Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of and in specific relates to an Aspergillus based fungal strain and use for degrading polyethylene (PE) films and method of cultivating the Aspergillus based fungus strain.
Background of the invention:
[0002] Enormous tons of plastic materials are produced around the world every year. Low density polyethylene (LDPE) is a long chain polyethylene that is the most widely used plastic material. LDPE is widely produced in the form of plastic bags for packaging materials due to their effectiveness and versatile properties, such as low-cost, durability, light-weight, energy efficiency, and ease.

[0003] Plastic bags are evaluated as one of the major plastic wastes causing global environmental pollution due to their lightness, high tensile strength, and resistance to water and microbial attacks. These plastic bags are generally released in the environment by dumping into landfills, rivers, and oceans. In dumping areas, terrestrial animals usually consume plastic bag wastes along with foodstuffs, which disrupts their digestive systems, resulting in the deaths of millions of animals.

[0004] At present, accumulated plastic bag wastes are degraded by physical, chemical, and photo-degradation methods. However, these methods are very expensive, and release pollutant organic compounds and toxic irritant products into the environment, such as furans and dioxins. The toxic compounds result in soil infertility, preventing degradation of normal substances, and depletion of underground water sources. Therefore, there is a need for an eco-friendly method for degradation of plastic bags.

[0005] Recently, biodegradation by microorganisms is a method used to degrade plastic bag wastes through anaerobic processes in soil and composts, producing carbon dioxide, water, and methane. Therefore, biodegradation is considered as an eco-friendly process for the removal of plastic bags which may be applied as an alternative method for plastic waste management. The biodegradation rate of microorganisms is dependent on various factors, such as substrates, environment, temperature, and the molecular weight of the plastic material. Their rates are increased through various reactions such as abiotic hydrolysis, photo-oxidation, and physical disintegration.

[0006] Various fungal genera used in the biodegradation of plastics, such as Gliocladium, Cunninghamella, Penicillium, Aspergillus, Fusarium, Mucor, and Mortierella. In addition, many filamentous fungi including A. niger, Aspergillus terreus, Aureobasidium pullulans, Paecilomyces varioti, Penicillium funiculosum, Penicillium ochrochloron, Scopulariopsis brevicaulis, and Trichoderma viride are reported to have LDPE-degrading abilities. The ability of fungi in the biodegradation of plastics is enhanced by producing special intracellular and extracellular enzymes for degrading polymers into small oligomers, dimers, and monomers. These products are used as carbon sources for fungi growth and are converted into water, carbon dioxide, or methane.

[0007] However, the existing method for biodegradation by microorganisms exhibit a poor degradation rate of LDPE, which could result in incorrect interpretations of the outcomes. For example, mixed cultures of several bacterial and fungal strains isolated from sludge, contaminated soil, and marine sediments show extremely slow biodegradation.

[0008] Therefore, there is a need to develop a more effective polyethylene (PE) degradation method and an improved biological system to increase the rate of polyethylene (PE) degradation. There is a need for a fungus strain that can produce extracellular enzymes laccase and manganese peroxidase to break down polyethylene (PE). There is a need for a microbe A. proliferans utilized for LDPE degradation and maximal weight loss.
Objectives of the invention:
[0009] The primary objective of the invention is to provide an Aspergillus proliferans based fungal strain and use for degrading polyethylene (PE).

[0010] Another objective of the invention is to provide an isolate fungal strain from polluted sites, for degrading Low-density and High-density polyethylene each of 50μ and 75μ.

[0011] The other objective of the invention is to provide a more effective polyethylene (PE) degradation method to increase the rate of polyethylene (PE) degradation so that the method can be used commercially.

[0012] The other objective of the invention is to screen extracellular enzymes laccase and manganese peroxidase which involve in the breakdown of polyethylene (PE).

[0013] Another objective of the invention is to provide a microbe A. proliferans utilized for LDPE degradation and maximal weight loss.
Summary of the invention:
[0014] The present disclosure proposes an Aspergillus Based Fungal Strain Culture for Degrading low density Polyethylene (LDPE) and Method Thereof. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0015] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem by cultivating the Aspergillus strain which is capable of degrading polyethylene (PE).

[0016] According to an aspect, the invention provides a fungal stain Aspergillus proliferans (A. proliferans) used for biodegradation of polyethylene (PE) films. In specific, the polyethylene films comprises low density polyethylene (LDPE) and high density poly ethylene (HDPE) each with different micron sizes 75μ and 50μ.

[0017] According to another aspect, the invention provides a method for cultivating the fungal stain A. proliferans. First, 0.1 mL of acclimatized culture is inoculated at 30°C for 5 days for enabling the formation of colonies to obtain sub-cultured fungi. Next, polyethylene (PE) powder is added to the mineral salt medium (MSM) to obtain a solution. Next, the obtained solution is subjected to sonication to obtain a sonicated mixture. Later, the sonicated mixture is autoclaved to obtain a sterilized mixture. Next, the sterilized mixture is solidified in petri plates and a sterilized well cutter well is used to cut the solidified sterilized wells. Next, 20 μL of the sub-cultured fungi is added to each solidified well, and incubated for 2 weeks. Further, growth of one or more colonies are observed.

[0018] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0019] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0020] FIG. 1 illustrates an exemplary flowchart of a method for cultivating the fungal strain Aspergillus proliferans (A. proliferans), in accordance to an exemplary embodiment of the invention.

[0021] FIG. 2A illustrates an exemplary SEM analysis of 50μ and 75μ untreated LDPE films, in accordance to an exemplary embodiment of the invention.

[0022] FIG. 2B illustrates an exemplary SEM analysis of 50μ and 75μ treated LDPE films by Aspergillus, in accordance to an exemplary embodiment of the invention.

[0023] FIGs. 3A-3B illustrate exemplary FTIR spectra of 50 μ LDPE film both untreated and treated by Aspergillus and FTIR spectra of 75 μ LDPE film both untreated and treated by Aspergillus, respectively, in accordance to an exemplary embodiment of the invention.

[0024] FIGs. 4A-4B illustrate exemplary XRD analyses of 50 μ and 75μ LDPE films untreated and treated with A. proliferans, respectively, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0025] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0026] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide an Aspergillus based fungal strain used for degrading polyethylene (PE) films and method of cultivating the Aspergillus based fungal strain.

[0027] According to an exemplary embodiment, the invention provides a fungal strain Aspergillus proliferans (A. proliferans) that is used for biodegradation of polyethylene (PE) films. In specific, the polyethylene films comprises low density polyethylene (LDPE) and high density poly ethylene (HDPE) each with different microns sizes 50μ and 75μ respectively.

[0028] According to another exemplary embodiment of the invention, soil samples are collected from different areas like garbage, dump yard, oil-contaminated and industrial wastes. Then, serial dilution of the soil samples is done by adding 1g of each soil sample to 10 mL of sterilized distilled water and kept shaking for 1 h. 9 test tubes are taken with 9 mL of sterile distilled water. To make a first test tube's total volume 10 mL, 1mL of sample is added from properly mixed samples. A first 10-1 dilution is obtained. Next, 1 mL of a mixture is now withdrawn from the 10-1 dilution and transferred into the second tube to obtain a second dilution 10-2. Above mentioned steps are continued for the other test tubes, with the cells being diluted to a final concentration of 10-6.

[0029] For acclimatization, in a conical flask, 500 mg of PE powder is mixed with 50 mL of Mineral Salt Medium (MSM) to obtain a media. After sonication of the media for an hour, 1mL of the serially diluted soil samples are added and then placed in an orbital shaking incubator at 30°C for 5 days.

[0030] According to another exemplary embodiment of the invention, FIG. 1 refers to an exemplary flowchart of a method 100 for cultivating the fungal stain A. proliferans. At step 102, 0.1 mL of acclimatized culture is inoculated at 30°C for 5 days for enabling the formation of colonies to obtain sub-cultured fungi. At step 104, polyethylene (PE) powder is added to the mineral salt medium (MSM) to obtain a solution. At step 106, the obtained solution is subjected to sonication to obtain a sonicated mixture. Later, the sonicated mixture is autoclaved to obtain a sterilized mixture. At step 108, the sterilized mixture is solidified in petri plates and a sterilized well cutter well is used to cut the solidified sterilized wells. At step 110, 20 μL of the sub-cultured fungi is added to each solidified well and incubated for 2 weeks. At step 112, one or more colonies formed capable of degrading polyethylene are observed.

[0031] In an embodiment, a comparative study is carried out on the biodegradation of PE films (LDPE & HDPE) each with different micron sizes such as 75μ and 50μ. First, LDPE film (50μ & 75μ) and HDPE film (50μ & 75μ) are cut into equal 5cm × 5cm pieces, sterilized with ethanol and air-dried to obtain disinfected sheets. The disinfected sheets are placed in 250 mL of an Erlenmeyer flask containing 100 mL of MSM. Later, inoculated with 5 mL of a mid-exponential-phase culture of fungal suspension. The flasks are incubated for 90 days in an orbital shaking incubator at 120 rpm, 30°C. After 90 days of incubation, weight loss%, dissolved carbon dioxide and the protein content of the films are measured.

[0032] For weight loss measurement, the films recovered are cleaned with 2 percent (w/v) aqueous Sodium Dodecyl Sulphate solution, then with distilled water and dried overnight on filter paper. For dissolved carbon dioxide analysis, carbonic acid is titrated against sodium hydroxide to measure the dissolved carbon dioxide in the culture flasks.

[0033] The protein content is determined by boiling the films for 30 min in 5 mL of 0.5M NaOH solution to obtain a mixture. The mixture is filtered followed by centrifugation at 4°C for 1 minute and the protein content is determined using the Bradford reagent technique. Later, the hydrophobicity of fungal surfaces is assessed. Further, potential LDPE degrading fungal strain is identified and characterized by 18s rRNA sequencing.

[0034] In an embodiment, the biodegradation of LDPE by A.proliferans is conducted at preliminary optimized parameters. The preliminary optimized parameters are 27°C temperature, pH 8, 60 days of the incubation period, and 13 days of photo-oxidation method, MnCl2 0.03 mmol/L, NaNO3 0.75 g/L, cellulose 2.0 g/L and ethanol at a concentration 0.5 g/L.

[0035] At the preliminary optimized parameters, maximum LDPE degradation obtained is 55.98% with dissolved CO2 of 2.95 g/L for 50μ LDPE film. Similarly, maximum LDPE degradation obtained is 45.02% with dissolved CO2 of 1.86 g/L for 75μ LDPE film. At the optimized parameters, maximum biodegradation is observed using 50μ LDPE film for A. proliferans.

[0036] For statistical optimization, pH, temperature and incubation period are optimized based on Central Composite Design (CCD) using Design expert 13.0 v software, for the degradation of LDPE using A. proliferans. The morphological, functional groups and crystallinity of LDPE films are investigated using scanning electron microscope (SEM), Fourier transform infrared (FTIR) and X-Ray diffraction analysis (XRD) analyses respectively. Further, enzymes like laccase, esterase and manganese peroxidase involved in the degradation of LDPE are investigated.

[0037] According to another exemplary embodiment of the invention, FIGs. 2A and 2B refer to exemplary SEM analysis of 50μ and 75μ untreated LDPE films, and SEM analysis of 50μ and 75μ treated LDPE films by Aspergillu, respectively. By performing SEM analysis proved that microbial depolymerizing enzymes and biofilm formation caused LDPE's surface abnormalities (craters and fissures), adhesion, cleavage (porous surface erosion) and morphology.

[0038] According to another exemplary embodiment of the invention, FIGs. 3A and 3B refer to exemplary FTIR spectra of 50 μ LDPE film both untreated and treated by Aspergillus and FTIR spectra of 75 μ LDPE film both untreated and treated by Aspergillus, respectively. From FTIR analysis, the formation of aldehyde and Thiol groups, bond stretching in carboxyl groups, change in bonds of C≡C, formation in halide groups, anhydrides like esters and ketones, carbonyl groups at different frequencies indicate degradation of LDPE. Along with the formation of new peaks, the disappearance of some functional groups is also observed in A. proliferans treated LDPE films of 75 μ and 50 μ.

[0039] According to another exemplary embodiment of the invention, FIGs. 4A and 4B refer to exemplary XRD analyses of 50 μ and 75μ LDPE films untreated and treated with A. proliferans, respectively.

[0040] The formation of additional peaks and an increase in peak strength are also used to assess crystallinity in the amorphous zone. The extracellular enzymes laccase and manganese peroxidase, which are essential to break down LDPE, can be produced by A. proliferans.

[0041] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, an Aspergillus based fungus strain, strain culture and use for degrading polyethylene (PE) films is disclosed and method of cultivating the Aspergillus based fungal strain.

[0042] The Aspergillus proliferans based fungal strain, is used for degrading polyethylene (PE). The proposed effective polyethylene (PE) degradation method increases the rate of polyethylene (PE) degradation. The fungal strain produces extracellular enzymes laccase and manganese peroxidase which are responsible for the breakdown of polyethylene (PE). The microbe A. proliferans utilized for LDPE degradation in terms of maximal weight loss & dissolved carbon dioxide.

[0043] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I / We Claim:
1. A method of cultivating a fungal strain Aspergillus proliferans (A. proliferans) for biodegradation of low density polyethylene, comprising:
inoculating 0.1 mL of acclimatized cultures at 30°C for at least 5 days for enabling formation of colonies to obtain sub-cultured fungi;
adding polyethylene (PE) powder to mineral salt medium (MSM) to obtain a mixture; and
subjecting the obtained mixture for sonication to obtain an agitated mixture, and then autoclaved.
2. The method of cultivating a fungal strain Aspergillus proliferans (A. proliferans) for biodegradation of low density polyethylene as claimed in claim 1, wherein the method comprises 0.1 mL of acclimatized culture is inoculated at 30°C for 5 days for enabling the formation of colonies to obtain sub-cultured fungi.
3. The method of cultivating a fungal strain Aspergillus proliferans (A. proliferans) for biodegradation of low density polyethylene as claimed in claim 1, wherein the sterilized media is poured into one or more sterilized plates and a sterilized well cutter wells is used to cut the wells.
4. The method of cultivating a fungal strain Aspergillus proliferans (A. proliferans) for biodegradation of low density polyethylene as claimed in claimed 1, wherein 20 μL of the sub-cultured fungi is added in each solidified well.
5. The method of cultivating a fungal strain Aspergillus proliferans (A. proliferans) for biodegradation of low density polyethylene as claimed in claimed 1, wherein the fungal stain A. proliferans is used for biodegradation of low density polyethylene (LDPE) films of 75μ and 50μ.