CN112481137B

CN112481137B - Cladosporium and application thereof in degradation of polyurethane plastic

Info

Publication number: CN112481137B
Application number: CN202011412031.XA
Authority: CN
Inventors: 董维亮; 刘嘉唯; 姜岷; 周杰; 许斌; 徐安明; 刘云龙
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-08-19
Anticipated expiration: 2040-12-04
Also published as: CN112481137A

Abstract

The invention discloses a Cladosporium and application thereof in degrading polyurethane plastics, wherein the Cladosporium is classified and named as Cladosporium (Cladosporum sp.), the strain name is P7, the Cladosporium has been preserved in China center for type culture collection, and the strain collection number is CCTCC NO: m2020627, with a preservation date of 2020, 10, 23. The strain P7 adopted by the invention can grow by using Impranil DLN as a unique carbon source, and the degradation rate of the waterborne polyurethane Impranil DLN in 7 days reaches 95.8%. In addition, the strain has a high degradation effect on polyurethane foam, and 86.6% of polyurethane foam can be degraded within 15 days. Therefore, the invention has extremely important application value for the biological treatment of the plastic waste.

Description

Cladosporium and application thereof in degradation of polyurethane plastic

Technical Field

The invention belongs to the technical field of biological treatment, and relates to cladosporium and application thereof in degradation of polyurethane plastics.

Background

Polyurethane (PU) is a polymer containing a urethane-bond repeating unit structure formed by the condensation of three components, namely polyisocyanate, polyol and a chain extender. It is a semi-crystalline thermosetting plastic in which the isocyanate constitutes the crystalline part, called the hard segment of PU, determining its hardness and tensile strength; the polyol and chain extender constitute the amorphous fraction, called soft blocks of the PU, which determines its elasticity and elongation characteristics. By varying the type and ratio of polyol and isocyanate, PUs of various properties can be produced. Therefore, PU products are more diversified than other plastics, and PU plastics are widely used in heat insulating building materials, packaging materials, automobile materials, synthetic leather, footwear materials, paints, medical materials, etc. due to their excellent mechanical properties. The widespread use of large amounts of PU products generates a lot of waste plastics, and the waste plastics are lack of reasonable recycling treatment, thereby causing serious damage and threat to the ecosystem.

PU has high hydrophobicity and is difficult to degrade under natural conditions, and it is very difficult to directly screen microorganisms or enzymes capable of efficiently degrading PU plastics by using the PU as a substrate. Most studies currently screen PU plastic degrading bacteria using Impranil DLN as a structural mimic. The Impranil DLN is called as a waterborne polyester type polyurethane dispersion, is in a milky white liquid state, is a nano particle with the size of 0.1-0.2 mu m, and can be uniformly dispersed in water. Due to the opaque nature of Impranil DLN, milky white, opaque plates can be made that screen for degrading microorganisms through the hydrolytic rings that are formed by hydrolysis. Currently, some microorganisms have been isolated that have a degrading effect on Impranil DLN: an endophytic fungus (Pestalotiopsis microspora E2712A) isolated from guava stems, which can utilize Impranil DLN as a unique carbon source and can degrade nearly 99% of Impranil DLN within two weeks; the Pseudomonas putida has strong hydrolytic activity to Impranil DLN, and the degradation rate can reach 92% within 4 days. But the microorganisms or enzymes for degrading PU plastics are few in types and still in the mining stage, and the degrading strains screened in the research can lay a good theoretical basis and reference for the research on the biodegradation of the PU plastics.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a cladosporium strain aiming at the defects of the prior art.

The invention also aims to solve the technical problem of providing the application of the cladosporium in degrading polyurethane plastics.

In order to solve the technical problem, the invention discloses a Cladosporium, which is classified and named as Cladosporium sp, has a strain name of P7, is preserved in China Center for Type Culture Collection (CCTCC), and has a strain preservation number of CCTCC NO: m2020627, the preservation date is 10/23/2020, and the preservation address is: wuhan, Wuhan university.

The screening method of the cladosporium is characterized in that an inorganic salt culture medium taking Impranil DLN as a unique carbon source is used as a medium, and strains with polyurethane plastic utilization capability are continuously enriched and domesticated from a refuse landfill, namely PU degrading strains in the refuse landfill soil are separated and screened. Specifically, taking landfill soil as a screening target, continuously enriching and domesticating strains with the utilization capacity of Impranil DLN by using inorganic salt culture media containing different concentrations of Impranil DLN as media, diluting and spreading the inorganic salt culture media on an inorganic salt solid culture medium containing 1% (v/v) of Impranil DLN, culturing at 30 ℃ for 7-14d, streaking and separating the strains with hydrolysis rings to obtain pure culture, and naming the pure culture as a strain P7; culturing the strain P7 in an incubator at 30 ℃ for 5 days to obtain a circular or oval bacterial colony, wherein the bacterial colony has dark villous hyphae on the surface, and the hyphae protrude upwards and have folds, and the edge and the back are black. The ITS sequence of strain P7 was aligned at NCBI and found to have the highest homology to Cladosporium sp and a similarity of up to 99.01% to Cladosporium pini-pondosa (NR _119730.1), and was therefore identified as Cladosporium sp.P7. Wherein the nucleotide sequence of ITS of the cladosporium is shown in SEQ ID No. 1.

A microbial liquid comprising the cladosporium above is also within the scope of the present invention.

The application of the cladosporium or the microbial solution in degrading the waterborne polyurethane (Impranil DLN) is also within the protection scope of the invention.

The application is that the seed liquid of the cladosporium is inoculated into an inorganic salt culture medium containing Impranil DLN and cultured, namely the Impranil DLN can be degraded.

The preparation method of the seed solution comprises inoculating the strain to PDA solid culture medium, culturing at 30 deg.C for 5 days, collecting 10mL sterile water, dripping onto flat plate, scraping surface mycelium and spore with coating rod, counting with blood count plate, and diluting to final concentration of 1 × 10 ⁶ spore/mL bacterial suspension was used as the seed solution for the degradation test.

Wherein the seed liquid of the cladosporium is inoculated in an inorganic salt culture medium containing Impranil DLN at a volume ratio of more than 1%.

Wherein the final concentration of the Impranil DLN in the inorganic salt medium containing the Impranil DLN is 0.25-1.5% v/v.

Wherein the culture is carried out at the temperature of 25-40 ℃ and under the condition of pH 6.0-8.0.

The application of the cladosporium or the microbial solution in the degradation of polyurethane is also within the protection scope of the invention.

Wherein, the application is that the seed liquid of the cladosporium is inoculated in a culture medium containing Polyurethane (PU) for culture, namely the polyurethane can be degraded.

Wherein the seed solution of the cladosporium is inoculated into a culture medium containing polyurethane at a volume ratio of 10%.

Wherein the final concentration of polyurethane in the culture medium containing polyurethane is 4 g/L.

Wherein the culture medium is 50% potato liquid culture medium.

Wherein the culture is carried out at 25-40 ℃ and 120 rpm.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention takes the landfill soil as a separation material, separates and purifies the garbage landfill soil into a microorganism which can efficiently degrade PU simulant Impranil DLN, and has practical significance and reference value for biodegradation of PU and other plastics. The bacterial strain has higher degradation effect on PU simulant Impranil DLN and biased real plastic PU foam, and is suitable for being applied to the biological treatment of PU garbage.

(2) The strain P7 adopted by the invention can grow by using Impranil DLN as a unique carbon source, and the degradation rate of the waterborne polyurethane Impranil DLN in 7 days reaches 95.8%. In addition, the strain has a high degradation effect on polyurethane foam, and 86.6% of polyurethane foam can be degraded within 15 days. Therefore, the invention has extremely important application value for the biological treatment of the plastic waste.

Drawings

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.

FIG. 1 is a standard curve of absorbance for different concentrations of Impranil DLN.

FIG. 2 is a phylogenetic tree of strain P7 and ITS sequences of related species.

FIG. 3 is a degradation curve of strain P7 on Impranil DLN.

FIG. 4 shows the change in extracellular supernatant esterase activity of strain P7 in degrading Impranil DLN.

FIG. 5 is a study of the degradation performance of strain P7 on Impranil DLN: (a) the effect of different temperatures on the degradation of Impranil DLN by strain P7; (b) effect of different pH on degradation of Impranil DLN by strain P7; (c) the effect of different substrate concentrations on the degradation of Impranil DLN by strain P7; (d) effect of different inoculum size on the degradation of Impranil DLN by strain P7.

Fig. 6 is a study of the degradation level of the strain P7 on polyurethane foam: (a) a degradation real object diagram of the foam after the strain P7 degrades the PU foam for 15 d; (b) degradation of PU foam by P7 is plotted as a function of time.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1: separation and screening of high-efficiency degradation Impranil DLN strain Cladosporium sp.P7 and strain identification

A5 g landfill soil sample was placed in 100mL enrichment medium containing 0.2% (v/v) Impranil DLN and cultured at 30 ℃ for 7 days at 180 rpm. Measuring the degradation condition of the enriched liquid on the Impranil DLN by using an ultraviolet spectrophotometer, after determining that the Impranil DLN is degraded, inoculating the degraded solution into an enrichment medium containing 0.4% (v/v) of the Impranil DLN in an inoculation amount of 10% (v/v), continuing to enrich and measuring the degradation condition, and carrying out the method until the concentration of the Impranil DLN is increased to 1% (v/v), and then carrying out passage for 4 times.

The enrichment solution of the Impranil DLN is subjected to gradient dilution and then coated on an inorganic salt flat plate with 1% (v/v) of the Impranil DLN as a unique carbon source, and the Impranil DLN is cultured in an incubator at 30 ℃ for 1-7 days respectively. Single colonies with different colony morphologies are respectively streaked on an LB (LB) plate for purification, inoculated in a liquid inorganic salt culture medium with 0.5% (v/v) Impranil DLN as a unique carbon source, subjected to shake culture at 30 ℃ and 180rpm for 7d, and then centrifuged to collect 1mL of sample, and the content of the Impranil DLN is detected by using an ultraviolet spectrophotometer. OD measurement by UV Spectrophotometer ₆₀₀ The OD of the supernatant of the strain numbered P7 was found to be comparable to the OD of a control shake flask without added bacteria ₆₀₀ The most significant decrease in value.

The inorganic salt culture medium is prepared by adding water into 1.0L of ammonium nitrate, 1.0g of sodium chloride, 0.5g of potassium dihydrogen phosphate and 1.5g of dipotassium hydrogen phosphate, adjusting the pH value to 7.0, adding 20.0g of agar into a solid culture medium, sterilizing at 121 ℃ for 20min, and adding Impranil DLN as a carbon source before use.

The LB medium is prepared from peptone 10.0g, yeast powder 5g, sodium chloride 5.0g, water to 1.0L, adjusting pH to 7.0, adding agar 20.0g into solid medium, and sterilizing at 121 deg.C for 15 min.

The ITS identification was performed on strain P7: using primer ITS 1: 5 '-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5 '-TCCTCCGCTTATTGATATGC-3' amplifying ITS sequence of P7, connecting to a cloning vector pMD19T by means of T/A cloning, constructing a recombinant cloning vector pMD19T-16S, transforming the recombinant cloning vector pMD19T-16S into a cloning host bacterium Escherichia coli DH5 alpha to obtain a recombinant microorganism Escherichia coli DH5 alpha (pMD19T-16S), sequencing the obtained recombinant microorganism exogenous fragment, comparing the ITS sequences by a NCBI database, constructing a phylogenetic tree of the strain P7 and ITS sequences of related species, identifying the strain P7 to Cladosporium on a molecular level (FIG. 2), wherein the nucleotide sequence of ITS is shown as SEQ ID No.1 in the sequence table.

Example 2: degradation curve of Cladosporium sp.P7 to Impranil DLN and change of esterase activity in degradation process

(1) Preparation of standard batches for Impranil DLN at different concentrations: impranil DLN mineral salt media (wherein 1% v/v equals 6g/L DLN) were prepared at concentrations of 0.2, 0.4, 0.6, 0.8, 1.0% (v/v), respectively, at OD _600nm The measurement was carried out, and a standard curve was plotted, as shown in FIG. 1.

The DLN degradation rate calculation is disclosed below:

DLN degradation rate = (initial concentration-final concentration)/initial concentration%

(2) Washing P7 mycelium with sterile water from PDA plate, scraping surface mycelium and spore with coating rod, counting with blood count plate to obtain final concentration of 1 × 10 ⁶ spore/mL concentration of bacterial suspension, i.e. seed liquid.

(3) Inoculating seed liquid into inorganic salt culture medium with 1% (v/v) Impranil DLN concentration, shake culturing at 30 deg.C and 150rpm, sampling once every 24 hr, centrifuging at 4000rpm and 4 deg.C for 5min (collecting thallus at bottom to reduce influence of thallus on transparency), and measuring OD of fermentation supernatant _600nm And the concentration of Impranil DLN was calculated according to the standard curve shown in fig. 1.

As can be seen in fig. 3, the control group (no inoculation) 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, and the degradation of Impranil DLN is started without obvious retardation; the degradation rate then gradually increased, reaching 95.8% on the sixth day, and then remained stable. The extracellular esterase activity of the strain P7 during the degradation process was detected, and the esterase activity was found to be present and reached a maximum of 1.24U/mL at 72h (FIG. 4), so that the esterase was presumed to play a very important role in the degradation process of Impranil DLN.

Example 3: study on degradation performance of Impranil DLN by Cladosporium sp.P7

Will be 1 × 10 ⁶ Spores/mThe seed liquid of the L is inoculated into an inorganic salt culture medium with the final concentration of 1% (v/v) of Impranil DLN in an inoculation amount of 10% (v/v), and is subjected to shake cultivation for 7d at 120rpm at 20 ℃, 25 ℃, 30, 37 and 42 ℃ respectively, the residual quantity of the Impranil DLN is detected, the degradation rate is calculated, and the influence of the temperature on the degradation of the Impranil DLN is studied. As shown in FIG. 5a, the optimal degradation temperature of the strain P7 is 30 ℃, and at the moment, the degradation rate of Impranil DLN is the highest and is 97%; the percentage degradation of strain P7 was 88% and 77% at 25 ℃ and 37 ℃ respectively; the degradation rate of the strain P7 to Impranil DLN was less than 40% at both lower (20 ℃) and higher (42 ℃) temperatures. This indicates that low temperature is not favorable for the strain P7 to degrade Impranil DLN.

Will be 1 × 10 ⁶ The spore/mL seed liquid is inoculated in an inorganic salt culture medium with the initial pH of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 in an inoculation amount of 10% (v/v), wherein the final concentration of the Impranil DLN is 1% (v/v), shaking culture is carried out at 30 ℃ and 120rpm for 7d, the residual quantity of the Impranil DLN is detected, the degradation percentage is calculated, and the influence of the pH on degradation of the Impranil DLN is researched. As shown in FIG. 5b, the optimal degradation pH of strain P7 was 7.0, at which time the degradation rate of Impranil DLN was the highest. The degradation percentage of the Impranil DLN is maintained to be above 70% in the pH range of 6.0-8.0, which indicates that the degradation of the Impranil DLN is not greatly influenced under the conditions of weak acid and weak base. And when the pH is less than 6 or more than 8, the degradation effect is significantly reduced.

Will be 1 × 10 ⁶ The spore/mL seed liquid is respectively inoculated in inorganic salt culture media with final concentrations of Impranil DLN of 0.5, 0.75, 1 and 2% (v/v) in an inoculation amount of 10% (v/v), shaking culture is carried out at 30 ℃ and 120rpm, samples are taken every 24h to detect the residual quantity of Impranil DLN, the degradation rate is calculated, and the influence of the substrate concentration on the degradation of Impranil DLN is researched. As shown in fig. 5c, initial concentrations of Impranil DLN had a significant effect on the degradation of strain P7. When the concentration of Impranil DLN is lower than 1% (v/v), the strain P7 can completely degrade Impranil DLN at 168 h. While the degradation rate of Impranil DLN was only 40.2% when the substrate concentration was 2% (v/v), indicating that the growth of strain P7 was inhibited when the substrate concentration was too high, thereby reducing the degradation effect on Impranil DLN.

Will be 1 × 10 ⁶ Inoculating the spore/mL seed liquid into an inorganic salt culture medium with the final concentration of 1% (v/v) of Impranil DLN at the inoculation amounts of 1% (v/v), 5% (v/v), 10% (v/v) and 20% (v/v), carrying out shake culture at 30 ℃ and 120rpm, sampling every 24h to detect the residual quantity of the Impranil DLN, calculating the degradation rate, and researching the influence of different inoculation amounts on degradation of the Impranil DLN. As shown in FIG. 5d, when the inoculation amount is 5%, 10% or 20%, the degradation rate of the strain P7 to Impranil DLN after 168h can reach more than 97%, and the degradation rate increases with the increase of the inoculation amount. The result shows that the strain P7 can be used for growth and metabolism by taking Impranil DLN as a unique carbon source, and the degradation effect is in direct proportion to the inoculation amount.

Through the experiments, the degradation strain P7 can utilize Impranil DLN as a unique carbon source, and the degradation rate of the Impranil DLN by the strain P7 at 168h can reach 97%.

Example 4: research on PU foam degradation capability of Cladosporium sp.P7

Cutting PU foam into 4 × 2.5 × 0.8cm (about 0.3 g), soaking in 75% ethanol for 10min, oven drying, and ultraviolet sterilizing for 30min before inoculation. Will be 1 × 10 ⁶ The spores/mL of the seed solution was inoculated at 10% (v/v) into a sterilized 50% potato liquid medium containing 0.2g of 50mL PU foam, and cultured with shaking at 30 ℃ and 120 rpm. Samples were taken at 5, 10 and 15 days, and the foam was removed with 2% SDS, rinsed with distilled water, dried at 50 ℃ in an incubator to constant weight and weighed using an analytical balance.

From fig. 6 it can be seen that the control has no significant mass loss in 15d, only 2.8% mass loss in 15 d; however, the strain P7 has a better degradation effect on the foam, and the mass loss reaches 15.23 percent, 40.45 percent and 86.6 percent respectively at 5 days, 10 days and 15 days. Since only the preliminary degradation experiment is explored, only the 15 th d is examined, and the degradation effect of the test sample is studied in the subsequent elongation period. The strain P7 can utilize PU foam, and the degradation rate of the strain P7 at 15d is up to 86.6%. Therefore, the strain can not only utilize waterborne polyurethane Impranil DLN, but also utilize PU foam which is closer to real plastic, lays a solid theoretical foundation for the biodegradation research of PU, and has huge potential in the biological treatment of PU and other plastics.

The invention provides a cladosporium and a method for degrading a polyurethane plastic by using the same, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

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Claims

1. Cladosporium species, its classification name is Cladosporium: (Cladosporiumsp.), the strain name is P7, the strain is preserved in China center for type culture Collection, and the preservation number of the strain is CCTCC NO: m2020627, the preservation date is 10/23/2020.

2. A microbial cell liquid comprising the Cladosporium sp.

3. Use of the cladosporium of claim 1 or the microbial inoculum of claim 2 for degrading aqueous polyurethane.

4. The use of claim 3, wherein the aqueous polyurethane is degraded by inoculating the seed solution of Cladosporium to an inorganic salt culture medium containing aqueous polyurethane at a volume ratio of 1% or more.

5. The use of claim 4, wherein the final concentration of the aqueous polyurethane in the inorganic salt medium containing the aqueous polyurethane is 0.25% to 1.5% v/v.

6. The use according to claim 4, wherein the culturing is carried out at 25-40 ℃ and pH 6.0-8.0.

7. Use of the cladosporium of claim 1 or the microbial broth of claim 2 for degrading polyurethane.

8. The use according to claim 7, wherein the seed solution of Cladosporium species is inoculated to a culture medium containing polyurethane at a volume ratio of 10%, i.e. capable of degrading polyurethane.

9. The use according to claim 8, wherein the final concentration of polyurethane in the polyurethane-containing medium is 4 g/L.

10. The use according to claim 8, wherein the culturing is carried out at 25-40 ℃ and 120 rpm.