Patent Application
20240254433
This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file contains the sequence listing entitled “PA1310182-SequenceListing.xml”, which was created on Feb. 28, 2024, and is 2,938 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety.
This invention, a marine-derived fungus and its application in degrading plastic waste, belongs to the field of biotechnology.
Plastics are a class of synthetic materials mainly consisting of polymers derived from petrochemicals (e.g., ethylene, propylene, and vinyl chloride) [1]. Since plastic materials generally have the characteristics of chemical resistance, good insulation, light weight, cheapness, low manufacturing cost, and convenient use, they have brought great convenience to human life and are widely used in construction, machinery, agriculture, food packaging, and other fields involve all aspects of human life. Over the past half-century, plastic has become one of the most used commodities in daily life and an essential part of modern society. However, conventional plastics have extremely high molecular weight and strong hydrophobicity, and lack functional groups recognized by microbial enzyme systems, which makes plastic waste take hundreds of years to be degraded in nature [2]. In addition, a large amount of plastic waste has not been reasonably disposed of, resulting in the accumulation of plastic waste in the environment, which has caused serious “white pollution” to the environment. Plastic pollution has become a major global environmental problem alongside global climate change, ozone depletion, acid rain, etc. According to statistics, by 2015, humans produced a total of 8.3 billion tons of plastic, of which 6.3 billion tons have become garbage. Of the plastic that becomes garbage, only 9% is recycled, 12% is incinerated, and 79% is landfilled or abandoned in the environment. If the current trend continues until 2050, there will be 12 billion tons of plastic waste in landfills and the environment [3]. Taking the ocean as an example, with the progress of human social production and industrial development, the amount of plastic waste entering the ocean every year has increased from about 45,000 tons in 1975 to more than 8 million tons, accounting for 80% of global marine waste [4]. At present, incineration, landfill, and recycling are the main methods adopted to deal with waste plastic pollution. However, landfills not only occupy scarce land resources but also cause secondary pollution. Incineration produces large amounts of toxic gases, and the recycling of plastics is technically demanding and economically inefficient. A further issue that should be considered is to truly achieve biodegradation, especially the degradation of non-degradable plastic products of petroleum origin. Microbial methods of degrading plastics are rather green and environmental protection compared to traditional treatment methods [3, 5, 6]. The development of microbial plastic degradation technology is more realistic and ecologically significant than the traditional ways, whose essence is the result of the action of enzymes secreted by microorganisms. The degradation begins when microbes contact with the plastic and form biofilm on the plastic surface. Then plastic pores are enlarged by the action of extracellular enzymes and/or compounds released into the extracellular space thus changing the physicochemical properties of the plastic [2]. At present, although a variety of microorganisms that can degrade plastics have been screened out, including fungi, bacteria, and actinomycetes, the degradation efficiency is limited and far from industrial application. At the same time, further research on their mechanism of action is still needed. In-depth research, especially its application, requires further expansion and industrialization. Therefore, it is imperative to screen microorganisms with the ability to efficiently degrade plastics and realize the industrialization of plastic microbial degradation. Bioremediation of plastic waste is an important area worthy of attention in the future.
The purpose of the present invention is to provide a marine-derived fungus and its application in the remediation of plastic-polluted environments.
In order to achieve the above objects, the technical solutions adopted by the present invention are: A marine fungus, characterized in that: the marine fungus is Alternaria alternata FB1. The strain is preserved in the Guangdong Provincial Microbial Culture Collection Center. The address is 5th Floor, Building 59, Courtyard, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation date is Jul. 7, 2021, with the collection number GDMCC No:61788.
Application of the marine fungus, i.e. the marine fungus adopted in degrading plastic pollutants in the environment.
The application of the bioactive substances produced by the marine fungus, genetically modified strains using this strain as the starting strain, or the bioactive substances produced by the genetically modified strains using this strain as the starting strain, in degrading plastic pollutants in the environment.
The plastics are polyethylene terephthalate (PET), polyethylene (PE), polyester polyurethane (PAUR), polyether polyurethane (PEUR), polypropylene (PP), polystyrene (PS), one or more of the common biodegradable plastics.
The common biodegradable plastics include one or more types of poly(butylene adipate-co-terephthalate) (PBAT), polybutylene adipate (PBA), polylactic acid (PLA), starch (St), and corn base. Furthermore, several common biodegradable plastics are the biodegradable plastics mixed with poly(butylene adipate-co-terephthalate), polylactic acid and starch (PBAT+PLA+St), the biodegradable plastics mixed with poly(butylene adipate-co-terephthalate) and polylactic acid (PBAT+PLA), the biodegradable plastics mixed with poly(butylene adipate-co-terephthalate), polylactic acid and corn base (PBA+PLA+corn-base).
The microbial inoculant for degrading plastics, which contains one or several following strains or substances, including the fungal strain, the genetically modified strain derived from this strain, the bioactive substances of the strain, and the bioactive substance of the genetically modified strain derived from this strain.
The microbial inoculant contains the culture fluid, the concentrate of culture fluid, or suspension of the cultured fungal strain.
The microbial inoculant culture fluid is made by the strain or genetically modified strain derived from the fungal strain in a culture medium (the culture medium is PDB liquid culture medium) and cultivating it at 30° C. for 2-3 days.
An application of the microbial inoculant in degrading plastic pollutants.
The plastics are polyethylene terephthalate (PET), polyethylene plastic (PE), polyester polyurethane (PAUR), polyether polyurethane (PEUR), polypropylene (PP), polystyrene (PS), one or more of the common biodegradable plastics.
When the microbial inoculant is applied to polyethylene plastic, the surface of the PE plastic is eroded (such as holes appearing), the hydrophilicity of the PE plastic is enhanced, and the molecular weight and crystallinity of the PE plastic are reduced.
When the microbial inoculant is applied to polyethylene terephthalate plastic, the surface of the PET plastic is eroded (such as holes appearing), the hydrophilicity of the PET plastic is enhanced, and the molecular weight and crystallinity of the PET plastic are reduced.
When the microbial inoculant is applied to polypropylene plastic, the surface of the PP plastic is corroded (such as holes appearing).
When the microbial inoculant is applied to polystyrene plastic, the surface of the PS plastic is corroded (such as holes appearing).
When the microbial inoculant is applied to polyester-type polyurethane plastic, the PAUR plastic is corroded (such as holes and/or cracks appearing).
When the microbial inoculant is applied to polyether-type polyurethane plastic, the surface of the PEUR plastic is corroded (such as holes appearing).
When the microbial inoculant is applied to several common biodegradable plastics: PBAT+PLA+St plastic, PBAT+PLA plastic, and PBA+PLA+corn-base plastic, these plastics are corroded (such as holes and/or cracks appearing).
The marine fungus in the present invention is isolated from the intertidal zone of Qingdao bathing beach and belongs to filamentous fungi. The sterile hyphae are creeping and separated, and the conidiophores are solitary or clustered. Most are unbranched, short, and almost indistinguishable from vegetative hyphae. The conidium is inverted rod-shaped, its tip lengthened into a pale brown beak, with dark brown brick-like partitions, and a constant number of them are in chains. It is a common saprophytic fungus in soil and air. This strain can efficiently degrade polyethylene terephthalate, polyethylene, polyester polyurethane, polyether polyurethane, polypropylene, polystyrene, and several common biodegradable plastics. It has potential application value in terms of developing microbial inoculants for environmental remediation and improving the ecological environment. At the same time, mixing it with other strains also has a certain effect on the degradation of plastics.
Compared with traditional plastic pollutant treatment methods, microbial degradation also has potential application value in solving the problem of “White Pollution”.
The present invention will be further described below regarding specific examples. These examples are only used to illustrate the invention and are not intended to limit the scope of the invention.
Isolation and identification of a marine derived fungus, Alternaria alternata: In mid-to-late July 2017, sediments with plastic garbage were collected from the intertidal zone of Qingdao No. 1 Bathing Beach. The samples contained beverage bottles (PET), fast food packaging boxes (PET and PE), plastic wrap (PE), and other plastic debris fragments and surrounding soil sediments, a total of 300 samples.
Sterile seawater with PET and PE plastics was the screening medium. The 300 samples collected were placed in the screening medium to screen plastic-degrading microorganisms at room temperature. After six months of screening, a strain that could effectively colonize two types of plastics and have a degradation effect was finally obtained. According to its morphological observation, it may be a fungus. The strain was further isolated and cultured on PDA medium in a 30° C. constant temperature incubator to obtain pure colonies and preserve the strains.
PDA medium components: 200 g potatoes, 20 g glucose, 15-20 g agar, 1 L water, pH 7.5.
The fungus was identified by ITS. Through comparison, it was found that the fungus was Alternaria alternata, with an identity of 99.81%.
Its ITS sequence is:
The preservation information of the above strains is as follows:
The strain Alternaria alternata obtained in the above embodiment was inoculated into PDB liquid medium (200 g potatos, 20 g glucose, 1 L water, pH 7.5) and cultured at 30° C. for 2 days. The resulting microbial suspension was obtained as the fungal inoculant.
Use the fungal inoculant obtained above to degrade plastics:
1) PET and PE plastics were placed at a constant temperature of 30° C. in a basic liquid medium (xylose 0.02 g/L, yeast powder 0.05 g/L, seawater 1 L, pH 7.5) with or without the above-mentioned fugal inoculants in an incubator. After two weeks of cultivation, take a piece of plastic sheet and prepare the biological sample to observe the biofilm colonization on its surface (
Method for preparing biological samples for scanning electron microscopy: wash the plastic sheet containing bacterial biofilm once with 0.1 mol/L PBS (pH7.0), soak the plastic sheet with 5% glutaraldehyde to fix the cells for 1 hour, wash the plastic sheet with sterile PBS, dehydrate with 30%, 50%, 70%, 90%, and 100% graded ethanol for 15 min respectively, and dry with CO2. The material is mounted on the pile and sputter coated with gold and platinum (10 nm) for 5 min using a Hitachi MC1000 ion sputterer, and then observed with a scanning electron microscope (Hitachi S-3400N). The scanning electron microscope observation voltage is 5 kV, the energy spectrum scanning voltage is 5 k eV, and the action time is 30 s.
Observing the colonization effect of fungi on plastics from
2) PET and PE plastics were placed at a constant temperature of 30° C. in a basic liquid medium (xylose 0.02 g/L, yeast powder 0.05 g/L, seawater 1 L, pH 7.5) with or without the above-mentioned inoculants in an incubator. PET was co-cultured with the above-mentioned microbial inoculants for two weeks for observation, and PE was co-cultured with the above-mentioned microbial inoculants for 120 days for observation. Take the plastic film and prepare non-biological samples to observe the surface degradation (see
Method for preparing non-biological samples for scanning electron microscopy: soak in a 50° C. constant-temperature water bath with 3% hydrogen peroxide for 12 hours, ultrasonically wash with 75% ethanol and distilled water for 30 minutes, and then fully dry. The material was mounted on the pile and sputter coated with gold and platinum (10 nm) for 5 min using a Hitachi MC1000 ion sputterer and observed with a scanning electron microscope (Hitachi S-3400N). The scanning electron microscope observation voltage is 5 kV, the energy spectrum scanning voltage is 5 keV, and the action time is 30 s.
From the electron microscope observation shown in
3) Add the microbial inoculant above to the basic liquid medium containing PET or PE plastics. The amount of the inoculant added was about 1 ml of the fungal suspension. After addition, culture for 2 weeks and 4 weeks. The medium without microbial inoculant was defined as the control group. Then the PE and PET plastic film colonized by fungi were washed away from the biofilm for infrared observation (
The above method of removing biofilm was to clean with 3% hydrogen peroxide, distilled water, and 75% ethanol in an ultrasonic cleaner for 30 minutes. After sufficient drying, the PE or PET film was scanned using a Nicolet-360 FTIR (Waltsam, USA) spectrometer with a wavelength range of 450-4000 cm−1 and a resolution of 1 cm−1, operating in ATR mode. Thirty-two scans were performed for each spectrum.
The results in
The results in
4) Add the microbial inoculant above to the basic liquid medium containing PE plastic. The amount of the inoculant added was about 1 ml of the fungal suspension. After addition, culture for 120 days. The medium without microbial inoculant was the control group. Then the plastic treated with the microbial inoculant was analyzed by high-temperature gel chromatography for its degradation ability (
The results in
5) Add the microbial inoculant above to the basic liquid medium containing PET or PE plastics. The amount of the inoculant added was about 1 ml of the fungal suspension. After addition, culture for four weeks. The medium without microbial inoculant was the control group. Then the X-ray diffraction pattern of the plastic was used to analyze the degradation ability of the microbial inoculant (
The results in
6) Put the four plastics of PP, PS, PAUR, and PEUR into rice medium (rice 700 g/L, yeast powder 5 g/L, peptone 3 g/L, corn steep liquor 2 g/L, MSG 6 g/L, seawater 1 L) with or without the above-mentioned inoculants. After culturing for 30 days in a 30° C. constant-temperature incubator, take out each plastic film and prepare non-biological samples to observe their surface degradation (
Method for preparing non-biological samples for scanning electron microscopy: After 30 days of culture, take out each plastic film and soak it in a 50° C. constant-temperature water bath with 3% hydrogen peroxide for 24 hours, then ultrasonically wash it with 75% ethanol and distilled water for 30 minutes, and then intensive drying. The material was mounted on the pile and sputter coated with gold and platinum (10 nm) for 5 min using a Hitachi MC1000 ion sputterer. Observe with a scanning electron microscope (Hitachi S-3400N). The scanning electron microscope observation voltage was 5 kV, the energy spectrum scanning voltage was 5 keV, and the action time was 30 s.
From the electron microscope observation in
7) Add the microbial inoculant above to the rice medium containing PAUR plastic. The amount of the inoculant added was about 1 ml of the fungal suspension. After addition, culture for 7 days, 14 days, 21 days, 28 days, and 35 days respectively. The medium without microbial inoculant was the control group. Then the biofilm was washed away from the PAUR membrane colonized by the bacterial inoculant for observation (
The above method of removing biofilm was to soak in 3% hydrogen peroxide in a constant temperature water bath at 50° C. for 24 hours, and ultrasonically clean with 75% ethanol and distilled water for 30 minutes respectively. After sufficient drying, observe.
8) Add the inoculants above to the rice medium (rice 700 g/L, yeast powder 5 g/L, peptone 3 g/L, corn steep liquor 2 g/L, monosodium glutamate 6 g/L, seawater 1 L) containing different conventional biodegradable plastics (biodegradable plastics containing poly(butylene adipate-co-terephthalate), polylactic acid and starch (PBAT+PLA+St)), biodegradable plastics containing poly(butylene adipate-co-terephthalate) and polylactic acid (PBAT+PLA), biodegradable plastics containing polybutylene adipate, polylactic acid and corn-base (PBA+PLA+corn-base)). The amount of the inoculant added was about 1 ml of the fungal suspension and cultured for 30 days. The medium without microbial inoculant was the control group. Then different conventional biodegradable plastic films colonized by fungi were washed away and observed (
The above method of removing biofilm was to soak in 3% hydrogen peroxide in a constant temperature water bath at 50° C. for 12 hours, and ultrasonically clean with 75% ethanol and distilled water for 30 minutes respectively. After sufficient drying, observe.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111002458.7 | Aug 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/098055 | Jun 2022 | WO |
| Child | 18590561 | US |
