CN111944704A

CN111944704A - Fungus strain for degrading polyurethane plastics, and culture method and application thereof

Info

Publication number: CN111944704A
Application number: CN202010879237.7A
Authority: CN
Inventors: 赛隆·可汗; 许建初; 桂恒; 赛迪亚·娜迪尔; 彼得·莫蒂默; 叶磊
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2020-11-17
Anticipated expiration: 2039-03-01
Also published as: CN111944704B; WO2020177175A1; CN109762744A

Abstract

The invention provides a fungus strain A.flavus G10 for degrading polyurethane plastics, wherein the microorganism preservation number is as follows: GDMCC 60537. Also provided is a method of culturing fungal strain a. flavus G10, comprising: isolating a fungal strain from the intestinal tract of cricket; culturing the fungus strain in a liquid culture medium by taking PU as a unique carbon source to obtain a culture solution; diluting the culture solution and spreading the culture solution on a solidified nutrient agar culture medium containing tetracycline antibiotics and a potato dextrose agar culture medium to obtain a culture growth; the culture growth was subcultured on fresh plates at 30 ℃ until a single fungal strain was obtained on each plate. The fungus strain A. flavus G10 has high speed of degrading polyurethane plastics.

Description

Fungus strain for degrading polyurethane plastics, and culture method and application thereof

Technical Field

The invention relates to the field of biodegradation, and particularly relates to a fungus strain for degrading polyurethane plastics, a fungus culture and application thereof.

Background

Plastic is a multifunctional material and can be almost used in every aspect of our daily life. The average human consumption of full-sphere synthetic plastics is about 38 kilograms per year. From 1950 to 2015, about 9150 ten thousand tons of plastic have been produced, resulting in about 6945 thousand tons of plastic waste on the earth's surface (Gyer et al, 2015). A very small fraction of the plastic waste produced can be recovered (9%) and incinerated (12%), with approximately 79% of the plastic still accumulating in landfills, other surfaces and marine environments (Gyer et al, 2015). It is estimated that the total quantity of municipal solid waste produced annually in the United states is 2.430 million tons, with waste plastics accounting for 13% (EPA report 2009, USA). In oasis, waste plastics account for 15-25% of 2.2 billion solid wastes (eurostat.2008). In China, waste plastics account for 12% of the total amount of municipal domestic waste ("China statistics yearbook", 2001-2007).

Most of the plastic waste in landfills is Polyurethane (PU). PU is a synthetic plastic used to make various objects in the industrial, automotive and medical fields. PU accounts for about 7% of the total global Plastic yield, which is about 1200 million tons per year, and most of it is finally Plastic garbage (Plastic Europe). Because of its negligible degradation in the environment, waste PU plastic waste accumulates at an ever increasing rate in soils, sediments and landfills and is therefore the main cause of plastic pollution in the environment. The lack of degradability of PU plastics and the increasing exhaustion of landfill sites and subsequent land pollution have prompted researchers to develop environmentally friendly biodegradable plastics, including biodegradable PU plastics. However, the fate of these degradable plastics in the environment and the time required for their complete mineralization are not fully understood. In addition, the main treatment method of the waste plastic products includes: landfill, incineration and recycling, but none of them solves the environmental persistence of PU Waste and its potential pollution problems (Macromolecules systems, 2006; water Management, 2009).

In the aspect of solving the problem of plastic waste, the biodegradation is simpler and the harm is less. Several microorganisms have been demonstrated to be able to biodegrade plastics (Kaplan et al, 1979; Otake et al, 1995; Nakamiya et al, 1997; Cacciari et al, 1993; Sivan et al, 2008; Arkatkar et al, 2009; Atiq et al, 2010; Zafar et al, 2013,2014; Yang et al, 2014; Khan et al, 2017). Researchers have discovered microorganisms that degrade PU from soil, landfills, and compost; wherein the fungus is found to be a dominant organism (Boubender 1993; Crabbe et al, 1994; Nakajima-Kambe 1995; Baumgartner et al, 1997; Blake and Howard 1998; Akutsu et al, 1998; Howard and Blake 1999; Allen et al, 1999; Manna and Paul, 2000; Yamada-Onodera et al, 2001; Zafar et al, 2013,2014; Khan et al, 2017;). Fungi are known to secrete different classes of hydrolases including esterases, ureases and proteases (Khan et al, 2017). It has been found that polyester-type PU is more susceptible to fungal hydrolysis than polyether-type PU (Tokiwa et al, 2009). The extent of biodegradation also varies with the chemical nature of the PU polymer and the fungal species. Some fungal species require the addition of some chemical inducer that induces the secretion of enzymes required for biodegradation. The colloidal polyester PU is biodegraded by a microbial consortium consisting of Curvularia senegalensis, Fusarium solani, Aureobasidium pullulans and Cladosporium sp. Among them, c. senegalensis is more efficient, and it produces extracellular polyurethane enzyme (PUase) enzyme (crabe et al, 1994).

However, the main problem with the use of plastics to degrade microorganisms is the slow rate of degradation of the plastics. For example, mixed cultures of several bacterial and fungal strains isolated from sludge, contaminated soil and marine sediments show extremely slow rates of biodegradation (Nadnda and Sahu,2010). In addition, in the test for degrading polystyrene using mixed microbial cultures isolated from sludge, soil, manure, garbage, it takes 11 weeks to measure 0.55% of its degradation using isotopic tracing (Kaplan et al, 1979). After 3 months of incubation in a liquid culture of the fungus Penicillium simplicissimum, a very slow degradation of the Polyester (PE) was observed (Yamada-Onodera et al, 2001). Recently, it has been reported that the biodegradation rate of the microbial consortium ethylene terephthalate (PET) plastic is 0.13mg/cm per day^-2(Yoshida et al, 2016). PU is generally difficult to biodegrade in the natural environment due to its polymeric nature, high molecular weight, complex structure.

Although there are reports on microorganisms degrading PU, the degradation rate has been reported to be slow so far. To date, no process has been found with biodegradable PUs produced at high rates (e.g., in less than 30 days) and on a large scale. Therefore, there is a need to develop more efficient PU degradation processes, as well as improved biological systems, that increase the rate of PU degradation so that the processes can be used commercially.

Disclosure of Invention

The invention mainly solves the technical problem of providing a fungus strain with higher degradation speed for degrading polyurethane plastics, a strain culture and application thereof.

A fungus strain a. flavus G10 for degrading polyurethane plastics, the microorganism deposit number of which is: GDMCC 60537.

The application of the fungus strain in polyurethane plastics.

In one embodiment, the polyurethane plastic is of a foam type or a transparent type.

A bioreactor containing a fungal strain a. flavus G10.

A strain culture of the fungal strain a. flavus G10 described above.

A method of culturing the fungal strain a. flavus G10 described above, comprising:

isolating a fungal strain from the intestinal tract of cricket;

culturing the fungus strain in a liquid culture medium by taking PU as a unique carbon source to obtain a culture solution;

diluting the culture solution and spreading the culture solution on a solidified nutrient agar culture medium containing tetracycline antibiotics and a potato dextrose agar culture medium to obtain a culture growth;

the culture growth was subcultured on fresh plates at 30 ℃ until a single fungal strain was obtained on each plate.

In one embodiment, the liquid medium is prepared by adding KH to deionized water₂PO₄、K₂HPO₄、MgSO₄·7H₂O、NH₄NO₃、NaCl、FeSO₄·7H₂O, and ZnSO₄·7H₂And O.

In one embodiment, the potato dextrose agar medium is prepared by adding potato starch, sugar and agar to deionized water.

In one embodiment, the nutrient agar medium is prepared by adding peptone, beef powder, NaCl and agar in deionized water.

In one embodiment, the liquid medium, nutrient agar medium, and potato dextrose agar medium are all autoclaved prior to use.

The fungus strain can effectively degrade polyurethane plastics in a short time. When PU films were cultured with fungal strain a. flavus G10, fungal strain a. flavus G10 could effectively degrade PU sheets, losing 4.4% weight per week.

Drawings

FIG. 1 is a schematic diagram of the growth of one embodiment of a fungal strain on a medium plate;

FIG. 2 is a photograph showing the morphology of mycelia and spores of the isolated fungal strain of the present invention, wherein a is the mycelia under an optical microscope; b is conidium under an optical microscope; c. d and e are conidia under SEM;

FIG. 3 is a schematic view of a normal PU film (a) and a PU film (b) after G10 treatment;

FIG. 4 is an SEM of a PU film, wherein a is an SEM of a normal PU film and b, c, d, e and f are SEM images of a PU film treated with G10;

FIG. 5 is a three-dimensional (3D) profile of the surface of a PU film using an Atomic Force Microscope (AFM), wherein A-represents a 3D surface scan of an untreated PU film. B, C and D represent 3D surface scans of PU films treated with G10 for 2,4 and 6 hours.

Detailed Description

The invention provides an embodiment of fungus strain Aspergillus flavus G10Aspergillus flavus G10 for degrading polyurethane plastic, wherein the microorganism deposit number is as follows: GDMCC 60537. The preservation center: guangdong province microbial strain preservation center, preservation unit address: guangzhou city, Jielizhou 100 college, building 59, floor 5, preservation date: year 2019, month 1, and day 16.

The fungus strain can be applied to degradation of polyurethane plastics.

Specifically, the polyurethane plastic is of a foam type or a transparent type.

Specifically, the polyurethane plastic is poly [4, 4-methylenebis (phenyl isocyanate) -salt-1, 4-butanediol/di (propylene glycol)/polycaprolactone-acetone ]. Specifically, PU of the formula poly [4, 4-methylenebis (phenylisocyanate) -salt-1, 4-butanediol/di (propylene glycol)/polycaprolactone-acetone ] was purchased as beads from Aldrich Chemical Company, Inc. (U.S.). These beads were used to prepare PU membranes according to the method of Khan et al. Briefly, approximately 1.25g of PU beads were dissolved in 100ml of tetrahydrofuran (PanreaceQuimica, SA) by shaking for 30min in a 250ml flask in a rotary shaker at 150 rpm. The PU solution was poured into four glass petri dishes and cured by holding a large plastic box with gel beads around the PU membrane plate for 48 hours at room temperature. After evaporation of the solvent, the dried PU film was separated from the petri dish and stored at room temperature.

A bioreactor containing fungal strain a. flavus G10, useful for the degradation of PU. The bioreactor allows control of environmental conditions to provide optimal growth of fungi. In particular, the bioreactor may comprise providing a strain of fungus and using the strain to produce an inoculum. The inoculum can be used to inoculate small, medium or large bioreactors containing petroleum-based plastics and minimal media. Internal environmental conditions and input and output streams may be monitored and controlled. Specifically, in one embodiment, the internal environmental conditions of the bioreactor including temperature, pH, carbon source and agitation speed are controlled. In particular, in an embodiment, the bioreactor may be a continuous feed bioreactor or a fed-batch bioreactor. In particular, in an embodiment, the bioreactor may be equipped with recirculation of liquid material. Specifically, in one embodiment, biodegradation can be monitored by sampling and monitoring while the plastic is in the bioreactor.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The embodiments are understood with reference to fig. 1-5.

Example 1: fungal strain a. flavus G10 isolation screening:

s110, separating fungus strains from intestinal tracts of crickets.

Specifically, the cricket may be cricket "Gryllus bimaculatus". Specifically, fungal strains are isolated from the intestinal tract of crickets.

S120, culturing the fungus strain in a liquid culture medium by taking PU as a unique carbon source to obtain a culture solution;

specifically, by dissolving each section of the intestinal tract of crickets in a sterile saline solution and incubating the intestine in a liquid medium.

The preparation method of the liquid culture medium comprises the following steps: 0.7g KH was added to 1000ml deionized water₂PO4、7.7g K₂HPO₄、0.7g MgSO₄·7H ₂0、1.0gNH₄NO₃、0.005g NaCl、 0.002g FeSO₄·7H₂O、0.002g ZnSO₄·7H₂O, 0.001 gxx. The liquid medium was autoclaved at 121 ℃ for 15 minutes to complete sterilization before use.

S130, diluting the culture solution containing the fungus strain, and spreading the culture solution on solidified nutrient agar medium containing tetracycline antibiotics and potato dextrose agar medium to obtain a culture growth.

Specifically, a Potato Dextrose Agar (PDA) medium was prepared by adding 5.0g/L of potato starch, 20.0g/L of sugar and 15g of agar to 1000mL of deionized water, and specifically, it was prepared in an Erlenmeyer flask. Specifically, sterilization was accomplished by autoclaving at 121 ℃ for 20 minutes prior to dispensing into petri dishes.

Specifically, 10g of peptone, 3g of beef powder, 5g of NaCl and 15g of agar were added to 1000mL of deionized water to prepare a nutrient agar medium. Specifically, sterilization was accomplished by autoclaving at 121 ℃ for 20 minutes prior to dispensing into petri dishes.

S140, subculturing the culture growth on fresh plates at 30 ℃ until a single fungal strain is obtained on each plate.

Example 2: identification of fungi

DNA of the degrading fungal strains was isolated at room temperature (25-28 ℃) and purified on wheat germ extract agar (MEA) plates. Morphological characterization was performed by optical microscopy and stereo electron microscopy. The PU degrading fungus separated by molecular morphology and phylogenetic analysis is identified as aspergillus flavus G10.

Specifically, the identification method comprises the following steps: morphological characterization was performed by optical microscopy and stereo electron microscopy. Molecular characterization was performed in the internal transcribed spacer (ITS5/ITS4), large subunit (LR0R/LR5), RNA polymerase II second large subunit (fRPB2-5F/fRPB2-7cR), calmodulin (CAL-228F/CAL 2Rd) and β -. Tubulin (T1/T2) sequence method. ITS sequence data generated in this study were BLAST searched in the nucleotide database of GenBank (www http:// BLAST. ncbi. nlm. nih. gov /) to determine their most likely closely related taxa. Single gene sequence alignments were performed using MAFFT v.7.215(Katoh and Standard 2016 (http:// MAFFT. cbrc. jp/alignment/server/index. html) and manually edited as necessary in BioEdit vv7.2.5(Hall 2004.) phylogenetic analyses of the compared data were based on Maximum Likelihood (ML). Phytograms were displayed using the FigTree v1.4.0 program (Rambaut 2012) and were displayed at Microsoft powerpoint (2007) and Standard (Inc.)

CS5 (version 15.0.0, Adobe) was recombined.

San Jose， CA)。

Example 3: the method for degrading the polyurethane plastic by adopting the fungus strain A. flavus G10 comprises the following steps:

the culture conditions are as follows: the conditions in the incubator are maintained at a temperature of 24 + -2 deg.C, a relative humidity of 75 + -2%, a light ratio of 8: under 10 conditions, 300 crickets were cultured, and a total of 6 small incubators were provided in one incubator, with 50 crickets per incubator.

Wherein, the control group: feeding 150 healthy adult crickets under the control conditions of 24 +/-2 ℃ of temperature, 75 +/-2% of relative humidity and 8:10 of illumination ratio, feeding wheat bran, wherein the proportion of wheat germs to yeast powder is respectively 10: 3: 1. additionally 0.2% of the total amount of vitamin powder and water was supplemented.

Experimental groups: another group of 150 healthy adult crickets were kept under control conditions of temperature 24. + -.2 ℃, relative humidity 75. + -.2% and illumination time 8:10, fed with a diet containing 4 g of PUF as sole carbon source.

The PU degradation of crickets was verified by examining their body weight, the percentage of body weight and feces per cricket. Three replicates per experiment, we determined the mean and standard error SE (n-3) for each treatment (experimental and control). The change in biomass was determined by randomly measuring the weight of five crickets. The feces of crickets were measured at zero time (starting point of the experiment) and after every three days for 18 days. The degradation of biodegradation was checked by the appearance and weight loss of the PU film.

To study the chemical digestion of crackets eating PU, the feces were analyzed by Scanning Electron Microscopy (SEM). After three days intervals, the feces of crickets were collected in sterilized sampling bags for 18 days and stored at-20 ℃ until further use. The collected faeces were dissolved in sterile water, separated, washed and stored separately in the excreted pieces of PUF in the faeces for SEM analysis.

The surface topology and cross-sectional top view of the PU sheet were determined using an Atomic Force Microscope (AFM), Dimension Icon, Veeco, USA. A.flavus G10 mycelia, mycelia and spores were pulverized in a mortar and pestle, spread on three PU films, and cultured at 37 ℃ for 2,4 and 6 hours. The PU discs were gently washed with sterile water and ethanol and stored at room temperature for AFM analysis. Degradation was confirmed using AFM and SEM methods, respectively. AFM was set to 2000nm and the three-dimensional picture was scanned. Scanning Electron Microscope (SEM) using ZEISS Sigma 300, acceleration voltage 3-7KV, magnification 50X-4500X, resolution 200 μm-200 nm. Chemical degradation was monitored using AT-FTIR analysis. The spectral range is 4000 to 600cm^-1Resolution of 4cm^-1. The sample was placed on the ATR spot of the additional ATR section and slowly pressed. Three replicates of each treatment and control sample were used for analysis. The AFM results showed roughness, cavities, pores, etc. of the PU surface. By using SEM microscopy, we can see the growth of fungi on the PU film surface and the formation of pores on the surface.

Experimental analysis shows that the fungus strain A. flavus G10 can degrade PU within 28 days in the experimental group. The PU quality in the control group did not change.

According to the experimental conditions described above, 5 sets of 5 films were simultaneously prepared, the first set terminating the reaction at the time of the first week, the second set terminating the reaction at the time of the second week … … and so on, and the fifth set terminating the reaction at the time of the fifth week, and the results are shown in Table 1 below. Experiments prove that when the PU film is cultured together with the fungus strain A. flavus G10, the fungus strain A. flavus G10 can effectively degrade PU sheets, and the weight is reduced by 4.4% every week after the PU film is stabilized.

TABLE 1 PU weight loss monitoring data in the experimental groups

Time of day	Initial weight (g)	Final weight (g)	Weight difference (g)	Weight loss percentage (g)
					0 th	0.349	0.0349	0.00	0.00％
First week	0.341	0.338	0.003	0.8％
					Second week	0.347	0.342	0.005	1.4％
The third week	0.374	0.366	0.008	2.2％
					The fourth side	0.340	0.325	0.015	4.4％

In addition, in another experiment it was found that biodegradation increased if the PU film was first pretreated with the fungus a. flavus G10. Experiments show that when the PU sheet is at 1% FeSO₄And NaCl salt solution for 24 hours and exposed to UV radiation for 5 minutes, its weight was reduced by 6.1% per week.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications made by the equivalent structures or equivalent processes in the contents of the present specification and the attached drawings, or applied directly or indirectly to other related technical fields are included in the scope of the present invention.

Claims

1. The application of the fungus strain in degrading polyurethane plastic is characterized in that the fungus strain is A.flavus G10, the microorganism preservation number of the fungus strain is GDMCC 60537, and the polyurethane plastic is poly [4, 4-methylenebis (phenyl isocyanate) -salt-1, 4-butanediol/di (propylene glycol)/polycaprolactone-acetone ].

2. A bioreactor comprising providing a strain of fungus and using the strain to produce an inoculum, the inoculum being useful for inoculating a bioreactor containing petroleum-based plastics and minimal media;

the fungus strain is A.flavus G10, and the microorganism deposit number is GDMCC 60537.

3. The bioreactor of claim 2, wherein the internal conditions of the bioreactor include temperature, pH, carbon source and agitation rate are controlled.

4. Bioreactor as claimed in claim 2, characterized in that the bioreactor can be a continuous feed bioreactor or a fed-batch bioreactor.

5. A method of culturing a fungal strain, comprising:

dissolving each section of the intestinal tract of cricket in a sterile saline solution and incubating the intestine in a liquid culture medium to obtain a culture solution;

subculturing the culture growth on fresh plates at 30 ℃ until a single fungal strain is obtained on each plate;

6. The method according to claim 5, wherein the liquid medium is prepared by adding KH to deionized water₂PO₄、K₂HPO₄、MgSO₄·7H₂O、NH₄NO₃、NaCl、FeSO₄·7H₂O, and ZnSO₄·7H₂And O.

7. The method of claim 5, wherein the potato dextrose agar medium is prepared by adding potato starch, sugar and agar to deionized water.

8. The method of claim 5, wherein the nutrient agar medium is prepared by adding peptone, beef powder, NaCl and agar in deionized water.

9. The method of claim 5, wherein the liquid medium, nutrient agar medium, and potato dextrose agar medium are all autoclaved prior to use.