss ratio of the polyethylene terephthalate plastic particles can reach 5.6%. Therefore, Microbacterium oleivorans JWG-G2 of the present disclosure has an extremely high application prospect in degradation of the polyethylene terephthalate.
Claims
1. A method for degrading polyethylene terephthalate and/or an intermediate of the polyethylene terephthalate, which comprises: inoculating Microbacterium oleivorans into a medium for cultivation, wherein the medium comprises polyethylene terephthalate (PET), an intermediate of the PET, or both; wherein the intermediate of the PET is monohydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), or both; and wherein the Microbacterium oleivorans is Microbacterium oleivorans deposited with the China Center for Type Culture Collection (CCTCC), with CCTCC deposit number M 2019416.
2. The method according to claim 1, wherein the medium is a liquid medium.
3. The method according to claim 1, wherein an inoculation quantity of Microbacterium oleivorans in the medium is not less than 1×10.sup.8 CFU/mL.
4. The method according to claim 1, wherein the medium comprises the PET, and a content of the PET is not greater than 2 g/L.
5. The method according to claim 1, wherein the medium comprises the intermediate of the PET, and a content of the intermediate of the PET is not greater than 0.2 g/L.
6. The method according to claim 1, wherein the medium comprises the PET and the intermediate of the PET, and a total content of the PET and the intermediate of the PET is not greater than 2.2 g/L.
7. The method according to claim 1, wherein the medium is an inorganic salt medium.
Description
BRIEF DESCRIPTION OF FIGURES
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DETAILED DESCRIPTION
(7) The present disclosure is further expounded below in combination with specific examples.
(8) Dimethyl terephthalate (DET), polyethylene terephthalate (PET) plastic particles, monohydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET) and terephthalic acid (TPA) involved in the following examples are purchased from Sigma Company.
(9) Mediums Involved in the Following Examples are as Follows:
(10) LB solid medium (g/L): 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 20 g of agar, and pH 7.0.
(11) LB liquid medium (g/L): 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, and pH 7.0.
(12) Inorganic salt liquid medium containing DET (g/L): 0.7 g of KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2 g of NH.sub.4Cl, 0.6 g of MgSO.sub.4.7H.sub.2O, 0.005 g of NaCl, 0.001 g of FeSO.sub.4.7H.sub.2O, 0.002 g of ZnSO.sub.4.7H.sub.2O, 0.001 g of MnSO.sub.4.H.sub.2O, and 2 g of the DET.
(13) Inorganic salt solid medium containing PET (g/L): 0.7 g of KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2 g of NH.sub.4Cl, 0.6 g of MgSO.sub.4.7H.sub.2O, 0.005 g of NaCl, 0.001 g of FeSO.sub.4.7H.sub.2O, 0.002 g of ZnSO.sub.4.7H.sub.2O, 0.001 g of MnSO.sub.4.H.sub.2O, 2 g of PET plastic particles, and 20 g of agar powder.
(14) Inorganic salt liquid medium containing PET (g/L): 0.7 g of KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2 g of NH.sub.4Cl, 0.6 g of MgSO.sub.4.7H.sub.2O, 0.005 g of NaCl, 0.001 g of FeSO.sub.4.7H.sub.2O, 0.002 g of ZnSO.sub.4.7H.sub.2O, 0.001 g of MnSO.sub.4.H.sub.2O, and 2 g of the PET.
(15) Inorganic salt liquid medium containing a PET intermediate (g/L): 0.7 g of KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2 g of NH.sub.4Cl, 0.6 g of MgSO.sub.4.7H.sub.2O, 0.005 g of NaCl, 0.001 g of FeSO.sub.4.7H.sub.2O, 0.002 g of ZnSO.sub.4.7H.sub.2O, 0.001 g of MnSO.sub.4.H.sub.2O, and 0.2 g of the PET intermediate (BHET or MHET).
(16) Inorganic salt solid medium without a nutrient source (g/L): 0.7 g of KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2 g of NH.sub.4Cl, 0.6 g of MgSO.sub.4.7H.sub.2O, 0.005 g of NaCl, 0.001 g of FeSO.sub.4.7H.sub.2O, 0.002 g of ZnSO.sub.4.7H.sub.2O, 0.001 g of MnSO.sub.4.H.sub.2O, and 20 g of agar powder.
(17) Detection Methods Involved in the Following Examples are as Follows:
(18) Detection Method of Changes of Functional Groups on Surfaces of Polyethylene Terephthalate (PET) Plastic Particles:
(19) The PET plastic particles treated by a strain are repeatedly cleaned with deionized water 3 to 4 times. The cleaned PET plastic particles are subjected to ultrasonic for 15 min at a power of 200 W and a frequency of 58 KHz. The PET plastic particles after ultrasonic are placed into a dryer to be dried for 6 h at 60° C. With the PET plastic particles not treated as control, a Fourier transform infrared spectrometer is utilized to detect the changes of the functional groups on the surfaces of the PET plastic particles not treated and the surfaces of the PET plastic particles treated by the strain.
(20) Detection Methods of Degradation Products and Contents Thereof:
(21) Standard treatment: Standards of TPA, MHET and BHET are weighed respectively to be dissolved in dimethylsulfoxide (DMSO) to prepare mother solutions, and the mother solutions are diluted into 0.1 mg/mL standard solutions by sterile water, filtered by a 0.22 μM filtering head, and injected into liquid phase bottles by an injector for HPLC detection.
(22) Sample treatment: A cultivation solution is subjected to still standing for 10 min, and 5 mL of a supernatant is taken, centrifuged for 8 min at 12,000 rpm, filtered by a 0.22 μM filtering head, and injected into a liquid phase bottle by an injector for HPLC detection.
(23) Detection Method of Weight Loss Ratio:
A weight loss ratio (%) of PET plastic particles=[(m2−m1)÷m2]×100.
(24) m1: The PET plastic particles treated by a strain are repeatedly cleaned with deionized water 3 to 4 times. The cleaned PET plastic particles are subjected to ultrasonic for 15 min at a power of 200 W and a frequency of 58 KHz, placed into a dryer to be dried for 6 h at 60° C., and then weighed.
(25) m2: The PET plastic particles before being treated by the strain are repeatedly cleaned with deionized water 3 to 4 times. The cleaned PET plastic particles are subjected to ultrasonic for 15 min at a power of 200 W and a frequency of 58 KHz, placed into a dryer to be dried for 6 h at 60° C., and then weighed.
A weight loss ratio (%) of a PET intermediate={[(c1−c2)×v2]÷(c1×v1)}×100.
(26) c1: A concentration of a PET intermediate in a reaction system before a reaction, mg/L.
(27) v1: A concentration of the PET intermediate in the reaction system before the reaction, L.
(28) c1: A concentration of a PET intermediate in a reaction system after the reaction, mg/L.
(29) v1: A concentration of the PET intermediate in the reaction system after the reaction, L.
Example 1: Screening and Identification of Microbacterium oleivorans
(30) Specific steps are as follows:
(31) 1. Screening
(32) With soil from the Taohuashan landfill in Wuxi as a sample, 1 g of landfill soil is taken, added into 9 mL of an inorganic salt liquid medium containing 2 g/L DET, and subjected to shaking enrichment culture for 48 h at 35° C. and 180 rpm. Then, 1 mL of above enrichment liquid is sucked, added into 9 mL of a new inorganic salt liquid medium containing 2 g/L DET and cultivated for 10 cycles at the same conditions. Cultivation solutions obtained after 10 cycles of cultivation are subjected to still standing for 15 min, 10.sup.−4, 10.sup.−5 and 10.sup.−6 diluents obtained by sequentially diluting 1 mL of supernatants and 10.sup.−4, 10.sup.−5 and 10.sup.−6 diluents obtained by diluting 200 μL of supernatants evenly coat inorganic salt solid mediums containing 2 g/L PET, and the mediums are placed in a 35° C. incubator for constant-temperature cultivation until bacterial colonies grow out. With an inorganic salt solid medium without a nutrient source as control, the bacterial colonies are picked to streak inorganic salt solid mediums containing 2 g/L PET and cultivated at 35° C., several times of repeated streaking are conducted to obtain non-autotrophic purified strains, and 4 non-autotrophic purified strains growing best are named a strain JWG-G2, a strain JWG-G5, a strain JWG-HD2 and a strain JWG-YR2 respectively.
(33) 2. Identification
(34) Total DNA of the strain JWG-G2, the strain JWG-G5, the strain JWG-HD2 and the strain JWG-YR2 is extracted for 16S rDNA amplification and sequencing (completed by Wuxi TianLin Biotechnology Co., Ltd.). Sequencing results show that the 16S rDNA similarity rate of the above 4 non-autotrophic purified strains is 100%. It can be seen that the above 4 non-autotrophic purified strains are all differentiated from 4 single bacterial colonies of the same strain. Therefore, the strain JWG-G2 is selected as an identification object for next step identification (the 16S rDNA sequence of the JWG-G2 is shown as SEQ ID NO: 1).
(35) Sequences obtained by sequencing are subjected to nucleotide sequence comparison in Genbank. It is found that the 16S rDNA sequence homology of the strain JWG-G2 to a Microbacterium is greater than 99%, and the 16S rDNA sequence similarity rate to Microbacterium oleivorans NBRC103075 reaches 99.5%. It can be seen that the strain JWG-G2 belongs to genus Microbacterium.
(36) The 16S rDNA sequence of the strain JWG-G2 and other high-similarity strains constitute a phylogenetic tree (see
Example 2: Cultivation of Microbacterium oleivorans
(37) Specific steps are as follows:
(38) A ring of Microbacterium oleivorans JWG-G2 obtained in Example 1 is scraped and inoculated into an LB solid medium for streaking cultivation. After cultivation for 36 h at 35° C., their bacterial colonies are observed, and it is found that their bacterial colonies are shaped like rounded raised protrusions, and are light red, not transparent, smooth in surface, wet and glossy, and regular in edge (specifically see
(39) Microbacterium oleivorans JWG-G2 obtained in Example 1 is observed under a microscope after Gram staining. It is found that Microbacterium oleivorans JWG-G2 is a Gram-positive bacterium.
(40) A ring of Microbacterium oleivorans JWG-G2 obtained in Example 1 is scraped and inoculated into LB liquid mediums with pH being 3 to 10 respectively to be cultivated. After cultivation for 36 h at 35° C., OD.sub.600 values in cultivation solutions are detected through a microplate reader. It is found that suitable growth pH of Microbacterium oleivorans JWG-G2 is 6.5 to 8.5, and the most suitable growth pH is 7.
(41) A ring of Microbacterium oleivorans JWG-G2 obtained in Example 1 is scraped and inoculated into LB liquid mediums with pH being 7 to be cultivated. After cultivation for 36 h at 20 to 50° C. respectively, OD.sub.600 values in cultivation solutions are detected through a microplate reader. It is found that a suitable growth temperature of Microbacterium oleivorans JWG-G2 is 25 to 40° C., and the most suitable growth temperature is 35° C.
(42) A ring of Microbacterium oleivorans JWG-G2 obtained in Example 1 is scraped and inoculated into LB liquid mediums with pH being 7 to be cultivated for 36 h at 35° C. During cultivation, OD.sub.600 values in cultivation solutions are detected through a microplate reader. It is found that Microbacterium oleivorans JWG-G2 has a quick propagation speed, and can enter a stable phase of growth after cultivation for 14 to 16 h.
Example 3: Degradation Abilities of Different Microbacteria and Microbacterium oleivorans to Polyethylene Terephthalate (PET) Plastic Particles
(43) Specific steps are as follows:
(44) Because Microbacterium oleivorans JWG-G2 belongs to a Microbacterium and the Microbacterium may be one of potential PET plastic particle degradation strain sources, 18 microbacteria with a close affinity to Microbacterium oleivorans JWG-G2 are collected and taken as test strains jointly with Microbacterium oleivorans JWG-G2.
(45) Single bacterial colonies of Microbacterium oleivorans JWG-G2 obtained in Example 1 and the 18 microbacteria are picked, inoculated into 100 mL of LB liquid mediums respectively, and subjected to shaking cultivation for 24 h at 35° C. and 180 rpm to obtain seed solutions A. The seed solutions A are transferred into 100 mL of fresh LB liquid mediums with an inoculation quantity of 10% (v/v), and subjected to shaking cultivation for 24 h at 35° C. and 180 rpm to obtain cultivation solutions A. The cultivation solutions A are centrifuged for 10 min at 8,000 rpm, and thalluses are collected. After the thalluses are washed with an inorganic salt medium for 2 times, bacterial suspensions with OD.sub.600 being 1.0 are prepared to be taken as seed solutions B. With inorganic salt liquid mediums not inoculated with the seed solutions B and containing 2 g/L PET as control groups, the seed solutions B are inoculated into the inorganic salt liquid mediums containing the 2 g/L PET with an inoculation quantity of 10% (v/v) and subjected to shaking cultivation for 16 d at 35° C. and 180 rpm. During shaking cultivation, sampling is conducted once every 1 d. OD.sub.600 of cultivation solutions B is determined to obtain growth curves of Microbacterium oleivorans JWG-G2 and the 18 microbacteria with the PET plastic particles as a unique nutrient source (see Table 1 for changes of the OD.sub.600 of Microbacterium oleivorans JWG-G2 and the 18 microbacteria before and after cultivation through the inorganic salt liquid mediums containing the 2 g/L PET, and see
(46) It can be seen from Table 1 and
(47) It can be seen from Table 2 and
(48) TABLE-US-00001 TABLE 1 Changes of OD.sub.600 before and after cultivation of Microbacterium oleivorans JWG-G2 and the 18 microbacteria through the inorganic salt liquid mediums containing the 2 g/L PET Strains OD.sub.600 Strains OD.sub.600 Microbacterium oleivorans JWG-G2 0.2 Microbacterium oleivorans NBRC103075 0.02 Microbacterium hibisci KACC18931 0.02 Microbacterium flavescens DSM20643 0.01 Microbacterium hominis NBRC15708 0.03 Microbacterium laevaniformans DSM20140 0.01 Microbacterium enclense DSM25125 0.03 Microbacterium dextranolyticum DSM8607 0.02 Microbacterium telephonicum KACC18715 0.01 Microbacterium saccharophilum 0.01 NBRC108778 Microbacterium ketosireducens 0.03 Microbacterium terrae JCM15516 0.01 DSM12510 Microbacterium flavum JCM15574 0.03 Microbacterium diaminobutyricum 0.01 DSM27101 Microbacterium schleiferi DSM20489 0.01 Microbacterium lacticum DSM20427 0.01 Microbacterium terregens JCM1342 0.01 Microbacterium aurum KACC15219 0.01 Microbacterium aoyamense JCM14900 0.01
(49) The changes of the OD.sub.600 before and after cultivation of Microbacterium oleivorans JWG-G2 and the 18 microbacteria through the inorganic salt liquid mediums containing the 2 g/L PET are obtained by subtracting OD.sub.600 values before cultivation of Microbacterium oleivorans JWG-G2 and the 18 microbacteria through the inorganic salt liquid mediums containing the 2 g/L PET from OD.sub.600 values after cultivation of Microbacterium oleivorans JWG-G2 and the 18 microbacteria through the inorganic salt liquid mediums containing the 2 g/L PET.
(50) TABLE-US-00002 TABLE 2 Contents of the degradation products MHET and TPA of the PET plastic particles in the cultivation solutions B obtained by cultivation of Microbacterium oleivorans JWG-G2 and the 18 microbacteria, changes of the structures of the functional groups on the surfaces of the PET plastic particles, and weight loss ratios of the PET plastic particles Contents of degradation Ester bond products (mg/L) PET weight functional Categories TPA MHET loss ratios (%) groups Microbacterium oleivorans JWG-G2 1.3 6.9 1 + Microbacterium oleivorans NBRC103075 − − − − Microbacterium hibisci KACC18931 − − − − Microbacterium flavescens DSM20643 − − − − Microbacterium hominis NBRC15708 − − − − Microbacterium laevaniformans DSM20140 − − − − Microbacterium enclense DSM25125 − − − − Microbacterium dextranolyticum DSM8607 − − − − Microbacterium telephonicum KACC18715 − − − − Microbacterium saccharophilum − − − − NBRC108778 − − − Microbacterium ketosireducens DSM12510 − − − − Microbacterium terrae JCM15516 − − − − Microbacterium flavum JCM15574 − − − − Microbacterium diaminobutyricum − − − − DSM27101 − − − Microbacterium schleiferi DSM20489 − − − − Microbacterium lacticum DSM20427 − − − − Microbacterium terregens JCM1342 − − − − Microbacterium aurum KACC15219 − − − − Microbacterium aoyamense JCM14900 − − − − “+”: detection is positive; and “−”: detection is negative.
Example 4: Degradation Abilities of Microbacterium oleivorans JWG-G2 to Polyethylene Terephthalate (PET) Plastic Particle Intermediate
(51) Specific steps are as follows:
(52) Single bacterial colonies of Microbacterium oleivorans JWG-G2 obtained in Example 1 and 18 microbacteria are picked, inoculated into 100 mL of LB liquid mediums respectively, and subjected to shaking cultivation for 24 h at 35° C. and 180 rpm to obtain seed solutions A. The seed solutions A are transferred into 100 mL of fresh LB liquid mediums with an inoculation quantity of 10% (v/v), and subjected to shaking cultivation for 24 h at 35° C. and 180 rpm to obtain cultivation solutions A. The cultivation solutions A are centrifuged for 10 min at 8000 rpm, and thalluses are collected. After the thalluses are washed with an inorganic salt medium for 2 times, bacterial suspensions with OD.sub.600 being 1.0 are prepared to be taken as seed solutions B. The seed solutions B are inoculated into inorganic salt liquid mediums containing 0.2 g/L MHET or 0.2 g/L BHET (the MHET and the BHET are both PET intermediates) respectively with an inoculation quantity of 10% (v/v) and subjected to shaking cultivation for 5 d at 35° C. and 180 rpm to obtain cultivation solutions B.
(53) The MHET and the BHET in the cultivation solutions B are taken out, and their weight loss ratios are detected. At the same time, changes of components in the cultivation solutions B are analyzed by HPLC (see
(54) It can be known from the analysis results of the weight loss ratios that after 5 d of treatment through Microbacterium oleivorans JWG-G2, the weight loss ratio of the MHET reaches 4.5%, and the weight loss ratio of the BHET reaches 11.2%. It can be seen that e Microbacterium oleivorans JWG-G2 can degrade the MHET and the BHET.
(55) It can be known from
(56) It can be known from
Example 5: Salt Resistance of Microbacterium oleivorans JWG-G2
(57) Specific steps are as follows:
(58) Single bacterial colonies of Microbacterium oleivorans JWG-G2 obtained in Example 1 are picked, inoculated into 100 mL of LB liquid mediums, and subjected to shaking cultivation for 24 h at 35° C. and 180 rpm to obtain seed solutions A. The seed solutions A are transferred into 100 mL of fresh LB liquid mediums with an inoculation quantity of 10% (v/v), and subjected to shaking cultivation for 72 h at 35° C. and 180 rpm to obtain cultivation solutions A. The cultivation solutions A are centrifuged for 10 min at 8,000 rpm, and thalluses are collected. After the thalluses are washed with an inorganic salt medium for 2 times, bacterial suspensions with OD.sub.600 being 1.0 are prepared to be taken as seed solutions B. The seed solutions B are inoculated into LB liquid mediums containing different concentrations of NaCl (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 g/L) respectively with an inoculation quantity of 10% (v/v) and subjected to shaking cultivation for 5 d at 35° C. and 180 rpm to obtain cultivation solutions B.
(59) It is found from results of determining OD.sub.600 of the cultivation solutions B that OD.sub.600 increments in the cultivation solutions B obtained after Microbacterium oleivorans JWG-G2 grows for 5 d in the LB liquid mediums containing 1 to 9 g/L NaCl are 0.11, 0.12, 0.18, 0.2, 0.23, 0.18, 0.15, 0.1 and 0.1 respectively. It can be seen that Microbacterium oleivorans JWG-G2 has excellent salt resistance.
Example 6: Abilities of Microbacterium oleivorans JWG-G2 to Degrade Starch and Liquidize Gelatin
(60) Specific steps are as follows:
(61) The abilities of Microbacterium oleivorans JWG-G2 to degrade the starch and liquidize the gelatin are detected through a plate transparent zone method according to a reference “Journal of Microbiology, 2014, 34(01): 28-32; Tang Yu, Southwest University, 2007”.
(62) It can be known from detection results that after Microbacterium oleivorans grows for 5 d on detection plates, there are obvious hydrolyzed transparent zones on surfaces of the plates, and diameters of starch and gelatin transparent zones reach 1.1 cm and 1.6 cm respectively. It can be seen that Microbacterium oleivorans JWG-G2 has the abilities to degrade the starch and liquidize the gelatin.