CN112522358B - System for evaluating polyurethane degradation - Google Patents
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Abstract
The invention belongs to the technical field of biological treatment, and particularly discloses a system for evaluating polyurethane degradation, which comprises the steps of inoculating seed liquid of polyurethane degrading bacteria into an inorganic salt culture medium containing polyurethane for culturing, degrading the polyurethane, and evaluating degradation effect by any one or a combination of a plurality of methods of mass loss, electron microscope scanning, infrared spectrum detection, gel chromatography detection, turbidity and degradation product identification; wherein the polyurethane is aqueous polyurethane or polyurethane film. The invention establishes a system for evaluating polyurethane degradation, namely, for two polyurethane substrates (PU simulants Imprandil DLN and PU film), the degradation condition of the polyurethane substrate is evaluated by means of microbial degradation based on turbidity change, mass loss, surface degradation condition and the like of physical characterization and means of surface functional group change, molecular weight change, degradation product identification and the like of chemical characterization, so that the degradation effect of polyurethane can be completely represented.
Description
Technical Field
The invention belongs to the technical field of biological treatment, and particularly relates to a system for evaluating polyurethane degradation.
Background
Polyurethane (PU) is a polymer with urethane bond repeating unit structure, and is formed by condensing three components of polyisocyanate, polyol and chain extender. Thus, various types of PUs can be synthesized by different types and proportions of polyols and isocyanates. Among them, polyurethanes are classified into two main types, namely polyester type and polyether type polyols, according to the type of polyol. The polyurethane product mainly comprises the following components: foam plastic, elastomer, fiber plastic, fiber, leather shoe resin, paint, adhesive, sealant and the like, wherein the foam plastic has the largest proportion and is widely applied to the fields of packaging, sound insulation, construction, automobiles, shoe industry, medical industry and the like.
According to statistics, the annual output of the global polyurethane plastic in 2016 is up to 24.2Mt (10.1 Mt in China), and the total output of the synthetic plastic is 7.5%, and the wide use of the polyurethane plastic leads to the unreasonable disposal of a large amount of wastes, thereby directly threatening the ecological environment and human health. The traditional waste plastic treatment method mainly comprises landfill, incineration, mechanical recovery and the like, has high economic and labor cost and can cause secondary pollution, so that the biodegradation method becomes a hot spot for global research. However, polyurethane has high antimicrobial degradability, and the current evaluation method for representing the degradation effect of polyurethane is uneven, so that the degradation effect of polyurethane plastic cannot be completely represented. The experiment establishes a standardized polyurethane plastic degrading bacterium function characterization system to study the degrading effect of Cladosporium sp.P7 screened in the earlier stage on two polyurethane substrates (Imprandil DLN and PU films). The establishment of the standardized polyurethane plastic degradation bacterium function characterization system provides a theoretical basis and a reference for the evaluation of a plastic biodegradation method and the characterization method of other strains on plastic degradation, and has practical application value.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a system for evaluating polyurethane degradation aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a system for evaluating polyurethane degradation, which is characterized in that seed liquid of polyurethane degrading bacteria is inoculated into an inorganic salt culture medium containing polyurethane for culturing, the polyurethane is degraded, and the degradation effect is evaluated by any one or a combination of a plurality of methods of mass loss, electron microscope scanning, infrared spectrum detection, gel chromatography detection (molecular weight change), turbidity and degradation product identification as shown in a figure 1.
Wherein the polyurethane is two polyurethane substrates, namely aqueous polyurethane (Imprandil DLN) or polyurethane film.
Preferably, the polyurethane degrading bacterium is Cladosporium sp, the strain name is P7, the polyurethane degrading bacterium is preserved in China center for type culture collection, and the strain preservation number is CCTCC NO: m2020627, the preservation date is 11/7 in 2020, the preservation address is Wuhan in China, and the nucleotide sequence of ITS of the Cladosporium is shown as SEQ ID No. 1.
The preparation method of the seed solution comprises inoculating strain P7 onto PDA solid culture medium, culturing at 30deg.C for 5d, collecting 10mL sterile water drop on flat plate, scraping mycelium and spore on surface with coating rod, counting with blood cell counting plate, and diluting to final concentration of 1×10 6 Bacterial suspension with spore/mL concentration is used as degradation test seed liquid.
Inoculating the seed solution of the cladosporium into an inorganic culture medium containing aqueous polyurethane or an inorganic culture medium containing a polyurethane film according to the volume ratio of 5-15%; preferably, the culture medium is inoculated into an inorganic culture medium containing aqueous polyurethane or an inorganic culture medium containing polyurethane film in a volume ratio of 10%.
Wherein, the content of the aqueous polyurethane in the inorganic culture medium containing the aqueous polyurethane is 0.2-2% v/v, preferably 1% v/v.
Wherein, in the inorganic culture medium containing the polyurethane film, the concentration of the polyurethane film is 3-10 mg/mL, preferably 6mg/mL.
Wherein the inorganic culture medium is prepared by adding 1.0g of ammonium nitrate, 1.0g of sodium chloride, 0.5g of monopotassium phosphate and 1.5g of dipotassium phosphate into 1.0L of water, adjusting the pH value to 7.0, adding 20.0g of agar into the solid culture medium, and sterilizing for 20min at 121 ℃.
Wherein the polyurethane is polyurethane itself or polyurethane film.
The preparation method of the polyurethane film comprises the following steps: dissolving PU solid with dichloromethane, spreading film again, specifically dissolving 2g PU solid in 20mL dichloromethane, pouring the dissolved solution into a glass plate, volatilizing solvent in a fume hood, slowly removing with forceps for 24 hr, and cutting into film with size of 2 x 4cm for use.
Wherein the culture is shake culture at 80-200 rpm and at 20-50 ℃; preferably, the culture is shake culture at 30℃and 120 rpm.
When the polyurethane is a polyurethane film, sampling is carried out in the culture process or after the culture is finished, thalli on the polyurethane are removed, cleaning and drying are carried out, the degraded polyurethane is obtained, and the quality loss is calculated by weighing; preferably, the sampling during or after the culture is sampling at weeks 2,4,6 and 8 of the culture. .
When the polyurethane is a polyurethane film, sampling is carried out in the culture process or after the culture is finished, thalli on the polyurethane are removed, cleaning and drying are carried out, the degraded polyurethane is obtained, the surface with proper size is cut out for metal spraying treatment, and an electron microscope Scanning (SEM) test is carried out to evaluate the surface morphology change of the polyurethane; preferably, the sampling during or after the culturing process is sampling at weeks 2,4 and 6 of the culturing process.
When the polyurethane is a polyurethane film, sampling is carried out in the culture process or after the culture is finished, thalli on the polyurethane are removed, cleaning and drying are carried out, the degraded polyurethane is obtained, infrared spectrum detection is carried out, and the change of the surface functional group of the polyurethane is analyzed; preferably, the sampling during or after the culture is performed at week 8 of the culture.
When the polyurethane is a polyurethane film, sampling is carried out in the culture process or after the culture is finished, thalli on the polyurethane are removed, cleaning and drying are carried out, the degraded polyurethane is obtained, the degraded polyurethane is dissolved, impurities are removed by filtration, gel chromatography detection is carried out, and the change of the molecular weight of the degraded polyurethane is analyzed; preferably, the sampling during or after the culture is sampling at 4 th and 8 th weeks of culture respectively; preferably, the solvent is dissolved with dimethylformamide.
Wherein in the above process (when the polyurethane is a polyurethane film), the cells on the polyurethane are removed by 2% SDS.
Wherein in the above process (when the polyurethane is a polyurethane film), the cleaning is by distilled water.
Wherein in the process (when the polyurethane is a polyurethane film), the drying is that of the incubator at 37 ℃.
Wherein, when the polyurethane is aqueous polyurethane, sampling is carried out during or after the culture, centrifuging, and the supernatant is subjected to OD 600nm Measuring turbidity; preferably, the sampling is performed every other day during or after the culturing process.
Wherein degradation products can also be used for evaluation; that is, when the polyurethane is a polyurethane film, sampling is performed during or after the completion of the culturing process, centrifuging is performed, and the supernatant is taken and used for High Performance Liquid Chromatography (HPLC) or Mass Spectrometry (MS) detection of degradation products.
When the polyurethane is aqueous polyurethane, sampling is carried out in the culture process or after the culture is finished, centrifuging is carried out, supernatant is dried, and the obtained solid is subjected to infrared spectrum detection; preferably, the sampling during or after the end of the culture is sampling on day 5 of the culture.
In the process (when the polyurethane is aqueous polyurethane), the centrifugation is carried out for 5min at 4000rpm and 4 ℃, so that thalli are gathered at the bottom, and the influence of the thalli on the transparency is reduced.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The invention establishes a system for evaluating polyurethane degradation, namely, for two polyurethane substrates (PU simulants Imprandil DLN and PU film), the degradation condition of the polyurethane substrate is evaluated by means of microbial degradation based on turbidity change, mass loss, surface degradation condition and the like of physical characterization and means of surface functional group change, molecular weight change, degradation product identification and the like of chemical characterization, so that the degradation effect of polyurethane can be completely represented.
(2) The strain P7 adopted by the invention can grow by using the Imprandil DLN as the only carbon source, the degradation rate of the water-based polyurethane Imprandil DLN reaches 95.8% within 7 days, and the strain P7 is a strain with higher PU degradation effect at present. By adopting the evaluation system for degradation of the strain, the evaluation efficiency of the whole system can be effectively improved.
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The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a schematic diagram of a standardized functional characterization system of a plastic degrading bacterium; the Imprandil DLN is taken as a waterborne polyurethane, is a simulant of polyurethane, and can provide guiding significance for the degradation of real PU by researching the degradation characterization method.
FIG. 2 is a characterization of degradation of Imprandil DLN by strain P7: (a) degradation profile of Impranil DLN; (b) Without strain P7, FTIR testing was performed on the Impranil DLN surface after 7 days of treatment; (c) After 7 days of treatment with strain P7, FTIR testing was performed on the Impranil DLN surface.
FIG. 3 characterization method of degradation of PU film by strain P7: (a) Mass loss curve of PU film after 2,4,6,8 weeks of strain P7 treatment; (b) After 2,4 and 6 weeks of treatment of the strain P7, SEM test is carried out on the surface of the PU film; (c) After 8 weeks of treatment with strain P7, FTIR testing was performed on the PU film surface; (d) After 4 and 8 weeks of treatment with strain P7, GPC testing was performed on PU films; (e) HPLC identification of degradation products when strain P7 is treated on the film for 2 weeks; (f) MS identification of degradation products when the strain P7 is treated with the film for 2 weeks.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
In the following examples, the seed solution was prepared by inoculating strain P7 to PDA solid medium, culturing at 30deg.C for 5d, collecting 10mL of sterile water droplets on a plate, scraping mycelia and spores from the surface with a coating rod, counting with a hemocytometer, and diluting to a final concentration of 1×10 6 Bacterial suspension with spore/mL concentration is used as degradation test seed liquid.
Example 1: degradation characterization method of strain P7 on aqueous polyurethane Imprandil DLN:
inoculating seed solution into inorganic salt culture medium with final concentration of 1% (v/v) of Imprandil DLN at 10% v/v, shake culturing at 30deg.C and 120rpm, sampling once every 1d, centrifuging at 4000rpm and 4deg.C for 5min (to reduce the influence of thallus on transparency), and collecting supernatant for OD 600nm Measuring turbidity and calculating degradation rate of DLN; lyophilizing the degraded fermentation supernatant of 5d and the fermentation supernatant of control (without inoculation) with a lyophilizing machine, and lyophilizing to obtain 2500cm solid -1 ～800cm -1 FTIR spectroscopic analysis was performed over the range.
As can be seen from fig. 2a, the control group (no bacteria) showed no signs of degradation, indicating that Impranil DLN did not spontaneously hydrolyze; in the treatment group, the strain P7 is inoculated into the culture medium for 1d, so that the degradation of the Imprandil DLN is started without obvious delay phenomenon; the degradation rate then gradually increased, reaching 95.8% on day seven, and then remained stable.
1735cm in the test sample (FIG. 2 c) compared to the control group (FIG. 2 b) by FTIR spectroscopy -1 Representing an ester carbonyl function, the disappearance of the absorbance peak indicates the hydrolysis of the ester bond; whereas 2938cm -1 Peak at the site representing aliphatic CH 2 The C-H vibration is also significantly reduced. Since the soft segment of the Imprandil DLN contains a large amount of aliphatic CH 2 The decrease in peak intensity may be indicative of chain scission at the soft segment of the polymer, and these characteristic peak changes are in agreement with the literature. Furthermore, no significant change in other peaks indicated that the key chemical bond amide bond in the Impranil DLN was relatively stable and did not degrade. Most of the current literature relates to the degradation mechanism of PU, which occurs in the cleavage of ester bonds in the polyol part, and the current analysis result is consistent with the literature.
Example 2: the degradation characterization method of the strain P7 on the PU film comprises the following steps:
inoculating seed solution into inorganic salt culture medium with final PU concentration of 0.3g/L according to 10% v/v inoculum size, shake culturing at 30deg.C and 120rpm, wherein the control group is not inoculated with bacteria, and culturing conditions are consistent. In the case of the 2 nd, 4 th,sampling for 6 and 8 weeks, removing thalli on the film by using 2% SDS, cleaning by using distilled water, drying in a 37-DEG incubator, weighing by using an analytical balance, and calculating weight loss; cutting out a film for weighing, and performing metal spraying treatment on the surface with proper size for Scanning Electron Microscope (SEM) test; the remaining film was tested by SEM at 2500-800 cm -1 FTIR spectroscopic analysis of the range; dissolving the rest film for FTIR test with dimethylformamide to obtain PU film, filtering to remove impurities, and testing with Gel Permeation Chromatography (GPC); the supernatant was centrifuged at 8000rpm for 2 weeks after incubation and used for HPLC and MS measurements.
After the strain P7 was cultured with the PU film for different weeks, it was found that the quality loss occurred in the control film, probably because the film was not measured by falling off a small amount of film during shake culture, and the quality loss described later was subtracted from the quality loss of the blank. The degradation of the film by the strain P7 reaches 26.99% in the second week; the rate of film degradation was then slowed by 4,6,8 Zhou Junzhu P7 and the mass loss reached a plateau of 35.42% at week 8 (fig. 3 a). SEM analysis is carried out on the surface of the PU film treated by the strain P7 with different weeks, and obvious holes and cracks are formed on the surface of the film in 2 weeks; holes and cracks deepened further at 4 and 6 weeks, which corresponds to the mass loss result (fig. 3 b). 1735cm in PU films after 8 weeks of degradation compared to the control group by FTIR spectroscopic analysis (FIG. 3 c) -1 Representing an ester carbonyl function, a significant decrease in absorbance peak intensity indicates hydrolysis of the ester bond. According to GPC (FIG. 3 d), the weight average molecular weight (Mw) and the number average molecular weight (Mn) of the films in both media were reduced to some extent, mw and Mn were reduced by 11.03% and 5.79% respectively during the period of 4 weeks of degradation, mw and Mn were further reduced by 15.55% and 21.17% respectively during the period of 8 weeks, indicating that degradation of the polymer caused a reduction in molecular weight. The degradation products are analyzed by HPLC, and the peak time of the treatment group is consistent with that of an adipic acid standard sample at 17.2min, so that the degradation products are presumed to be adipic acid; at the same time, by MS analysis, it was identified that the M/z was 145.04 (M+H), respectively + Chemical structure of bound PU and molecule thereofThe stability of each chemical bond, thus identified as adipic acid, further illustrates the hydrolysis of the ester bond that is part of the PU soft segment.
Through a standardized plastic degradation characterization system established based on the physical and chemical characterization means, the P7 is found to have a degradation effect on PU structure simulative Imprandil DLN and a certain degradation effect on real solid PU, lays a solid theoretical foundation for the biodegradation research of PU, and has great potential in the biological treatment of PU or other plastics. The establishment of the standardized polyurethane plastic degradation bacteria function characterization system not only can comprehensively and uniformly evaluate the biodegradation degree of PU, but also provides theoretical basis and reference for the biodegradation characterization method of other microorganisms on plastics, and can greatly promote the biodegradation process of plastics.
The invention provides a system thought and a method for evaluating polyurethane degradation, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Sequence listing
<110> university of Nanjing Industrial science
<120> a System for evaluating polyurethane degradation
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<213> Cladosporium sp)
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Claims (9)
1. A system for evaluating polyurethane degradation is characterized in that seed liquid of polyurethane degrading bacteria is inoculated into an inorganic salt culture medium containing polyurethane for culture, the polyurethane is degraded, and the degradation effect is evaluated by any one or a combination of a plurality of methods of quality loss, electron microscope scanning, infrared spectrum detection, gel chromatography detection, turbidity and degradation product identification; wherein the polyurethane is aqueous polyurethane or polyurethane film;
the polyurethane degrading bacteria are cladosporiumCladosporium sp. the strain is named P7 and is preserved in China Center for Type Culture Collection (CCTCC) NO: m2020627, the preservation date is 11/7 in 2020.
2. The system for evaluating polyurethane degradation according to claim 1, wherein the seed solution of cladosporium is inoculated into an inorganic medium containing aqueous polyurethane or an inorganic medium containing a polyurethane film at a volume ratio of 5% -15%.
3. The system for evaluating polyurethane degradation according to claim 2, wherein the inorganic medium containing the aqueous polyurethane has a content of 0.2% -2% v/v; in the inorganic culture medium containing the polyurethane film, the concentration of the polyurethane film is 3-10 mg/mL.
4. The system for evaluating degradation of polyurethane according to claim 1, wherein when the polyurethane is a polyurethane film, sampling is performed during or after the cultivation, removing the cells on the polyurethane, washing, drying, obtaining the degraded polyurethane, weighing, and calculating the mass loss.
5. The system for evaluating polyurethane degradation according to claim 1, wherein when the polyurethane is a polyurethane film, sampling is performed during or after the cultivation, removing the cells on the polyurethane, washing, drying, obtaining the degraded polyurethane, cutting, spraying gold, and performing electron microscope scanning.
6. The system for evaluating polyurethane degradation according to claim 1, wherein when the polyurethane is a polyurethane film, sampling is performed during or after the cultivation, removing the thalli on the polyurethane, cleaning, drying, obtaining the degraded polyurethane, and performing infrared spectrum detection; when the polyurethane is aqueous polyurethane, sampling is carried out in the culture process or after the culture is finished, centrifuging is carried out, supernatant is dried, and the obtained solid is subjected to infrared spectrum detection.
7. The system for evaluating degradation of polyurethane according to claim 1, wherein when the polyurethane is a polyurethane film, sampling is performed during or after the cultivation, removing the cells on the polyurethane, washing, drying, obtaining the degraded polyurethane, dissolving, filtering, and performing gel chromatography detection.
8. The system for evaluating degradation of polyurethane according to claim 1, wherein when the polyurethane is an aqueous polyurethane, sampling is performed during or after the completion of the cultivation, centrifuging, and subjecting the supernatant to OD 600nm Turbidity was measured.
9. The system for evaluating degradation of polyurethane according to claim 1, wherein when the polyurethane is an aqueous polyurethane, the sample is taken during or after the completion of the culture, centrifuged, and the supernatant is measured by HPLC or MS to identify degradation products.
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| CN109762744A (en) * | 2019-03-01 | 2019-05-17 | 中国科学院昆明植物研究所 | Fungal bacterial strain and its cultural method and purposes for degradable polyurethane plastics |
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| Optical sensing for evaluating the severity of disease caused by Cladosporium sp. in barley under warmer conditions;Dohyeok Oh et al.;《Plant Pathol J》;第236-240页 * |
| 枝孢属的形态分类概述;李旭;《安徽农业科学》;第6254-6255页 * |
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