CN111690553B - Efficient degradation bacterium for polyethylene and PBAT plastic mulching films and application thereof - Google Patents
Efficient degradation bacterium for polyethylene and PBAT plastic mulching films and application thereof Download PDFInfo
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- CN111690553B CN111690553B CN202010421381.6A CN202010421381A CN111690553B CN 111690553 B CN111690553 B CN 111690553B CN 202010421381 A CN202010421381 A CN 202010421381A CN 111690553 B CN111690553 B CN 111690553B
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a polyethylene and PBAT plastic mulching film degrading bacterium and application thereof; the polyethylene and PBAT plastic mulching film degrading bacteria arePseudomonas knackmussiiN1-2, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191053; the preservation time is 12 months and 16 days in 2019. The degrading bacteria N1-2 are obtained by separation and screening, so that the polyethylene plastic mulching film and the PBAT plastic mulching film both reach higher weight loss rates, the polyethylene plastic mulching film and the PBAT plastic mulching film are efficiently degraded, the production cost is low, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a high-efficiency degradation bacterium for polyethylene and PBAT plastic mulching films and application thereof.
Background
The plastic products are largely used in human life due to low cost, uneasiness to rot, wide application range and the like, but simultaneously, the problem of plastic pollution in natural environment is more serious, particularly in agriculture, the plastic mulching film is widely applied, and the problem of serious white pollution is also generated in farmlands while the agricultural yield is improved.
At present, most of the disposal methods for waste plastics are landfill, incineration, secondary processing and the like, and the disposal methods have great pollution to the environment and high disposal cost; in comparison, the process of degrading plastics by biological strains has the characteristics of mild degradation conditions and no pollution of degradation products, is environment-friendly, and has fewer degradation methods and more or less ideal effects on plastic mulching films at present; meanwhile, the degradation strain resource of the mulching film is less.
The traditional polyethylene plastic mulching film is the most widely applied mulching film in the farmland at present, but the degradability of the traditional polyethylene plastic mulching film is low, and the problem that the degradation of polyethylene is difficult to overcome all the time is solved, so that the finding of an efficient polyethylene plastic mulching film degrading strain is very important. In recent years, the pollution problem of polyethylene mulching films in farmlands is reduced to a certain extent due to the appearance of biodegradable mulching films, and Poly (butylene-co-terephthalate) (PBAT) is used as a biodegradable material in farmlands, but the degradation condition of PBAT in the actual application process is general, the production cost is high, and the comprehensive popularization and use cannot be realized. Therefore, it is also very important to further search for strains with high PBAT degradation efficiency.
Disclosure of Invention
The invention provides a high-efficiency degradation bacterium for polyethylene and PBAT plastic mulching films and application thereof, aiming at the technical problems of low degradability and high cost of the existing plastic mulching films, and particularly provides a high-efficiency degradation bacterium for polyethylene and PBAT plastic mulching films and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a high-efficiency degradation bacterium for polyethylene and PBAT plastic mulching film is Pseudomonas knackmussi N1-2, which is preserved in China center for type culture Collection with the preservation number of M20191053; the preservation time is 12 months and 16 days in 2019.
The 16S rDNA gene sequence of the polyethylene and PBAT plastic mulching film efficient degrading bacteria is a sequence table No. 1.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading mulching films.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading plastic mulching films.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading polyethylene plastic mulching films.
An application of efficient degrading bacteria of polyethylene and PBAT plastic mulching films in degrading the PBAT plastic mulching films.
A method for degrading polyethylene plastic mulching films by efficient degradation bacteria of polyethylene and PBAT plastic mulching films comprises the following steps:
1) cutting the polyethylene plastic mulching film into membranes, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) sequentially soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating high-efficiency degradation bacteria of polyethylene and PBAT plastic mulching film into an LB culture medium to be cultured to a logarithmic phase, transferring into a centrifuge tube to be centrifuged, removing supernatant, and washing precipitated bacteria with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli obtained in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane sterilized in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
A method for degrading a PBAT plastic mulching film by using efficient degradation bacteria of polyethylene and PBAT plastic mulching film comprises the following steps:
1) cutting the PBAT plastic mulching film into films, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) sequentially soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating high-efficiency degradation bacteria of polyethylene and PBAT plastic mulching film into an LB culture medium to be cultured to a logarithmic phase, transferring into a centrifuge tube for centrifugation, removing supernatant, and washing precipitated bacteria with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane subjected to sterilization treatment in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator at 28 ℃ and 180rpm for 8 weeks.
The beneficial effects of the invention are: the strain obtained by separation and screening is subjected to biological identification analysis, and the strain is Pseudomonas knackmussi N1-2; the N1-2 bacteria has good degradation effect on polyethylene and PBAT plastic mulching films, obvious biological erosion holes are formed in the surfaces of the polyethylene and PBAT films after 8 weeks of biodegradation treatment, the hydrophilicity of the films is obviously improved, and new polar functional groups appear; meanwhile, the weight loss rate of the polyethylene mulching film can reach 5.949 +/-0.033% (CK: 0.964 +/-0.066%), the weight loss rate of the PBAT mulching film can reach 6.490 +/-0.006% (CK: 4.470 +/-0.035%), new resources are provided for biodegradation of the polyethylene plastic mulching film and the PBAT plastic mulching film, the production cost is saved, and the application prospect is wide.
Drawings
FIG. 1 is a colony morphology of degrading bacteria N1-2 of the present invention;
FIG. 2 is a scanning electron microscope observation result of the surface topography of the degrading bacteria N1-2 of the present invention;
FIG. 3 is an atomic force microscope observation result of the surface topography of the degrading bacteria N1-2 of the present invention;
FIG. 4 is a phylogenetic tree of degrading bacteria N1-2 of the present invention;
FIG. 5 is a scanning electron microscope characteristic diagram of the polyethylene plastic mulching film and the PBAT plastic mulching film after the degrading bacteria N1-2 provided by the invention degrade for 8 weeks;
FIG. 6 is an atomic force microscope characteristic diagram of the polyethylene plastic mulching film and the PBAT plastic mulching film after the degrading bacteria N1-2 provided by the invention degrade for 8 weeks;
FIG. 7 is an infrared spectroscopic analysis feature diagram of the polyethylene plastic mulching film and the PBAT plastic mulching film after the degradation of the degrading bacteria N1-2 for 8 weeks, which is provided by the invention;
FIG. 8 is a water contact angle change diagram of the polyethylene plastic mulching film and the PBAT plastic mulching film after the degradation of the degrading bacteria N1-2 for 8 weeks.
Detailed Description
The present invention will now be described in detail with reference to the accompanying drawings and examples.
A high-efficiency degradation bacterium for polyethylene and PBAT plastic mulching film is Pseudomonas knackmussi N1-2, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191053; the preservation time is 12 months and 16 days in 2019.
A16S rDNA gene sequence of efficient degradating bacteria for the polyethylene and PBAT plastic mulching film is sequence table No. 1.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading mulching films.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading plastic mulching films.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading polyethylene plastic mulching films.
An application of efficient degrading bacteria for polyethylene and PBAT plastic mulching films in degrading PBAT plastic mulching films.
A method for degrading polyethylene plastic mulching films by efficient degradation bacteria of polyethylene and PBAT plastic mulching films comprises the following steps:
1) cutting the polyethylene plastic mulching film into membranes, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) sequentially soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating high-efficiency degradation bacteria of polyethylene and PBAT plastic mulching film into an LB culture medium to be cultured to a logarithmic phase, transferring into a centrifuge tube for centrifugation, removing supernatant, and washing precipitated bacteria with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane sterilized in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
A method for degrading PBAT plastic mulching films by high-efficiency degradation bacteria of polyethylene and PBAT plastic mulching films comprises the following steps:
1) cutting the PBAT plastic mulching film into films, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) with 75% ethanol and 95% ethanol in sequence for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating a polyethylene and PBAT plastic mulching film efficient degrading bacterium to an LB culture medium for culturing to a logarithmic phase, transferring the LB culture medium into a centrifuge tube for centrifugation, removing supernatant, and washing precipitated thalli with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli obtained in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane subjected to sterilization treatment in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator at 28 ℃ and 180rpm for 8 weeks.
EXAMPLE 1 isolation and screening of degrading bacterium N1-2
(1) Adding 1g of soil sample into a test tube containing 9mL of trace carbon source culture medium;
in this example, the trace carbon source medium was: 1000mL of deionized water was added with 0.5g of Yeastextract, NH4SO42.0g and 100mL of a mother solution of trace elements.
In this example, the mother liquor of trace elements (g.L)-1)：1.0gFeSO4·7H2O、1.0gMgSO4·7H2O、0.1gCuSO4·5H2O、0.1gMnSO4·H2O and 0.1g ZnSO4·7H2O; the pH value is about 7.0;
(2) adding a2 x 1.5cm polyethylene plastic mulching film membrane into the test tube, and performing shake culture at 180rpm and 28 ℃ for 10 days;
(3) then transferring the membrane into 10mL of new trace carbon source culture medium to be cultured for 10 days;
(4) then vibrating and cleaning the membrane by using PBS solution to prepare bacterial suspension, and transferring all the bacterial suspension into a 30mL liquid inorganic salt culture medium; simultaneously, adding about 0.1g of polyethylene plastic mulching film membrane pieces according to the proportion of 0.35% (w/v), shaking and culturing for 30 days at 180rpm and 28 ℃ in a shaking way;
in this example, the inorganic salt medium formulation was: KH (Perkin Elmer)2PO4 0.7g、K2HPO4 0.7g、MgSO4·7H2O 0.7g、NH4NO3 1.0g、NaCl 0.005g、FeSO4·7H2O 0.002g、ZnSO4·7H2O 0.002g、MnSO4·H20.001g of O; weighing the reagent, dissolving the reagent in 1000mL of deionized water, and adjusting the pH value to 7.0 by using 1 mol/LNaOH;
(5) after the culture is finished, separating the strains by using a dilution plate coating method, and diluting the bacterial liquid concentration to 10-4Sucking 50 mu L of bacterial liquid, coating the bacterial liquid into an LB culture medium, and streaking, purifying and separating the bacterial liquid for multiple times to obtain a bacterial strain;
(6) through the separation and screening work and multiple separation and purification, a strain of quick-growing bacteria N1-2 with the capacity of degrading the polyethylene plastic mulching film is obtained.
In the embodiment, the screened strain Pseudomonas knackmussi N1-2 is preserved in China center for type culture Collection; the address is as follows: eight Lopa in Wuchang region of Wuhan city, Hubei province; the preservation number is CCTCC NO: M20191053; the preservation time is as follows: 12 and 16 in 2019.
In this embodiment, during the separation and screening of the strains, a2 × 1.5cm PBAT plastic mulching film membrane can be added into the test tube, and then the bacteria N1-2 having a degradation effect on the PBAT plastic mulching film can be obtained through culturing, separation and purification.
Example 2 degradation of polyethylene Plastic mulch film with N1-2
1) Cutting a diaphragm of the polyethylene plastic mulching film into a size of 2 x 1.5cm, weighing, and placing the diaphragm into a sterile 10mL centrifuge tube for sterile treatment;
the specific treatment process comprises the following steps:
1.1) soaking the membrane in 2% SDS solution for more than 4 hours, and performing ultrasonic treatment for 30min during the soaking period;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for more than 4 hours in sequence, and performing ultrasonic treatment for 30min during soaking;
2) inoculating the bacterium N1-2 separated and purified in the embodiment 1 into an LB culture medium to be cultured to a logarithmic phase, transferring the bacterium into a 10mL centrifuge tube, centrifuging the bacterium at 4 ℃ and 8000rpm for 10min, removing supernatant, and washing precipitated thalli for 3 times by using 0.01mol/L phosphate buffer solution to remove the culture medium on the surface of the thalli;
3) adding 30mL of inorganic salt culture medium into the conical flask, adding thalli to carry out heavy suspension and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) 0.1g of membrane with sterilized surface is added into the conical flask, and the conical flask is placed in a constant temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
Example 3 degradation of PBAT Plastic mulch film with N1-2
1) Cutting the PBAT plastic mulching film into 2 x 1.5 cm-sized films, weighing, and placing in a sterile 10mL centrifuge tube for sterile treatment;
the specific treatment process comprises the following steps:
1.1) soaking the membrane in 2% SDS solution for more than 4 hours, and performing ultrasonic treatment for 30min during the soaking period;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for more than 4 hours in sequence, and performing ultrasonic treatment for 30min during the soaking period;
2) inoculating the bacterium N1-2 separated and purified in the embodiment 1 into an LB culture medium to be cultured to a logarithmic phase, transferring the bacterium into a 10mL centrifuge tube, centrifuging the bacterium at 4 ℃ and 8000rpm for 10min, removing supernatant, and washing precipitated bacteria for 3 times by using 0.01mol/L phosphate buffer solution to remove the culture medium on the surface of the bacteria;
3) adding 30mL of inorganic salt culture medium into a conical flask, adding thalli to carry out heavy suspension, adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) 0.1g of PBAT plastic mulching film membrane with a sterilized surface is added into the conical flask, and the conical flask is placed in a constant-temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
Control group 1(CK1)
1) Cutting the polyethylene plastic mulching film into 2 x 1.5 cm-sized films, weighing, and placing the films into a sterile 10mL centrifuge tube for sterile treatment;
the specific treatment process comprises the following steps:
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) with 75% ethanol and 95% ethanol in sequence for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) 30mL of inorganic salt culture medium is added into the conical flask, then 0.1g of polyethylene plastic mulching film membrane with a sterilized surface is added, and the conical flask is placed in a constant-temperature shaking incubator for culture for 8 weeks at 28 ℃ and 180 rpm.
Control group 2(CK2)
1) Cutting the PBAT plastic mulching film into 2 x 1.5 cm-sized films, weighing, and placing the films into a sterile 10mL centrifuge tube for sterile treatment;
the specific treatment process comprises the following steps:
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) sequentially soaking the membrane soaked in the step 1.2) in 75% ethanol and 95% ethanol for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) 30mL of inorganic salt culture medium is added into the conical flask, 0.1g of PBAT plastic mulching film membrane with a sterilized surface is added, and the conical flask is placed in a constant-temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
Furthermore, in order to explain the type characteristics of the degrading bacteria separated and screened by the invention and the degrading effect of the degrading bacteria on the polyethylene plastic mulching film and the PBAT plastic mulching film, the physiological characteristics and the molecular biology of the degrading bacteria are identified, and the results of the degrading bacteria on the polyethylene plastic mulching film and the PBAT plastic mulching film are compared and verified.
Through the separation and screening work, a degradation bacterium is obtained through multiple separation and purification, the shape of the strain is observed on an LB (lysogeny broth) flat plate, and the result is shown in figure 1.
As can be seen from fig. 1: the bacterial colony of the degrading bacteria on an LB flat plate is light yellow, the shape of the bacterial colony is circular, the surface of the bacterial colony is smooth and wet, and the edge of the bacterial colony is neat.
Test 2
The strains isolated, screened and purified in example 1 were observed under a scanning electron microscope, and the results are shown in FIG. 2.
Test 3
The strains isolated, screened and purified in example 1 were observed under an atomic force microscope, and the results are shown in FIG. 3.
Test 4
The strain obtained by separation, screening and purification in example 1 was subjected to a gram stain test, and the gram stain was red, and the strain was gram-negative bacteria.
Test 5
The 16S rDNA gene sequence of the strain obtained in example 1 was subjected to amplification sequencing, specifically, DNA of a single strain isolated in example 1 was extracted with a kit, and PCR amplification was performed using bacterial universal primers 27F and 1492R as amplification primers.
16SrDNA sequence primer 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3', 1492R: 5'-GGT TAC CTT GTT ACG ACT T-3' are provided.
PCR amplification system (50. mu.L): template DNA 2. mu.L, Mix (2X) 25. mu.L, primer 27F 2. mu.L, primer 1492R 2. mu.L, ddH2O 19μL。
And (3) amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 58 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; finally, the extension is carried out for 10min at 72 ℃, and the product is stored at 4 ℃.
In this experiment, the sequence of the 16SrDNA sequencing result is shown in the sequence table No. 1.
And (3) carrying out electrophoresis detection on the amplification product, and sequencing by the corporation of Weituosheng bioengineering (Shanghai). BLAST analysis is carried out on the obtained sequence and sequences existing in NCBI database, strains with similar homology are selected, MEGA X software is adopted to construct a phylogenetic tree, and the construction method is Neighbor-join. The results are shown in FIG. 4.
As can be seen from fig. 4: n1-2 was 99% similar to Pseudomonas knackmussi B13(NR121733) and was relatively close in evolutionary distance, and, in combination with the physiological and biochemical characteristics of the strain, initially identified N1-2 as Pseudomonas knackmussi N1-2.
Test 6 weight loss ratio measurement test
The weight loss of the membrane is the most basic phenomenon of the degradation of the membrane, so the weight loss rate of the membrane is tested.
In particular, the method comprises the following steps of,
treatment group: two films before and after degradation of polyethylene and PBAT in example 2 and example 3;
control group: two membranes before and after degradation of polyethylene and PBAT in the control group 1 and the control group 2;
the membrane is respectively processed according to the following procedures: taking out the membrane by using a sterile forceps, placing the membrane into a sterile 10mL centrifuge tube, soaking the membrane for more than 4 hours by using a 2% SDS solution, performing ultrasonic treatment for 30min during soaking, then soaking the membrane for 10min by using 30% hydrogen peroxide, and finally soaking the membrane for more than 4 hours by using 75% ethanol and 95% ethanol respectively in sequence, and performing ultrasonic treatment for 30min during soaking; the washed membrane was dried on filter paper and weighed.
In this experiment, 3 replicates of treatment were set for each set of membranes.
In this test, example 2 was compared with control 1, and the results are shown in Table 1;
in this test, example 3 was compared with control 2, and the results are shown in Table 2.
The weight loss ratio (%) of the membrane sheet is (initial mass of the membrane sheet-mass after degradation)/initial mass of the membrane sheet x 100%.
TABLE 1 weight loss results of PE polyethylene plastic mulch film compared to control 1
| Treatment of | Film original weight | Film weight after 8 weeks | ΔR | Percent weight loss | Average weight loss ratio% |
| N1-2(1) | 0.1003 | 0.0944 | -0.0059 | 5.88235 | 5.9488 |
| N1-2(2) | 0.1003 | 0.0943 | -0.0060 | 5.98205 | |
| N1-2(3) | 0.1003 | 0.0943 | -0.0060 | 5.98205 | |
| CK1(1) | 0.1004 | 0.0993 | -0.0011 | 1.09562 | 0.9644 |
| CK1(2) | 0.1004 | 0.0995 | -0.0009 | 0.89641 | |
| CK1(3) | 0.1003 | 0.0994 | -0.0009 | 0.9012 |
As can be seen from table 1: after 8 weeks of culture, the weight loss rate of the polyethylene mulching film of the treatment group reaches 5.949 +/-0.033 percent, and the weight loss rate of the polyethylene mulching film of the control group 1 reaches 0.964 +/-0.066 percent. (the reason why the weight loss of the membrane occurred in the control group 1 may be that the membrane was mechanically damaged during shaking oscillation and during the test operation)
TABLE 2 comparison of weight loss results of PBAT plastic mulch film with control group 2
From table 2 it can be seen that: after 8 weeks of culture, the weight loss rate of the PBAT mulching film of the treated group reaches 6.489 +/-0.006 percent, and the weight loss rate of the PBAT plastic mulching film of the control group 2 reaches 4.470 +/-0.035 percent. (the reason why the weight loss of the membrane occurs in the control group 2 may be that the membrane is mechanically damaged in the shaking oscillation process and the test operation process.)
Experiment 7 observation test of scanning electron microscope
Treatment group: two films before and after degradation of polyethylene and PBAT in example 2 and example 3;
control group: two membranes before and after degradation of polyethylene and PBAT in the control group 1 and the control group 2;
the specific test process is as follows: the membrane is cleaned and naturally dried, and the microscopic morphology characteristics of the membrane are observed under a scanning electron microscope (S-4800; Hitachi, Japan) after the gold spraying is fixed, wherein the magnification is 3000 times. The test results are shown in FIG. 5.
Wherein FIG. 5(a1) shows an untreated virgin polyethylene film sheet of example 2; FIG. 5(a2) is a control 1 polyethylene film without bacterial treatment; FIG. 5(a3) is a polyethylene film sheet obtained by sterilization treatment in example 2;
wherein FIG. 5(b1) shows the raw PBAT membrane of example 3 without treatment; FIG. 5(b2) shows PBAT membrane of control group 2 without bacterial treatment; FIG. 5(b3) shows the PBAT membrane after the bacterial treatment of example 3.
From fig. 5, it can be derived:
(1) in fig. 5(a1), the polyethylene mulch film (not tested) has smooth and flat surface; in fig. 5(a2), the surface of the polyethylene mulch film of control 1 showed very fine roughness; in FIG. 5(a3), after 8 weeks of cultivation, the treated polyethylene mulch had a rough surface with significant holes and ravines; the strain has good degradation effect on the polyethylene mulching film.
(2) In fig. 5(b1), the PBAT raw film (not tested) has a smooth and flat surface; in fig. 5(b2), the surface of the PBAT geomembrane of control group 2 appeared with very fine roughness points; in fig. 5(b3), after 8 weeks of cultivation, the treated PBAT mulch had a rough surface with significant ravines; the strain of the invention is proved to have good degradation effect on PBAT mulching film.
Test 8 Observation test of atomic force microscope
Treatment group: two films before and after degradation of polyethylene and PBAT in example 2 and example 3;
control group: two membranes before and after degradation of polyethylene and PBAT in the control group 1 and the control group 2;
the specific test process is as follows: the membrane sheets were cleaned and then naturally dried, and the surface characteristics of the plastic mulching film were scanned at room temperature using an atomic force microscope (Multimode-8; Bruker, USA), and the resulting images were analyzed by NanoScope Analysis software. The test results are shown in fig. 6.
Wherein FIG. 6(a1) shows an untreated virgin polyethylene film sheet of example 2; FIG. 6(a2) is a control 1 polyethylene film without bacterial treatment; FIG. 6(a3) is a polyethylene film sheet after bacterial treatment in example 2;
wherein FIG. 6(b1) represents the raw PBAT membrane of example 3 without treatment; FIG. 6(b2) shows PBAT membrane of control group 2 without bacterial treatment; FIG. 6(b3) shows the PBAT membrane after the bacterial treatment of example 3.
From fig. 6, it can be derived:
(1) in fig. 6(a1), the polyethylene mulch film raw film (not tested) has a smooth and flat surface; in fig. 6(a2), the surface roughness of the polyethylene mulch film of control 1 was very light; in FIG. 6(a3), after 8 weeks of cultivation, the surface roughness of the treated polyethylene mulch was deep and obvious ravines appeared; the strain has good degradation effect on the polyethylene mulching film;
(2) in fig. 6(b1), the PBAT raw film (not tested) has a smooth and flat surface; in fig. 6(b2), the PBAT geomembrane of control group 2 had very light surface roughness; in fig. 6(b3), after 8 weeks of culture, the surface roughness of the treated PBAT mulch was deeper; the strain of the invention is proved to have good degradation effect on PBAT mulching film.
Run 9 analysis of the Infrared Spectrum
Test groups: two degraded films of polyethylene and PBAT in example 2 and example 3;
the specific process is as follows: the two groups of membranes are respectively cleaned and naturally dried, the functional groups on the surfaces of the membranes are measured by a Fourier infrared spectrometer (Vetex 70; Bruker, Germany), and the scanning wavelength range is 400-4000cm-1. The results are shown in FIG. 7.
FIG. 7(a) is a polyethylene film sheet of example 2 after the sterilization treatment; FIG. 7(b) shows the PBAT membrane after the bacterial treatment of example 3.
As can be seen from FIG. 7(a), after 8 weeks of culture, many oxygen-containing functional groups such as carbonyl groups (1716 cm) were newly introduced into the chemical structure of the polyethylene mulch film-1Para), carboxyl (-1558 cm)-1At) and-1436 cm-1C-OH bending vibration and the like show that the strain N1-2 has degradation effect on the polyethylene mulching film.
As can be seen from FIG. 7(b), after 8 weeks of culture, carbonyl groups (. about.1714 cm) were introduced into the chemical structure of PBAT mulching film-1) Carbon-nitrogen triple bond (-2355 cm)-1) Carbon-carbon double bond (-3099 cm)-1) And hydroxyl (. about.3292 cm)-1) And an isocolar functional group. The strain N1-2 is shown to have degradation effect on PBAT mulching film.
Therefore, when the strain N1-2 provided by the invention degrades plastics, the strain can introduce polar functional groups on the surface of the plastics, so that the instability of the film structure is increased, and the later degradation of the plastic film is facilitated.
Test 10 Water contact Angle
Treatment group: two films before and after degradation of polyethylene and PBAT in example 2 and example 3;
control group: two membranes before and after degradation of polyethylene and PBAT in the control group 1 and the control group 2;
the specific test process is as follows: the membrane sheet was cleaned and dried naturally, and then a change in the hydrophobicity of the surface of the mulching film was analyzed by measuring a hydrostatic contact angle (n: 3) using a water contact angle meter (JC2000D 1; powerarch, China). The test results are shown in fig. 8.
Wherein FIG. 8(a1) shows an untreated virgin polyethylene film sheet of example 2; FIG. 8(a2) is a control 1 polyethylene film without bacterial treatment; FIG. 8(a3) is a polyethylene film sheet obtained after the sterilization treatment in example 2;
wherein FIG. 8(b1) represents the raw PBAT membrane of example 3 without treatment; FIG. 8(b2) shows PBAT membrane of control group 2 without bacterial treatment; FIG. 8(b3) shows the PBAT membrane after the bacterial treatment of example 3.
From fig. 8 it can be derived:
(1) in fig. 8(a1), the polyethylene mulch film was raw (not tested), and the water contact angle of the film sheet was 101.7 ± 1.3 °; in fig. 8(a2), the water contact angle of the polyethylene mulch film sheet of control 1 was 99.7 ± 1.9 °; in fig. 8(a3), after 8 weeks of culture, the water contact angle of the treated polyethylene mulch film sheet was 87.9 ± 0.7 °;
(2) in fig. 8(b1), PBAT membrane raw film (not tested), the water contact angle of the membrane sheet was 98.5 ± 1.6 °; in fig. 8(b2), the water contact angle of the PBAT geomembrane membrane patch of control 2 was 98.4 ± 0.9 °; in fig. 8(b3), after 8 weeks of incubation, the water contact angle of the PBAT mulch film pieces of the treated group was 73.1 ± 1.4 °.
The results show that the degrading bacteria N1-2 can obviously reduce the hydrophobicity of the polyethylene plastic mulching film membrane and the PBAT plastic mulching film membrane, so that the polyethylene plastic mulching film membrane and the PBAT plastic mulching film membrane are easier to be attached by microorganisms.
The tests show that the strain N1-2 separated and screened by the invention has good degradation effect on polyethylene and PBAT plastic mulching films. After 8 weeks of biodegradation treatment, the surfaces of the polyethylene film and the PBAT film have obvious bioerosion holes, the hydrophilicity of the film is obviously improved, new polar functional groups appear, after 8 weeks, the weight loss rate of the polyethylene mulching film can reach 5.949 +/-0.033%, the weight loss rate of the PBAT mulching film can reach 6.489 +/-0.006%, the degradation effect is good, the production cost is low, and the application prospect is wide.
Claims (5)
1. A polyethylene and PBAT plastic mulching film degrading bacterium is characterized in that: the polyethylene and PBAT plastic mulching film degrading bacteria arePseudomonas knackmussii N1-2, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191053; the preservation time is 12 months and 16 days in 2019.
2. The use of the polyethylene and PBAT plastic mulch degrading bacteria of claim 1 in degrading polyethylene plastic mulch.
3. The use of the polyethylene and PBAT plastic mulch degrading bacteria of claim 1 in degrading PBAT plastic mulch.
4. The method for degrading the polyethylene plastic mulching film by the polyethylene and PBAT plastic mulching film degrading bacteria based on the claim 1 is characterized in that: the method comprises the following steps:
1) cutting the polyethylene plastic mulching film into membranes, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) with 75% ethanol and 95% ethanol in sequence for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating the polyethylene and PBAT plastic mulching film degrading bacteria of claim 1 into an LB culture medium to be cultured to a logarithmic phase, transferring into a centrifuge tube to be centrifuged, removing supernatant, and washing precipitated bacteria with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli obtained in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane sterilized in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator for 8 weeks at 28 ℃ and 180 rpm.
5. The method for degrading the PBAT plastic mulching film by the polyethylene and PBAT plastic mulching film degrading bacteria based on the claim 1 is characterized by comprising the following steps: the method comprises the following steps:
1) cutting the PBAT plastic mulching film into membranes, and performing aseptic treatment;
1.1) soaking the membrane in 2% SDS solution for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
1.2) soaking the membrane soaked in the step 1.1) in 30% hydrogen peroxide for 10 min;
1.3) soaking the membrane soaked in the step 1.2) with 75% ethanol and 95% ethanol in sequence for not less than 4 hours, and performing ultrasonic treatment for 30min during soaking;
2) inoculating the polyethylene and PBAT plastic mulching film degrading bacteria of claim 1 into an LB culture medium to be cultured to a logarithmic phase, transferring into a centrifuge tube to be centrifuged, removing supernatant, and washing precipitated bacteria with 0.01mol/L phosphate buffer solution;
3) mixing 30mL of inorganic salt culture medium with the precipitated thalli in the step 2), resuspending and adjusting the concentration of the bacterial liquid, and keeping the OD of the bacterial liquid600The value is 0.8 to 1.0;
4) adding the membrane subjected to sterilization treatment in the step 1) into the bacterial liquid in the step 3), and placing the membrane in a constant-temperature shaking incubator at 28 ℃ and 180rpm for 8 weeks.
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