CN117645930A - Application of rhodopenicillium rubrum in preparation of urea-formaldehyde plastic degradation biological agent - Google Patents
Application of rhodopenicillium rubrum in preparation of urea-formaldehyde plastic degradation biological agent Download PDFInfo
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- CN117645930A CN117645930A CN202311371496.9A CN202311371496A CN117645930A CN 117645930 A CN117645930 A CN 117645930A CN 202311371496 A CN202311371496 A CN 202311371496A CN 117645930 A CN117645930 A CN 117645930A
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- 229920001807 Urea-formaldehyde Polymers 0.000 title claims abstract description 129
- ODGAOXROABLFNM-UHFFFAOYSA-N polynoxylin Chemical compound O=C.NC(N)=O ODGAOXROABLFNM-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 229920003023 plastic Polymers 0.000 title claims abstract description 89
- 239000004033 plastic Substances 0.000 title claims abstract description 89
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 61
- 230000015556 catabolic process Effects 0.000 title claims abstract description 59
- 239000003124 biologic agent Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 title abstract description 5
- 241000228143 Penicillium Species 0.000 claims abstract description 15
- 241001280501 Penicillium rubens Species 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 22
- 239000012153 distilled water Substances 0.000 claims description 20
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 18
- 239000002861 polymer material Substances 0.000 claims description 18
- 229940088594 vitamin Drugs 0.000 claims description 18
- 235000013343 vitamin Nutrition 0.000 claims description 18
- 239000011782 vitamin Substances 0.000 claims description 18
- 229930003231 vitamin Natural products 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 239000012452 mother liquor Substances 0.000 claims description 14
- 239000002689 soil Substances 0.000 claims description 13
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000002994 raw material Substances 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 239000010413 mother solution Substances 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- -1 compound vitamin Chemical class 0.000 claims description 9
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 8
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 8
- 239000002351 wastewater Substances 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000011734 sodium Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 6
- 241000606161 Chlamydia Species 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 241000195597 Chlamydomonas reinhardtii Species 0.000 claims description 4
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 4
- 241000233866 Fungi Species 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 229930003270 Vitamin B Natural products 0.000 claims description 4
- 229960004050 aminobenzoic acid Drugs 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 238000001125 extrusion Methods 0.000 claims description 4
- 239000011790 ferrous sulphate Substances 0.000 claims description 4
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 4
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229960003512 nicotinic acid Drugs 0.000 claims description 4
- 235000001968 nicotinic acid Nutrition 0.000 claims description 4
- 239000011664 nicotinic acid Substances 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 4
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 4
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 4
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 235000010344 sodium nitrate Nutrition 0.000 claims description 4
- 239000004317 sodium nitrate Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 235000019156 vitamin B Nutrition 0.000 claims description 4
- 239000011720 vitamin B Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000000593 degrading effect Effects 0.000 abstract description 11
- 241000894006 Bacteria Species 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 6
- 230000004580 weight loss Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241001540751 Talaromyces ruber Species 0.000 description 4
- 238000002441 X-ray diffraction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000006065 biodegradation reaction Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000007773 growth pattern Effects 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229920000426 Microplastic Polymers 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 244000007853 Sarothamnus scoparius Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- MGJURKDLIJVDEO-UHFFFAOYSA-N formaldehyde;hydrate Chemical compound O.O=C MGJURKDLIJVDEO-UHFFFAOYSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000011810 insulating material Substances 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229920001187 thermosetting polymer Polymers 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- Y—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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Abstract
The invention provides application of rhodopenicillium rubrum in preparation of urea-formaldehyde plastic degradation biological agents, and belongs to the technical field of application of bacteria. In particular to application of Penicillium erythrorhizon (Penicillium rubens) PSRF-507 in preparing urea-formaldehyde plastic degradation biological agents. The invention discovers that the rhodopenicillium rubrum (Penicillium rubens) PSRF-507 has the function of degrading macromolecular urea-formaldehyde plastics for the first time, has the degradation performance on a heat-resistant and acid-resistant macromolecular main chain in urea-formaldehyde resin, contains urea-formaldehyde plastics degrading enzyme in fermentation liquor, can be applied to biological treatment of urea-formaldehyde plastics, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of application of strains, and particularly relates to application of penicillium erythrotoxidanum in degradation of urea-formaldehyde plastics.
Background
Urea-formaldehyde plastic is a plastic with urea-formaldehyde resin as basic component, and is a thermosetting plastic with smooth and hard surface, bright colour, arc-resistance, flame-resistance and good electric insulating property, and can be extensively used for making various electric appliances, mechanical parts, fittings of electric and instrument industries, various handles and ornaments, religious articles, various buttons and bottle caps, etc. and also can be used for making sound-insulating and heat-insulating materials of vehicles, ships and buildings, etc.
The urea-formaldehyde plastic is mass produced, applied and abandoned in a short time, which can cause white pollution and cause huge pressure to the environment. Urea-formaldehyde plastics are easy to form garbage bands in soil and marine environments, not only attack living habitats, but also the pollution of micro plastics to the environment is increasingly serious.
In the prior art, researchers find out microorganisms and degrading enzymes of degradable PET foam plastics for degradation research of plastic pollution, but have few reports on biodegradation of urea-formaldehyde plastics.
Disclosure of Invention
The invention provides application of Penicillium erythropoieticum in degradation of urea-formaldehyde plastics, which can effectively realize degradation of urea-formaldehyde plastics.
The invention provides application of Penicillium erythrorhizon (Penicillium rubens) PSRF-507 in preparation of urea-formaldehyde plastic degradation biological agents.
Further, the penicillium erythrotovorum (Penicillium rubens) PSRF-507 is preserved in China general microbiological culture Collection center (CGMCC) at the year 2021, month 09 and 13, and has a strain preservation number of CGMCC No.23229.
Further, the urea-formaldehyde plastic degradation biological agent is prepared by the following steps:
(1) Inoculating Penicillium erythroproducing fungus (Penicillium rubens) PSRF-507 into a Chlamydomonas reinhardtii culture medium, and performing first culture to obtain an activated strain;
(2) Inoculating the activated strain into a 1# urea formaldehyde culture medium, and performing secondary culture to obtain bacterial liquid;
(3) Transferring the bacterial liquid into a 2# urea formaldehyde culture medium liquid fermentation medium according to the volume ratio of 1-15%, and performing third culture to obtain a fermentation liquid;
(4) Centrifuging the fermentation liquor, taking supernatant, and concentrating in vacuum to half of the original volume to obtain the concentrated urea-formaldehyde plastic degradation biological agent.
Further, in the step (1), the first culture is shaking culture at 100-300 rpm, and the culture is carried out for 3-8 d at 20-30 ℃;
in the step (1), the Chlamydia medium is prepared from the following raw materials: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate and MgSO 4 ·7H 2 0.5g of O, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and distilled water to 1000mL.
Further, in the step (2), the second culture is performed for 10-30 days at 20-30 ℃ and 100-300 rpm;
in the step (2), the 1# urea formaldehyde culture medium is prepared from the following raw materials: 1-5g of urea formaldehyde polymer material, 5-20g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.05-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
Further, the preparation method of the inorganic salt mother solution comprises the following steps:
weighing FeCl 2 ·4H 2 O 0.2-1.8g，CoCl 2 ·6H 2 O 0.1-0.25g，NiCl 2 ·6H 2 O 0.01g，CuCl 2 ·2H 2 O 0.01g，MnCl 2 ·4H 2 O 0.1-0.70g，ZnCl 2 0.1g，H 3 BO 3 0.1-0.5g，Na 2 MoO 4 ·2H 2 O 0.01-0.03g，Na 2 SeO 3 ·5H 2 0-0.01g of O, pouring the mixture into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4 ℃ for later use;
the preparation method of the vitamin complex mother solution comprises the following steps:
weighing 0.01-0.1g of biotin, 0.05-0.2g of p-aminobenzoic acid, 0.1-0.4g of nicotinic acid, 0.05g of vitamin B and 0.1g of pyridoxine hydrochloride respectively by using an electronic day, pouring into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4deg.C for use.
Further, in the step (3), the third culture is carried out at a temperature of 20-30 ℃ and at a speed of 100-300 rpm for 10-30 hours;
in the step (3), the 2# urea formaldehyde culture medium is prepared from the following raw materials:
1-15g of urea formaldehyde polymer material, 0-10g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.5-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
Further, in the step (2) and the step (3), the preparation method of the urea formaldehyde polymer material comprises the following steps:
uniformly mixing urea and formaldehyde according to a molar ratio of 1:1.5-4, and adding 0.3-1 mol/L sodium hydroxide to adjust the pH value of a reaction system to 8-10;
heating the reaction system to 50 ℃ and reacting for 1-3 h, extruding the obtained reactant from extrusion equipment to obtain a strip-shaped object, and drying to constant weight to obtain the urea formaldehyde polymer material;
the molecular formula of the obtained urea formaldehyde polymer material is as follows:
wherein m=1 to 8, n=1 to 10, and m=1 to 1000.
Further, the application method of the urea-formaldehyde plastic degradation biological agent comprises the following steps:
the urea-formaldehyde plastic degradation biological agent is added into the waste water containing urea-formaldehyde plastic or the soil polluted by the urea-formaldehyde plastic to degrade.
Further, in the wastewater containing urea-formaldehyde plastics, the addition amount of the urea-formaldehyde plastics degradation biological agent is 3% -15%;
in the soil polluted by the urea-formaldehyde plastics, the addition amount of the urea-formaldehyde plastics degradation biological agent is 5-20%.
The invention has the following advantages:
the invention discovers that the rhodopenicillium rubrum (Penicillium rubens) PSRF-507 has the function of degrading macromolecular urea-formaldehyde plastics for the first time, has the degradation performance on a heat-resistant and acid-resistant macromolecular main chain in urea-formaldehyde resin, contains urea-formaldehyde plastics degrading enzyme in fermentation liquor, can be applied to biological treatment of urea-formaldehyde plastics, and has wide application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a diagram (400-fold) showing the morphology of Penicillium rubrum in example 1 of the present invention;
FIG. 2 shows colony morphology of 20d strain grown on Chlamydia medium in example 1 of the present invention;
FIG. 3 shows the growth pattern of the strain of example 1 of the present invention on 2# urea formaldehyde broth for 20 d;
FIG. 4 is a weight loss diagram of the degradation agent in test example 1 of the present invention for degradation of urea formaldehyde plastics;
FIG. 5 is a LCMS analysis of intermediate metabolites degraded before and after degradation in test example 2 of the present invention;
FIG. 6 is an X-ray diffraction pattern of urea-formaldehyde polymer according to the different treatment modes in test example 3 of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The urea-formaldehyde plastic high-efficiency degradation bacteria are an important link for starting the biodegradation of the urea-formaldehyde plastic. By utilizing the microbial degradation effect, urea-formaldehyde plastics in soil or sea can be degraded gradually, and each component in the urea-formaldehyde plastics is used as a nitrogen source, a carbon source and the like of microorganisms for absorption and utilization, so that the harm to the environment is reduced.
The structural formula of the urea-formaldehyde plastic is shown as formula I. The urea-formaldehyde plastic is doped with adhesive and the like in the reaction process of the urea-formaldehyde plastic, and the urea-formaldehyde plastic without phosphate groups has no charge-dense parts and is difficult to degrade.
The inventor discovers that activating and screening urea-formaldehyde plastic degrading microorganisms has important significance for bioremediation of soil or water body polluted by the urea-formaldehyde plastic. At present, no biological strain related to urea-formaldehyde plastic degradation is reported.
An embodiment of the invention provides application of Penicillium erythrorhizon (Penicillium rubens) PSRF-507 in preparation of urea-formaldehyde plastic degradation biological agents.
Penicillium rubrum is a microorganism of Penicillium genus, and the colony has a velvet shape; the edge is pure round; the bacterial colony is blue and green; the surface is provided with regular radial grooves; there is a large amount of yellow exudates. Conidiophores occur in the matrix, the stems are separated, the diameter is 2.0-3.8 mu m, and the wall is smooth; two-wheel or three-wheel broom-shaped branches, even four-wheel broom, usually have 1-2 auxiliary branches, 9.0-20×2.5-3.5 μm; 2-4 stems per round, 8.0-13×2.0-3.0 μm; 3-6 bottle stems per round, needle shape, 5.5-9.5X1.5-2.3 μm; the conidium is nearly spherical or elliptic, 2.3-3.8X2.2-3.3 μm, and the wall is smooth.
In the embodiment of the invention, the penicillium erythrotoxidanum (Penicillium rubens) PSRF-507 is preserved in China general microbiological culture Collection center (CGMCC) at the year 2021, month 09 and 13, and the address is CGMCC No.23229 of China academy of sciences of national academy of sciences of China, north Chen, west Lu No. 1, the Korean region of Beijing.
The penicillium rubrum provided by the embodiment of the invention is a degrading bacterium screened from potted soil samples obtained by continuously applying modified urea formaldehyde PSRF fertilizer (fertilized 1 time per year) from North university for 2 years. Firstly, PSRF is used as a unique carbon source and a unique nitrogen source to design a screening culture medium, a strain of PSRF degrading bacteria is directionally screened and separated from 16 samples, and the strain is identified as penicillium erythropoieticum through morphology and ITS molecular biology; the strain preservation is carried out in China general microbiological culture collection center (CGMCC) with a strain number of Penicillium rubens 23229. The strain has been proved to degrade heat-resistant urea formaldehyde polymer fertilizer containing phosphorus and potassium elements.
In one embodiment of the invention, the urea-formaldehyde plastic degradation biological agent is prepared by the following steps:
(1) Inoculating Penicillium erythroproducing fungus (Penicillium rubens) PSRF-507 into a Chlamydomonas reinhardtii culture medium, and performing first culture to obtain an activated strain;
(2) Inoculating the activated strain into a 1# urea formaldehyde culture medium, and performing secondary culture to obtain bacterial liquid;
(3) Transferring the bacterial liquid into a 2# urea formaldehyde culture medium liquid fermentation medium according to the volume ratio of 1-15%, and performing third culture to obtain a fermentation liquid;
(4) Centrifuging the fermentation liquor, taking supernatant, and concentrating in vacuum to half of the original volume to obtain the concentrated urea-formaldehyde plastic degradation biological agent.
Further, in the step (1), the first culture is shaking culture at 100 to 300rpm, and the culture is performed at 20 to 30 ℃ for 3 to 8 days.
Further, in the step (1), the preparation method comprises the following steps: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate and MgSO 4 ·7H 2 0.5g of O, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and distilled water to 1000mL.
Further, in the step (2), the second culture is a shaking culture at 100-300 rpm and 20-30 ℃ for 10-30 d.
Further, in the step (2), the 1# urea formaldehyde culture medium is prepared from the following raw materials: 1-5g of urea formaldehyde polymer material, 5-20g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.05-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
Specifically, the preparation method of the inorganic salt mother solution comprises the following steps:
weighing FeCl 2 ·4H 2 O 0.2-1.8g，CoCl 2 ·6H 2 O 0.1-0.25g，NiCl 2 ·6H 2 O 0.01g，CuCl 2 ·2H 2 O 0.01g，MnCl 2 ·4H 2 O 0.1-0.70g，ZnCl 2 0.1g，H 3 BO 3 0.1-0.5g，Na 2 MoO 4 ·2H 2 O 0.01-0.03g，Na 2 SeO 3 ·5H 2 0-0.01g of O, pouring the mixture into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4deg.C for use.
Specifically, the preparation method of the vitamin complex mother solution comprises the following steps:
weighing 0.01-0.1g of biotin, 0.05-0.2g of p-aminobenzoic acid, 0.1-0.4g of nicotinic acid, 0.05g of vitamin B and 0.1g of pyridoxine hydrochloride respectively by using an electronic day, pouring into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4deg.C for use.
Further, in the step (3), the third culture is performed at a temperature of 20 to 30℃and at a speed of 100 to 300rpm for 10 to 30 hours.
Further, in the step (3), the 2# urea formaldehyde culture medium is prepared from the following raw materials:
1-15g of urea formaldehyde polymer material, 0-10g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.5-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
Specifically, in the step (2) and the step (3), the preparation method of the urea formaldehyde polymer material comprises the following steps:
uniformly mixing urea and formaldehyde according to a molar ratio of 1:1.5-4, and adding 0.3-1 mol/L sodium hydroxide to adjust the pH value of a reaction system to 8-10;
and (3) heating the reaction system to 50 ℃ and reacting for 1-3 hours, extruding the obtained reactant from extrusion equipment to obtain a strip-shaped substance, and drying the strip-shaped substance to constant weight to obtain the urea formaldehyde polymer material.
The molecular formula of the obtained urea formaldehyde polymer material is as follows:
wherein m=1 to 8, n=1 to 10, and m=1 to 1000.
Further, in the step (4), the centrifugation time is 1min.
In the step (4) of the embodiment of the invention, the supernatant comprises urea formaldehyde degrading bacteria, spores, degrading enzymes and the like.
The embodiment of the invention provides an application method of urea-formaldehyde plastic degradation biological agent, which comprises the following steps:
the urea-formaldehyde plastic degradation biological agent is added into the waste water containing urea-formaldehyde plastic or the soil polluted by the urea-formaldehyde plastic to degrade.
Specifically, in the wastewater containing urea-formaldehyde plastics, the addition amount of the urea-formaldehyde plastics degradation biological agent is 3% -15%; preferably, the addition amount of the urea-formaldehyde plastic degradation biological agent in the wastewater containing the urea-formaldehyde plastic is 8%. The addition amount of the inorganic salt and the vitamin adjuvant is 0-5 percent.
Specifically, in the soil polluted by the urea-formaldehyde plastics, the addition amount of the urea-formaldehyde plastics degradation biological agent is 5-20%. The addition amount of the inorganic salt and the vitamin adjuvant is 0-3 percent.
Further, the degradation time is 10 to 200 days. The degradation temperature is 25-35 ℃.
For example, in practical application, urea-formaldehyde plastic degradation biological agent is added into waste water containing urea-formaldehyde plastic, and inorganic salt and vitamin adjuvant are added at the same time, and degradation is carried out for 15-150 days. Thus, the urea formaldehyde plastic can be degraded by 10 to 50 percent.
Wherein the addition amount of the urea-formaldehyde plastic degradation biological agent is 3-15%. The addition amount of the inorganic salt and the vitamin adjuvant is 0-5 percent.
For example, in practical application, urea-formaldehyde plastic degradation biological agent is added into the polluted soil containing urea-formaldehyde plastic, and inorganic salt and vitamin adjuvant are added at the same time, soil is turned once every week, the water content of the soil is maintained to be 20-50wt%, and degradation is carried out for 50-150 days.
Wherein the addition amount of the urea-formaldehyde plastic degradation biological agent is 5-20%. The addition amount of the inorganic salt and the vitamin adjuvant is 0-3 percent.
The present invention will be described in detail below with reference to the accompanying drawings.
The urea-formaldehyde plastic degradation biological agent of the embodiment 1 is prepared by the following steps:
(1) Inoculating Penicillium erythroproducing fungus (Penicillium rubens) PSRF-507 into a Chlamydomonas reinhardtii culture medium, and performing first culture to obtain an activated strain; wherein the first culture is shaking culture at 200rpm, and culturing at 28deg.C for 5d;
wherein, the Chlamydia medium is prepared from the following raw materials: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate and MgSO 4 ·7H 2 0.5g of O, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and adding distilled water to 1000mL;
(2) Inoculating the activated strain into a 1# urea formaldehyde culture medium according to the volume ratio of 5%, and performing shake culture at 28 ℃ and 200rpm for 12d to obtain bacterial liquid 1;
wherein, the 1# urea formaldehyde culture medium is prepared from the following raw materials: urea formaldehyde polymer material 5g, glucose 10g, inorganic salt mother liquor 0.5mL, compound vitamin mother liquor 0.25mL, distilled water to 1000mL;
the preparation method of the urea formaldehyde polymer material comprises the following steps: firstly, uniformly mixing urea and formaldehyde according to a molar ratio of 1:2, and regulating the pH value of the system to 9 by using 0.5mol/L sodium hydroxide solution. The reaction system temperature was raised to 50℃and maintained for 2h. Extruding the reactants from extrusion equipment, and then placing the strips in an oven for drying to constant weight;
the preparation method of the inorganic salt mother solution comprises the following steps:
weighing FeCl 2 ·4H 2 O 1.0g，CoCl 2 ·6H 2 O 0.2g，NiCl 2 ·6H 2 O 0.01g，CuCl 2 ·2H 2 O0.01g，MnCl 2 ·4H 2 O 0.70g，ZnCl 2 0.1g，H 3 BO 3 0.5g，Na 2 MoO 4 ·2H 2 0.03g of O is poured into a beaker, and 1000mL of distilled water is measured; dissolving, mixing, pouring into a reagent bottle, and preserving at 4deg.CAnd storing for standby.
The preparation method of the compound vitamin mother solution comprises the following steps:
the method comprises the steps of weighing 0.05g of biotin, 0.1g of p-aminobenzoic acid, 0.2g of nicotinic acid, 0.05g of vitamin B, 0.1g of pyridoxine hydrochloride and pouring the mixture into a beaker, and weighing 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4deg.C for use.
(3) Transferring the bacterial liquid 1 into a 2# urea formaldehyde culture medium liquid fermentation culture medium according to the volume ratio of 10%, performing shake culture at 28 ℃ and 200rpm for 10d, and performing shake culture at 30 ℃ and 200rpm for 15d to obtain bacterial liquid 2;
wherein, the 2# urea formaldehyde culture medium is prepared from the following raw materials:
10g of urea formaldehyde polymer material, 0.2mL of inorganic salt mother solution, 1.0mL of compound vitamin mother solution, and distilled water to 1000mL;
(4) Centrifuging the fermentation broth 2, and vacuum concentrating the supernatant to half of the original volume to obtain the concentrated urea-formaldehyde plastic degradation biological agent.
Wherein, FIG. 1 is the morphology diagram (400 times) of Penicillium rubrum produced in the step (1); FIG. 2 shows the colony morphology of the strain grown for 20d on the Chlamydia medium in the step (1); FIG. 3 shows the growth pattern of the strain in step (3) on 2# urea formaldehyde broth for 20 d.
The application method of the urea-formaldehyde plastic degradation biological agent in the embodiment 2 comprises the following steps:
the degradation biological agent obtained in the example 1 is added into an aqueous solution containing urea-formaldehyde plastics according to the proportion of 5 percent, and the temperature is kept for 30 days at 28 ℃.
Test example 1 weight loss test of Urea-formaldehyde plastics
In example 2, urea-formaldehyde plastic degradation biological agent is added into urea-formaldehyde plastic containing solution according to the proportion of 5%, the sample is split-packed into 10 parts, the temperature is kept at 28 ℃, and one part of sample is taken every 3 days. Filtering with quick filter paper after sampling, and discarding the filtrate; the precipitate was washed with deionized water and the filtrate was discarded. And (3) drying the residual undegraded precipitate in a drying oven at 40 ℃ for 24 hours, weighing, calculating the weight loss rate according to the following formula, and drawing a weight loss graph.
Weight loss ratio = Δw/w 0 Wherein Deltaw is the weight of the urea-formaldehyde plastic; w (w) 0 Is the weight of the original urea-formaldehyde plastic. As can be seen from fig. 4, the urea-formaldehyde plastic loses weight up to 35%.
Test example 2 degradation products and metabolite analysis
The degradation agent obtained in the example 1 is added into the solution containing urea-formaldehyde plastics according to the proportion of 5%, the solution is treated for 15d, the solution is taken to pass through a 0.22 mu m water-based filtering membrane, and the filtrate is taken for high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. HPLC conditions: the solution was separated using a chromatographic column (Waters C18 1.7 μm, 2.1X100 mm) with a mobile phase formaldehyde-water solution (volume ratio 9:1) at 40℃and a flow rate of 0.75mL/min. The end of the column was introduced into a mass spectrometer (Shimadzu LCMS-8050), MS conditions: ESI+ ion source, positive ion in scanning mode, interface voltage of 4.0kV, interface temperature of 400deg.C, detector voltage of 1.76kV, and IG vacuum degree of 2.0X10 -3 Pa, PG vacuum of 1.3X10 g 2 Pa, CID gas of 2.7X10 5 Pa, the scanning range is 50-400m/z.
Results and analysis:
LCMS analysis of intermediate metabolites before and after degradation is shown in fig. 5 and table 1. As a result, the number and intensity of peaks were significantly changed, and (3), (8), (9), and (I) were newly appeared as compared with the control group,The corresponding peaks, (1), (2), (4), (5), (6), (7) increase in intensity. And (3) analyzing an intermediate metabolite formed by degrading urea-formaldehyde plastics based on a mass spectrum diagram corresponding to a peak, wherein the m/z value corresponding to a peak (7) is 205, the corresponding degraded intermediate metabolite is dimethyl triurea, the m/z value corresponding to a peak (d) is 277, and the corresponding degraded intermediate metabolite is dimethyl triurea. Fracture involving C-N in amide bond, -CH during degradation 2 -NH-cleavage of C-N, -CH 2 -NH-in-NH is converted to-OH.
TABLE 1 major degradation products in degradation Process using liquid analysis
Test example 3XRD analysis of crystallinity before and after treatment
In example 2, the crystal forms of urea-formaldehyde plastic samples before and after degradation were characterized by X-ray diffractometry (XRD), respectively. The scanning temperature range is 10-80 degrees, and the scanning speed is 1 degree/min. Sample crystallinity was determined by MDI jack 6 and HighScore Plus analysis.
The X-ray diffraction patterns of urea-formaldehyde plastics before and after degradation are shown in FIG. 6, and crystallinity calculations were performed on all samples using MDI jade 6 and HighScare Plus. The results showed that the crystallinity of the sample before degradation was 26.21% and the crystallinity after degradation by biological agents was 29.44%. The crystallinity is improved, which shows that in the degradation process, urea formaldehyde molecules regularly arranged in the crystallization area are partially degraded, and the biodegradation has better degradation efficiency on the urea formaldehyde plastic.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. Application of Penicillium erythrorhizon (Penicillium rubens) PSRF-507 in preparing urea formaldehyde plastic degradation biological agent.
2. The use according to claim 1, wherein the strain of penicillium erythrotoxidanum (Penicillium rubens) PSRF-507 is deposited in China general microbiological culture collection center (CGMCC) at the institute of microbiological culture collection center, national institute of sciences, national academy of sciences, no. 3, of the south-facing area, beijing, with a strain deposit number of CGMCC No.23229.
3. The use according to claim 1, wherein,
the urea-formaldehyde plastic degradation biological agent is prepared by the following steps:
(1) Inoculating Penicillium erythroproducing fungus (Penicillium rubens) PSRF-507 into a Chlamydomonas reinhardtii culture medium, and performing first culture to obtain an activated strain;
(2) Inoculating the activated strain into a 1# urea formaldehyde culture medium, and performing secondary culture to obtain bacterial liquid;
(3) Transferring the bacterial liquid into a 2# urea formaldehyde culture medium liquid fermentation medium according to the volume ratio of 1-15%, and performing third culture to obtain a fermentation liquid;
(4) Centrifuging the fermentation liquor, taking supernatant, and concentrating in vacuum to half of the original volume to obtain the concentrated urea-formaldehyde plastic degradation biological agent.
4. The use according to claim 1, wherein,
in the step (1), the first culture is shaking culture at 100-300 rpm, and the culture is carried out for 3-8 d at 20-30 ℃;
in the step (1), the Chlamydia medium is prepared from the following raw materials: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate and MgSO 4 ·7H 2 0.5g of O, 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and distilled water to 1000mL.
5. The use according to claim 1, wherein,
in the step (2), the second culture is shaking culture at 100-300 rpm and 20-30 ℃ for 10-30 d;
in the step (2), the 1# urea formaldehyde culture medium is prepared from the following raw materials: 1-5g of urea formaldehyde polymer material, 5-20g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.05-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
6. The use according to claim 5, wherein,
the preparation method of the inorganic salt mother solution comprises the following steps:
weighing FeCl 2 ·4H 2 O 0.2-1.8g，CoCl 2 ·6H 2 O 0.1-0.25g，NiCl 2 ·6H 2 O 0.01g，
CuCl 2 ·2H 2 O 0.01g，MnCl 2 ·4H 2 O 0..1-0.70g，ZnCl 2 0.1g，H 3 BO 3 0.1-0.5g，Na 2 MoO 4 ·2H 2 O 0.01-0.03g，Na 2 SeO 3 ·5H 2 0-0.01g of O, pouring the mixture into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4 ℃ for later use;
the preparation method of the vitamin complex mother solution comprises the following steps:
weighing 0.01-0.1g of biotin, 0.05-0.2g of p-aminobenzoic acid, 0.1-0.4g of nicotinic acid, 0.05g of vitamin B and 0.1g of pyridoxine hydrochloride respectively by using an electronic day, pouring into a beaker, and measuring 1000mL of distilled water; dissolving, mixing, and pouring into a reagent bottle, and preserving at 4deg.C for use.
7. The use according to claim 1, wherein,
in the step (3), the third culture is carried out for 10 to 30 hours at a temperature of between 20 and 30 ℃ and at a speed of between 100 and 300 rpm;
in the step (3), the 2# urea formaldehyde culture medium is prepared from the following raw materials:
1-15g of urea formaldehyde polymer material, 0-10g of glucose, 0.01-1mL of inorganic salt mother liquor, 0.5-7.5mL of compound vitamin mother liquor and 1000mL of distilled water.
8. The use according to claim 1, wherein,
in the step (2) and the step (3), the preparation method of the urea formaldehyde polymer material comprises the following steps:
uniformly mixing urea and formaldehyde according to a molar ratio of 1:1.5-4, and adding 0.3-1 mol/L sodium hydroxide to adjust the pH value of a reaction system to 8-10;
heating the reaction system to 50 ℃ and reacting for 1-3 h, extruding the obtained reactant from extrusion equipment to obtain a strip-shaped object, and drying to constant weight to obtain the urea formaldehyde polymer material;
the molecular formula of the obtained urea formaldehyde polymer material is as follows:
wherein m=1 to 8, n=1 to 10, and m=1 to 1000.
9. The use according to claim 1, wherein,
the application method of the urea-formaldehyde plastic degradation biological agent comprises the following steps:
the urea-formaldehyde plastic degradation biological agent is added into the waste water containing urea-formaldehyde plastic or the soil polluted by the urea-formaldehyde plastic to degrade.
10. The use according to claim 9, wherein,
the addition amount of the urea-formaldehyde plastic degradation biological agent in the wastewater containing the urea-formaldehyde plastic is 3-15%; in the soil polluted by the urea-formaldehyde plastics, the addition amount of the urea-formaldehyde plastics degradation biological agent is 5-20%.
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