CN114410485B - Fungus strain for degrading polyurethane in soil and application thereof - Google Patents
Fungus strain for degrading polyurethane in soil and application thereof Download PDFInfo
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Abstract
The invention discloses a fungus strain for degrading polyurethane in soil and application thereof, and belongs to the technical field of microorganisms. By utilizing the method provided by the invention, the strain obtained by separating and purifying the soil polluted by explosive substances is collected, the culture conditions are optimized, and the soil fungi with degradable polyurethane are screened out, so that the method can be applied to microbial degradation of waste polyurethane materials. The method has simple process, visual effect and reliable result, and can provide reference for degrading other high polymer materials. Can be applied to the microbial degradation of waste polyurethane materials.
Description
Technical Field
The invention belongs to the technical field of microorganisms. The utility model relates to a fungus for degrading polyurethane in soil and application thereof.
Background
Polyurethane is a synthetic polymer formed by reacting polyol and polyisocyanate, and conventionally disposal of waste polyurethane causes environmental pollution. Most fungi in nature realize degradation function by decomposing lignin and soil organic matters which are difficult to digest or consuming monosaccharides, and have higher level of biological degradation enzyme activity, and various enzymes play an important role in the biological degradation of polymers, especially extracellular enzymes degrade polyurethane substrates by cutting ester bonds and actively participate in the biological degradation of polymers. Therefore, it is highly desired to directly separate fungi having the ability to degrade polyurethane from soil, and solve the problem of environmental pollution caused by the disposal of polyurethane waste.
At present, no method for separating fungi with the capability of degrading polyurethane from soil is reported in the prior art.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for degrading polyurethane in soil, which is used for separating, purifying and screening fungi with degrading high molecular materials from the soil and can degrade the polyurethane in the soil. And further analyzing physiological and biochemical process mechanisms in the microbial degradation process of the polyurethane through enzyme activity detection on the basis of obtaining the soil fungi by the method. Can effectively solve the problem of environmental pollution caused by the conventional treatment method of the polymer material waste.
The invention realizes two ideas, and firstly provides a screening method for degrading polyurethane soil fungi. Species names were determined by morphological and molecular biological identification of strains by combining polygenic sequence alignment with phylogenetic tree analysis. And secondly, analyzing physiological and biochemical process mechanisms in the microbial degradation process of polyurethane through enzyme activity detection on the basis of obtaining soil fungi by the method.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
fungus strain for degrading polyurethane in soil, wherein the strain is FusariumFusarium solani) Deposited with the collection of microorganism strains of Guangdong province under the deposit number GDMCC 62182.
The invention also provides application of the fungus strain for degrading polyurethane in soil in degrading polyurethane in soil.
According to the application, the application adopts the following steps:
(1) Optimizing a culture medium: respectively taking SNA+polyurethane, modified PDA+polyurethane, MEA+polyurethane and WA+polyurethane culture medium as matrixes, and measuring the weight change of the polyurethane film after the culture at room temperature for 30 days;
(2) Optimizing the culture temperature: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively culturing for 30 days at 15 ℃,25 ℃ and 30 ℃ as culture conditions, and measuring the change of the weight of the polyurethane film;
(3) Optimizing the pH value condition: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively adjusting the pH value of a culture medium to be 5,6,7 and 8, and culturing in a constant temperature incubator at 30 ℃ for 30 days, and measuring the change of the weight of the polyurethane film;
(4) Degrading polyurethane in soil: the strain of claim 1 for degrading polyurethane, the strain of claim 1 is inoculated into a modified pda+ polyurethane matrix having a PH of 5 and cultured in a constant temperature incubator at 30 ℃ for 30 days, and the change in polyurethane film weight is measured;
the culture medium in the step (4) is modified potato dextrose agar, the culture temperature is 30 ℃, the pH value of the culture medium is 5, and the formula of the modified potato dextrose agar culture medium is as follows: 150g of potato, 20g of glucose, 20g of agar, 1000mL of water and pH value of 5.
The invention further provides a method for degrading polyurethane in soil, which comprises the following steps: collecting soil polluted by explosive substances, separating and purifying to obtain the fungus strain with the capability of degrading polyurethane, optimizing culture conditions, screening out soil fungus with the capability of degrading polyurethane, and using the fungus strain to degrade polyurethane in the soil.
The method for degrading polyurethane in soil comprises the following steps:
(1) Sampling:
collecting soil samples polluted by explosive substances, taking soil 5cm deep from a surface layer by using a disinfection auger, collecting 5 samples at each test point, placing the collected soil samples into a disinfection bag, thoroughly mixing, conveying to a laboratory, and storing in a refrigerator at 4 ℃;
(2) Fungal isolation:
1g of the soil sample was taken and shaken in 9mL of sterile water for 1 hour, and then centrifuged at 2000 rpm for 10 minutes, and the supernatant was serially diluted under sterile operating conditions, namely: suction 10 -1 1mL of the fungus solution is placed into a centrifuge tube filled with 9mL of sterile water, and the concentration is 10 after shaking evenly -2 In the same way, the fungal solutions are diluted 4 times in turn and marked as 10 respectively -1 、10 -2 、10 -3 、10 -4 Finally, sucking 20 mu L of diluent with a pipette, placing in a culture dish containing Potato Dextrose Agar (PDA), uniformly smearing the diluent on the surface of the culture dish by using a sterile glass rod, inverting the culture dish, culturing in a dark incubator at 28 ℃ for 3-5 days, transferring the cultured fungus colony into a new PDA culture dish for 3 days, and performing secondary culture and purification again to obtain a pure culture, namely a strain H14;
(3) Screening of degradation strains:
selecting a fungus culture with a diameter of about 2mm by using a disinfection needle, placing the fungus culture in a malt extract agar Medium (MEA), covering a polyurethane film with a diameter of 80mm on the surface of the MEA culture medium, culturing the culture in a sterile culture room at room temperature (26+/-2 ℃) for 30 days in parallel 3 times, collecting the polyurethane film, thoroughly cleaning the polyurethane film by using distilled water, weighing the polyurethane film after drying in the shade, and calculating the weight loss condition of the polyurethane film according to the collected data, wherein the result shows that in the fungus separation step in the step (2), the obtained strain H14 has the highest degradation rate on the polyurethane film;
degrading polyurethane in soil:
optimizing a culture medium: respectively taking SNA+polyurethane, modified PDA+polyurethane, MEA+polyurethane and WA+polyurethane culture medium as matrixes, and measuring the weight change of the polyurethane film after the culture at room temperature for 30 days;
optimizing the culture temperature: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively culturing for 30 days at 15 ℃,25 ℃ and 30 ℃ as culture conditions, and measuring the change of the weight of the polyurethane film;
optimizing the pH value condition: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively adjusting the pH value of a culture medium to be 5,6,7 and 8, and culturing in a constant temperature incubator at 30 ℃ for 30 days, and measuring the change of the weight of the polyurethane film;
degrading polyurethane in soil: the strain of claim 1 for degrading polyurethane. The strain of claim 1 was inoculated into a modified pda+ polyurethane matrix at PH 5 and incubated in a constant temperature incubator at 30 ℃ for 30 days to measure the change in polyurethane film weight.
According to the method for degrading polyurethane in soil, in the step (4) of degrading polyurethane in soil, the culture medium is modified potato dextrose agar, the culture temperature is 30 ℃, and the pH value of the culture medium is 5.
According to the method for degrading polyurethane in soil, in the step (4) of degrading polyurethane in soil, the formula of the modified potato dextrose agar medium is as follows: 150g of potato, 20g of glucose, 20g of agar, 1000mL of water and pH value of 5.
In addition, the invention provides a method for degrading polyurethane in soil, which applies the strain of claim 1 to degrade polyurethane in soil, comprising the following steps:
(1) Optimizing a culture medium: respectively taking SNA+polyurethane, modified PDA+polyurethane, MEA+polyurethane and WA+polyurethane culture medium as matrixes, and measuring the weight change of the polyurethane film after the culture at room temperature for 30 days;
(2) Optimizing the culture temperature: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively culturing for 30 days at 15 ℃,25 ℃ and 30 ℃ as culture conditions, and measuring the change of the weight of the polyurethane film;
(3) Optimizing the pH value condition: inoculating the strain of claim 1 into a modified PDA+polyurethane matrix, respectively adjusting the pH value of a culture medium to be 5,6,7 and 8, and culturing in a constant temperature incubator at 30 ℃ for 30 days, and measuring the change of the weight of the polyurethane film;
(4) Degrading polyurethane in soil: the strain of claim 1 for degrading polyurethane, the strain of claim 1 is inoculated into a modified pda+ polyurethane matrix having a PH of 5 and cultured in a constant temperature incubator at 30 ℃ for 30 days, and the change in polyurethane film weight is measured;
the culture medium in the step is modified potato dextrose agar, the culture temperature is 30 ℃, and the pH value of the culture medium is 5;
in the step (4) of degrading polyurethane in soil, the formula of the modified potato dextrose agar medium is as follows: 150g of potato, 20g of glucose, 20g of agar, 1000mL of water and pH value of 5.
Compared with the prior art, the invention has the following advantages:
1. the invention provides fungus strains with the capability of degrading polyurethane from soil, which are not reported in the prior art, and a method thereof for the first time, and fills up the technical blank.
2. The separation method is simple and feasible, convenient to operate, high in yield, environment-friendly and safe, and high in feasibility.
3. The fungus with the capability of degrading polyurethane obtained by the method has the capability of degrading polyurethane in soil, the strain is inoculated into an improved PDA+polyurethane matrix with the pH value of 5 and is placed in a constant temperature incubator with the temperature of 30 ℃ for 30 days, the degradation rate of polyurethane reaches 25.8 percent, and the method has great superiority and benefits as follows!
4. The method can effectively solve the problem of environmental pollution caused by the conventional treatment method of the high polymer material waste.
Drawings
FIG. 1 shows the strain H14 of the present inventionFusarium solani) In the figure, strains a-d are on PDA medium; e-g hyphae; h-k spore-forming cells and conidia; l-q conidia. Scale bar e ‒ k=20 μm, l ‒ q=15 μm;
FIG. 2 is a schematic diagram based on LSU, ITS,tef1-alpha andrpb2, performing Bayesian inference analysis (Bayesian Inference analysis) on the generated phylogenetic tree. In the figure, the support value of the maximum likelihood (maximum likelihood) is equal to or greater than 80%, the Bayesian posterior probability (Bayesian posterior probabilities) is greater than or equal to 0.95, and the numerical value is placed above the node. Strain H14 of the present inventionFusariumsolani) Indicated in bold.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection scope of the present invention is not limited to the following embodiments. The following specific examples further illustrate the gist of the present invention, but are not intended to limit the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conditions conventional in the art or as recommended by the manufacturer.
Example 1
1. The screening method of the fungus strain comprises the following steps:
the fungus strain of the invention is screened by adopting a dilution coating flat plate method. 1g of the soil sample was taken and shaken in 9mL of sterile water for 1 hour, and then centrifuged at 2000 rpm for 10 minutes, and the supernatant was serially diluted under sterile operating conditions, namely: suction 10 -1 1mL of the fungus solution is placed into a centrifuge tube filled with 9mL of sterile water, and the concentration is 10 after shaking evenly -2 In the same way, the fungal solutions are diluted 4 times in turn and marked as 10 respectively -1 、10 -2 、10 -3 、10 -4 Finally, 20 mu L of diluent with various concentrations is sucked by a pipette, placed in a culture dish containing potato dextrose agar culture medium (PDA), uniformly smeared on the surface of the culture dish by a sterile glass rod, the culture dish is inverted to be cultured in a dark incubator at 28 ℃ for 3-5 days, the cultured fungus colony is transferred to a new PDA culture dish to be cultured for 3 days, and then the culture dish is subjected to secondary culture and purification to obtain a pure culture, namely the strain H14 of the inventionFusarium solani）。
Selecting a fungus pure culture with a diameter of about 2mm by using a disinfection needle, placing the fungus pure culture in a malt extract agar Medium (MEA), covering a polyurethane film with a diameter of 80mm on the surface of the MEA medium, culturing the culture in a sterile culture room at room temperature (26+/-2 ℃) for 30 days in parallel 3 repetitions, collecting the polyurethane film, thoroughly cleaning the polyurethane film by using distilled water, weighing the polyurethane film after drying in the shade, calculating the weight loss condition of the polyurethane film according to the collected data, and displaying that the obtained strain H14 has the highest degradation rate on the polyurethane film in the step of obtaining the pure culture by fungus separation;
2. identification of the fungal strains of the invention.
The fungus strain identification of the invention adopts a method combining morphological identification and molecular identification.
2.1 morphological identification. Strain H14 was inoculated on PDA medium and cultured in a dark incubator at 28 ℃ for 30 days, and species identification was performed based on the morphology, color, size and morphological characteristics of the spore-forming cells and conidia produced by the mycelium on the medium. The morphological characteristics of the strain are as follows: the mycelium is white, dense, radial, circular, conical in center, smooth in texture, and orange-yellow in reverse side. The spore-producing cells are single cells and are placed at the top of hypha and transparent. Conidia of 14-25×3.5-6 (x ̅ =18.6×5.1, n=30) μm in size, acronymous, elliptic or kidney-shaped, colorless, upright or curved with 2-3 transverse septa, see fig. 1. FIG. 1 shows the strain H14 of the present inventionFusarium solani) A-d strains on PDA medium; e-g hyphae; h-k spore-forming cells and conidia; l-q conidia. Scale bar e ‒ k=20 μm, l ‒ q=15 μm.
2.2 molecular identification. A small amount of mycelia was taken and DNA was extracted using Biospin fungal genomic DNA extraction kit (BioFlux). Using the genes ITS (ITS 4/ITS 5), LSU (LR 0R/LR 5),tef1-alpha (983F/2218R) andrpb2 (fRPB 2-5F/fRPB2-7 cR) and sequencing. The related sequences are downloaded from GenBank (http:// www.ncbi.nlm.nih.gov /), data are processed by BioEdit7.2.3, MAFFTv.7.110 and other software, and Maximum Likelihood (ML) and Bayesian posterior probabilities (BYPP) analysis is carried out by combining multiple genes, so that a species phylogenetic tree is constructed, and the strain H14 is seen from the phylogenetic treeFusarium solaniSee fig. 2. FIG. 2 is based on LSU, ITS,tef1-alpha andrpb2, performing Bayesian inference analysis (Bayesian Inference analysis) on the generated phylogenetic tree. The support value of the maximum likelihood (maximum likelihood) is equal to or greater than 80%, the Bayesian posterior probability (Bayesian posterior probabilities) is greater than or equal to 0.95, and the numerical value is placed above the node. Strain H14 of the present inventionFusariumsolani) Indicated in bold.
3. The preservation condition, time, place, unit and number of the strain.
H14（Fusarium solani) Deposited under the accession number GDMCC 62182 in the Guangdong province microorganism strain deposit on 1 and 4 of 2022.
Any mutant of the strain is within the scope of protection of the present patent, given that the strain may be altered or engineered due to mutation (spontaneous, physical or chemical), protoplast fusion, transformation or otherwise by biotechnology.
Example 2
A method for degrading polyurethane in soil. The method comprises the following steps of:
(1) Sampling
Soil samples contaminated with explosive substances were collected in the middle of china. Soil collection soil was taken 5cm deep from the surface using a sterile auger and 5 samples were taken at each test point. The collected soil samples were placed in a sterilization bag, thoroughly mixed, and transported to a laboratory, and stored in a refrigerator at 4 ℃.
(2) Fungal isolation
1g of the soil sample was taken and shaken in 9mL of sterile water for 1 hour, and then centrifuged at 2000 rpm for 10 minutes, and the supernatant was serially diluted under sterile operating conditions, namely: suction 10 -1 1mL of the fungus solution is placed into a centrifuge tube filled with 9mL of sterile water, and the concentration is 10 after shaking evenly -2 In the same way, the fungal solutions are diluted 4 times in turn and marked as 10 respectively -1 、10 -2 、10 -3 、10 -4 Finally, 20 mu L of the diluted solution with each concentration is sucked by a pipette, placed in a culture dish containing potato dextrose agar medium (PDA), and uniformly smeared on the surface of the culture dish by a sterile glass rod. The plates were inverted and incubated in a dark incubator at 28℃for 3-5 days. Transferring the cultured fungus colony into a new PDA culture dish, culturing for 3 days, and performing secondary culture and purification to obtain a pure culture, namely the strain H14 of the inventionFusarium solani）。
(3) Screening of degradation strains
Medicine for treating common coldThe toxin needle picks up approximately 2mm of the fungal culture and places it in Malt Extract Agar (MEA) and a polyurethane film 80mm in diameter is applied to the MEA surface (3 replicates). The cultures were incubated in a sterile incubator at room temperature (26.+ -. 2 ℃) for 30 days. The polyurethane film is collected, thoroughly washed with distilled water, dried in the shade and weighed. The results showed that in the step (2) of fungal isolation, strain H14 was obtainedFusarium solani) The degradation rate of the polyurethane reaches 22.1 percent.
(4) Degradation of polyurethane in soil
Optimization of the culture medium. The change in polyurethane film weight WAs measured after 30 days incubation at room temperature using sna+polyurethane, modified pda+polyurethane, mea+polyurethane, wa+polyurethane medium as substrate, respectively. The result shows that the improved PDA culture medium has the greatest influence on the degradation rate of polyurethane, and the degradation rate can reach 12.6%.
Optimization of the culture temperature. Strain H14%Fusarium solani) Inoculating into modified PDA+polyurethane matrix, culturing at 15deg.C, 25deg.C and 30deg.C for 30 days, and measuring polyurethane film weight change. The results show that the degradation rate of polyurethane is highest and can reach 19.1% at 30 ℃.
Optimization of pH conditions. Strain H14%Fusarium solani) Inoculating in modified PDA+polyurethane matrix, regulating pH value of culture medium to 5,6,7,8, and culturing in 30 deg.C incubator for 30 days to obtain the final product with highest polyurethane degrading rate up to 25.8%.
Degrading polyurethane in soil.Fusarium solani(the strains screened for polyurethane degradation) the optimal laboratory conditions for polyurethane degradation are: modified potato dextrose agar medium (pH 5) at 30 ℃. The formula of the modified potato dextrose agar medium is as follows: 150g of potato, 20g of glucose, 20g of agar, 1000mL of water and pH value of 5.
The fungus with the capability of degrading polyurethane obtained by the method has the capability of degrading polyurethane in soil, and the strain H14 is used for preparing the microbial inoculumFusarium solani) Inoculated into a modified PDA+polyurethane matrix with a pH of 5 and placed in 3The degradation rate of polyurethane reaches 25.8% after culturing in a constant temperature incubator at 0 ℃ for 30 days, which proves that the method has great benefit.
Claims (2)
1. A fungus strain for degrading polyurethane in soil is characterized in that the strain is fusariumFusarium solani) Deposited with the collection of microorganism strains of Guangdong province under the deposit number GDMCC 62182.
2. Use of a fungal strain for degrading polyurethane in soil according to claim 1 for degrading polyurethane in soil.
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