Method for enhancing biodegradability of polyolefin materials

Abstract

The present invention relates to a method for enhancing the biodegradability of a polyolefin material, the method comprising the steps of: providing a polyolefin material; mixing the polyolefin material with at least one fatty agent; heating the polyolefin material mixed with at least one fatty agent to the melting temperature of said polyolefin material to obtain a molten material; allowing said molten material to cool at room temperature for a sufficient time to obtain a solidified product; culturing the solidified product with at least one fungal mycelium selected from fungal strains that excrete unspecific peroxygenases (UPOs) in the presence of at least one fungal culture medium.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for enhancing the biodegradability of polyolefin materials. BACKGROUND

[0002] Synthetic polymers have flooded our lives in a wide range of applications over the last 50 years, with their advantageous features such as light weight, tunable physical properties, low cost, easy processing, etc. In particular, polyolefins (POs) constitute the most widely used polymeric material and almost account for 60% of the total plastic content of municipal solid waste. The PO recovery rate worldwide is less than 10%, and the remaining waste material mainly comes from disposable packaging that is improperly discarded. Because of the widespread disposal and the disadvantage of biopersistence of plastic materials, plastic materials are found to accumulate in the environment all over the world, causing a devastating impact on wildlife. Therefore, the necessity of developing a strategy to avoid the accumulation of polymeric waste in the environment has become urgent.

[0003] Biotechnology-based strategies can achieve enhanced biodegradation and obtain specific high-value products by taking advantage of the specificity and selectivity of enzyme catalysis. Alshehrei F. [Alshehrei F. 2017. Journal of Applied & Environmental Microbiology, Vol. 5 (1): 8] discloses the potential of inducing the biodegradation of plastics by the attack of microbial enzymes; however, the efficiency of these methods is still low and cannot yet constitute a reliable method for large-scale reduction of plastic waste.

[0004] Restrepo-Flórez et al. [Restrepo-Flórez et al. Int. Biodeterior. Biodegrad. 2014, 88, 83-90] describe the resistance of polyolefins to microbial attack. Sheik et al. [Sheik et al. International Biodeterioration & Biodegradation. 105 (2015): 21] describe that low-density polyethylene (LDPE) forms carbonyl groups after exposure to gamma radiation at doses of 200 to 1000 kGy, while lower doses were ineffective, and there is evidence that fungi are effective at plastic degradation of LDPE strips irradiated with the highest dose. The absorbed doses employed in the experiments are very high compared to the doses commonly used in the materials industry, i.e. 10-30 kGy for sterilization, 20-50 kGy for catalytic polymerization and grafting, and 50-150 kGy for inducing crosslinking. Due to the high levels of energy required, this pre-treatment can be difficult to apply on an industrial level for the treatment of large amounts of waste polymer materials. Therefore, the necessity to develop alternative sustainable pre-treatment methods remains crucial. The value of pre-treatment methods aimed at increasing the biodegradability of polyolefins is strictly related to the reliability of the soon-to-be transferred to industry.

[0005] A second challenge faced in order to biodegrade PE is the identification of effective biotechnological tools (enzymes / microorganisms). It is worth noting that the enzymes used to induce the degradation of other commercially diffused plastics, such as polyethylene terephthalate (PET) or nylon, are completely useless for the degradation of polyolefins. In fact, the former contain hydrolysable ester or amide bonds, while the polyolefin chain is composed of carbon and hydrogen atoms only. In comparison with the former, C-C and C-H bonds are high in energy and difficult to break. In fact, due to the lack of active carbons, C-C and C-H bonds are not easily hydrolyzed by the nucleophilic attack of oxygen of water molecules.

[0006] Microorganisms used to induce PE biodegradation are usually selected "blindly" by screening unknown microbial communities and assessing their degradation potential. Polymers in the form of films or powders are incubated with soil collected at landfills that contain microbial consortia. Microorganisms able to grow using PE as sole carbon source are isolated and possibly further characterized for the enzymes involved in biodegradation. The work of Yang [Yang et al., Environ. Sci. Technol. 2014, 48, 13776] and Bertocchini [Bombelli et al., Current Biology 27, R283-R293, April 24, 2017] represent an example of this approach, which independently identified some earthworms that feed on polyolefin materials. The main experimental evidence is that PE is fragmented and swallowed by earthworms. In addition, the authors present weak evidence of potential biodegradation activity. In terms of details, Yang isolated some bacterial strains from earthworm guts, but did not identify any putative relevant enzymes. Bertocchini detected some activity after smearing earthworm homogenate on polyethylene films. But no specific microorganism was isolated and no enzyme was identified. Moreover, Weber et al. argue that Bertocchini's experiments lack comparison with negative controls to demonstrate that the detected signal can be attributed to biodegradation of PE and not to biological material from earthworm homogenate. In conclusion, the apparent macroscopic consumption of plastic material can be attributed to chewing and swallowing by earthworms, rather than to real biodegradation. Moreover, it must be considered that this fragmentation activity generates microplastics that are much more harmful to the environment and wildlife than large plastic waste.

[0007] The only approach described in the literature aiming at rationally selecting a biotechnological tool for the biodegradation of PE is to consider the ability of laccases to degrade lignin, the most persistent natural biopolymer, and to try to employ laccases. These enzymes are secreted by white-rot fungi and are able to catalyze the oxidation of (poly)phenolic substrates such as lignin. Despite the low redox potential of laccases (0.5 to 0.8 V), which would limit the activity of these enzymes to only phenolic species, this class of enzymes has been successfully used to degrade non-phenolic aromatic persistent pollutants such as dyes, pesticides, polycyclic aromatic hydrocarbons, hormonal chemicals, etc. [Xiaoting Jin et al. 2016. Conditions Optimizing and Application of Laccase-Mediator System (LMS) for the Laccase catalyzed Pesticide Degradation. Scientific reports. 6: 35787]. This application is allowed by the so-called laccase mediator system (LMS) which requires the addition of an oxidoreductase mediator. The limitation associated with this LMS is the high cost of the mediator and the need for high mediator / substrate ratios (from 5:1 to 100:1 or more). Moreover, considering the significantly lower oxidizability of saturated polyolefin chains than that of aromatic compounds, the oxidation of polyethylene with a system like LMS developed for the oxidation of aromatic substrates is expected to have very low efficiency. In fact, the potential of LMS disclosed by Mayer and Staples [Laccase: new functions for an old enzyme. Phytochemistry 60 (2002) 551-565] to oxidize not only aromatic compounds but also aliphatic compounds is explicitly limited to polyunsaturated and aromatic allylic alcohols characterized by intensive unsaturation and electron delocalization, high reactivity due to the stability of the free radical reaction intermediates. Therefore, laccases (and the microorganisms secreting them) do not represent a promising candidate for the biodegradation of polyolefin materials.

[0008] Therefore, there is a need in the market for a method for enhancing the biodegradability of polyolefin materials which requires little energy, low cost reagents, environmental sustainability, which is efficient, suitable for the treatment of large amounts of polyolefin materials, which uses efficient biotechnological tools able to degrade polyolefin materials. SUMMARY

[0009] Therefore, the object of the present application is to provide a method for enhancing the biodegradability of polyolefin materials, which requires little energy, low cost reagents, environmental sustainability, is highly efficient, suitable for the treatment of large quantities of polyolefin materials, which uses highly efficient biotechnological tools capable of degrading polyolefin materials.

[0010] This object is achieved by the method outlined in the appended claims, the limits of which constitute an integral part of the present patent application.

[0011] In particular, the first object of the present application relates to a method for enhancing the biodegradability of polyolefin materials, comprising the steps of:

[0012] a. providing a polyolefin material;

[0013] b. mixing the polyolefin material with at least one fatty reagent, in a weight ratio between 1 / 5 and 1 / 1 of fatty reagent / polyolefin material;

[0014] c. heating the polyolefin material mixed with at least one fatty reagent to the melting temperature of said polyolefin material, to obtain a molten material;

[0015] d. allowing said molten material to cool at room temperature for a sufficient time to obtain a solidified product;

[0016] e. incubating said solidified product with at least one fungal mycelium selected from fungal strains secreting non-specific peroxygenases (UPOs) (EC 1.11.2.1) in the presence of at least one fungal culture medium.

[0017] According to a preferred embodiment of the method of the present application, said polyolefin material is preferably selected from the group consisting of: low-density polyethylene (LDPE), polypropylene (PP), high-density polyethylene (HDPE). More preferably, said polyolefin material is low-density polyethylene (LDPE).

[0018] According to a preferred embodiment of the method of the present application, said at least one fatty reagent is preferably selected from the group consisting of: fatty acids (FA), vegetable oils. More preferably, it is selected from the group consisting of: oleic acid (OA), olive oil (OO).

[0019] According to a preferred embodiment of the method of the present application, said at least one fungal mycelium is preferably Agrocybe aegerita mycelium.

[0020] According to a preferred embodiment of the method of the present application, the fungal medium preferably comprises sodium, potassium, magnesium, iron (II) cations, nitrate, hydrogenphosphate, sulfate, chloride anions and further the fungal medium is preferably free of any carbon source. The term "carbon source" refers to those substances from which carbon can be obtained by metabolic reactions. A non-limiting example of a carbon source is a carbohydrate.

[0021] As known to the person skilled in the art, carbon sources can either have a structural function (forming components of the biomass) or be used as an energy source. While autotrophic organisms like plants or cyanobacteria can utilize atmospheric or dissolved CO2 as inorganic carbon source, mycelia and other heterotrophic organisms rely on organic compounds.

[0022] More preferably, the fungal medium is a modified Czapek-Dox broth medium comprising sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, ferrous (II) chloride. As well known to the person skilled in the art, Czapek-Dox broth is a commercially known fungal growth medium, but the Czapek-Dox broth medium according to the present application is modified by mixing only the inorganic salts (sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, ferrous (II) chloride) without mixing any carbon source to force the mycelia to consume the polymer as the only available carbon source.

[0023] According to a preferred embodiment of the method of the present application, preferably between step c and step d, the method further comprises the following steps:

[0024] • once the melting temperature of the polyolefin material is reached, the temperature is maintained for a time period of 4 minutes to 6 minutes; more preferably 5 minutes.

[0025] According to a preferred embodiment of the method of the present application, preferably in step d of the method, the molten material is cooled down at room temperature for a time period of 4 minutes to 6 minutes. More preferably 5 minutes.

[0026] According to a preferred embodiment of the method of the present application, preferably in step e of the method, the incubation takes place at a temperature of 23 °C to 29 °C, a relative humidity (RH) of 60 % to 90 %, for a time period of 1 month to 5 months. More preferably, the temperature is 26 °C, the RH is 70 % to 80 % and the time period is 3 months.

[0027] According to an alternative embodiment of the method of the present application, steps c and d of the method are preferably repeated 1 to 5 times in succession.

[0028] The present invention aims at developing an effective method to solve the environmental problem of plastic pollution, exploiting chemical and biotechnological approaches to enhance the potential of polyolefin biodegradation. In particular, the claimed method focuses on a double strategy, which includes the design of a pre-treatment step, using a fatty reagent, to help the material colonization by fungal mycelium, and the selection of suitable fungal strains that can effectively oxidize polyolefin materials.

[0029] Among those strains expressing enzymes (mainly monooxigenases and dioxygenases) able to oxidize and cleave the inactivated C(sp3)-H bond of saturated hydrocarbons, fungal strains for the degradation of mixed LDPE biota are sought.

[0030] Among the potential enzymes and fungal strains, preferred according to the present invention is the fungus A. aegiritus [Gupta et al., BMC Genomics (2018) 19:48; Liers et al., FEMS Microbiol Ecol (2011) 78:91].

[0031] This selection is due to the combination of three different aspects:

[0032] - Aae expresses a dioxygenase (AaeUPO) able to oxidize alkanes;

[0033] - Some bacteria are known to express cytosolic or membrane-bound oxidases (CYT450 or AlkB family), the property of being cytosolic or membrane-bound enzymes means that they can only exert their biocatalytic activity inside the microbial cell; on the contrary, fungal degrading enzymes are secreted, which eliminates the need for internalization of the substrate. This property is determinant for the biodegradation of polyolefins, since it allows to attack long chains, which exceed the length limit of ~500 Da (equivalent to a PE of 40 C long), which is the maximum weight able to cross the cell membrane.

[0034] - Like the cells of most fungi, Aae cells grow as elongated, filamentous structures, i.e. hyphae, which form a dense network called mycelium; this can also exert a mechanical action by "digging" the polyolefin material, similar to the action of plant roots.

[0035] These properties allow a synergistic biodegradation action through the combination of the mechanical pressure of the fungal mycelium and the biocatalytic power of the secreted oxidizing enzymes.

[0036] More specifically, steps a to d of the method of the present invention are directed at loosening and energizing the chain packing of the polyolefin material, which limits the attack of the enzymes, as the mobility and accessibility of the target moieties is very low. Indeed, in order for the enzymatic action of the biocatalysis to occur, the polyolefin chain of the substrate must enter the catalytic pocket and fold properly to fit it and subsequently form an activated complex with the enzyme. This process leads to an increase in the energy demand as the conformational freedom of the substrate chain decreases. A specific treatment of the polyolefin, aimed at loosening the tight packing of the polyolefin chains prior to the enzyme attack, can be the key to facilitate the subsequent enzymatic cleavage. The method of the present invention successfully addresses this challenge by mixing the polyolefin material with a fatty agent (FA or vegetable oil) at a temperature slightly above the melting temperature (Tm) of the polyolefin. The fatty agent has a hydrocarbon tail whose conformation and stiffness depend on its length and on the presence and position of unsaturation. This tail will intercalate in the bulk of the polyolefin and interfere with the chain packing. Moreover, thanks to its polar head (carboxyl or ester group), its integration within the polyolefin matrix is able to disrupt the interactions between the chains, leading to swelling and softening of the polyolefin material.

[0037] The method of the present invention has the advantage of requiring a small amount of energy, as it only requires heating the polyolefin material above its Tm, while the fatty agent can be sourced from waste of the food and oleochemical industries.

[0038] According to a preferred embodiment of the method of the present invention, the preferred fungal mycelium is that of the white-rot basidiomycete: Pleurotus ostreatus (Aae). Aae expresses and secretes a peroxygenase enzyme (AaeUPO) which is able to oxidize alkanes and, as demonstrated by the example given below, has now been advantageously proven to be also able to degrade polyolefins.

[0039] Moreover, the method of the present invention advantageously employs a mycelial network instead of a unicellular fungal suspension, in order to exploit the mechanical action of the mycelium.

[0040] Each of the technical features described above leads to a specific advantage:

[0041] - the step with the fatty agent aims at loosening the polyolefin chain packing, facilitating the mycelial colonization and the enzymatic action;

[0042] - the cultivation with the fungal mycelium allows a deep penetration into the solidified product and has a prominent ability to oxidize the polyolefin chains. BRIEF DESCRIPTION OF DRAWINGS

[0043] - Figure 1It is shown: a) Growth of Ganoderma lucidum (Gl) mycelium in the presence of Potato Dextrose Broth (PDB). Although the mycelium covered the available space in the Petri dish and consumed the PDB, the mycelium did not grow on the original LDPE sheet (left panel), while it colonized the 1 : 1 mixed OA:LDPE and the space around it (right panel). b) Growth of Gl mycelium in the presence of medium without any carbon source (Modified Czapek-Dox Broth, without sucrose). The mycelium can use the mixed material completely as a carbon source: the mycelium did not grow on the original LDPE sheet nor in the Petri dish (left panel), while it colonized the 1 : 1 mixed OA:LDPE and the space around it (right panel).

[0044] - Figure 2 It is shown Gl mycelium grown on LDPE mixed with olive oil (ratio 1 : 1). a) Growth in the presence of PDB; the mixed LDPE is colonized by the mycelium. b) Growth in the presence of medium without any carbon source (Modified Czapek-Dox Broth, without sucrose); the mycelium can use LDPE mixed 1 : 1 with OO as the sole carbon source.

[0045] - Figure 3 It is shown Gl mycelium grown in PDB (a), PDB supplemented with 20% of oleic acid (b) or olive oil (c). Fatty acids and oils inhibit the growth of the mycelium.

[0046] - Figure 4 It is shown mycelium grown on mixed polyolefin material. a) Cross section of the material and mycelium after cultivation. The mycelium grew deep and consumed part of the treated material; (rectangular dashed line - material cross section before cultivation; arcuate dashed line - interface cross section between material and mycelium after mycelium growth). b) Trace left on the material after removal of the mycelium.

[0047] - Figure 5 It is shown SEM images of samples obtained from cultivation of fungal mycelium on LDPE mixed with oleic acid. The samples were collected after isolation of the biological component from the polyolefin material to emphasize the deep interaction between the mycelium and the treated material. a) 2000X magnification of the surface of Gl mycelium grown in direct contact with the polyolefin material. Fragments of polyolefin material trapped within the mycelial network are clearly visible. b) and c) show 1000X and 500X magnification of the polyolefin material cultivated with Gl and Aae mycelium, respectively, and after removal of the biological material. Some mycelium hyphae penetrated into the material (tubular structures with circular cross section).

[0048] - Figure 6A comparison of the ATR-FTIR spectra of oleic acid (OA), raw LDPE (LDPE), OA- mixed LDPE (OA-mixed LDPE), and OA-mixed LDPE after incubation with Aae (OA-mixed LDPE with Aae) is shown.

[0049] - Figure 7 A comparison of the ATR-FTIR spectra of oleic acid (OA), raw LDPE (LDPE), OA- mixed LDPE (OA-mixed LDPE), and OA-mixed LDPE after incubation with Aae (OA-mixed LDPE with Aae) is shown. Figure 6 A zoom of the carbonyl region (1600-1800 cm -1 ) is shown; the CH peak at 1465 is indicated as a reference.

[0050] - Figure 8 A zoom of the ATR-FTR spectra of Pleurotus eryngii and Ganoderma lucidum mycelium in the carbonyl region 1600-1800 cm -1 is shown. The spectrum presents a broad peak with two maxima at about 1645 cm -1 and 1720 cm -1 . No signal is shown at 1750 cm -1 .

[0051] The following examples of embodiments are provided solely for the purpose of illustrating the present application and should not be construed as limiting the scope of the protection defined by the appended claims. Particular embodiments

[0052] Example 1

[0053] LDPE pellets were ground into 3 mm size particles with a dry grinder. 500 mg of ground LDPE was mixed with a fatty agent (pure oleic acid or vegetable oil) in a final material with a fatty agent / LDPE ratio of 1 / 1. Subsequently, the sample was heated to the melting temperature of LDPE (about 120°C) for 5 minutes using a hot plate and was allowed to cool and solidify at room temperature for 5 minutes, repeating the melting / solidification 5 cycles. Melting was easily detected because the polyolefin material became transparent and indistinguishable from the liquid fat. During the melting / solidification cycles, the liquid fat was incorporated into the solid material and the two phases became indistinguishable in the resulting material, which finally solidified into a sheet (1.0 mm thickness). A control sample of raw LDPE was subjected to the same temperature cycles. The sheets after heat treatment were kept in a sterile environment to avoid contamination, cut into squares of 1 cm side and placed in Petri dishes of 5 cm diameter.

[0054] Subsequently, 8 mm diameter discs were punched out from Poploliomyces mycelial mats grown on Potato Dextrose Broth (PDB, a commercially known medium for fungal growth as known by the skilled person) and placed on the surface or in the proximity of the polyolefin sample. Mycelia were grown in PDB or in a modified Czapek-Dox medium prepared by mixing only the inorganic salts (sodium nitrate, potassium hydrogen phosphate, magnesium sulfate, potassium chloride, ferrous (II) chloride) without any carbon source in the presence of the polyolefin material. Growth conditions were maintained at 26°C and 70-80% RH in a plant growth chamber (Mennert). Mycelia were grown for 3 months.

[0055] At the same time, Ganoderma lucidum (Gl), another white-rot basidiomycete, was chosen as a reference strain, which secretes lignin-modifying enzymes such as laccases, manganese-dependent peroxidases and lignin peroxidases [D'souza et al., Appl. Environ. Microbiol. (1999) 65(12) 5307-5313]. The comparison between the action of Aae and Gl aimed at demonstrating that Poploliomyces has a higher oxidative power on the LDPE chain than other lignin-degrading mycelia (such as Gl).

[0056] The biodegradation of LDPE was evaluated by monitoring the formation of biofilms and the changes in the surface morphology of the polyolefin substrate using a scanning electron microscope (SEM). The growth of the mycelia of the tested strains only on the pretreated (FA-containing) LDPE confirmed the decisive role of the mixing with fatty acids to overcome the well-known microbial inertia of polyolefins. Figure 1 and Figure 2 The characteristic images of Gl mycelia grown on LPDE and OA-mixed LPDE are shown. In particular, mycelia did not grow on the control (raw LDPE) in the presence of alternative carbon sources (PDB) ( Figure 1 a) and in the absence of ( Figure 1 b) they grew abundantly on the polyolefin material modified with the fatty agent. The fact that mycelia could not grow on raw LDPE, despite the availability of all nutrients that allowed the mycelia to grow on the free space of the Petri dish ( Figure 1 a), confirmed that the modification induced by the treatment of LDPE with the fatty agent was the decisive factor for the feasibility of fungal mycelium colonization. Moreover, the results shown in Figure 1 b demonstrated that mycelia could grow on the mixed (with FA) LDPE as the only carbon source.

[0057] Figure 2Gl mycelium growing on LPDE and olive oil-mixed LPDE is shown. This behavior is consistent with the observed colonization of LDPE with OA but not with olive oil. Aae mycelium showed the same behavior as Gl mycelium shown in Figure 1. Figures 1-2

[0058] In this case, it is worth noting that the ability of fatty agents to promote LDPE colonization upon insertion of the material is contrary to the well-known antimicrobial action of these molecules (see Figure 3 ). In fact, Figure 3 Gl mycelium growing in PDB (a), PDB supplemented with 20% of oleic acid (b) or olive oil (c) is shown. As expected, fatty acids and oils inhibited the growth of mycelium.

[0059] Mycelial colonization is not limited to the surface of the treated LDPE, as the hyphae penetrate the material, macroscopically altering its morphology. Figure 4 a shows a cross-section of an OA-mixed LDPE (ratio 1 : 1) sample after incubation with Gl. Before incubation, the sample presented a rectangular cross-section (dotted rectangle), while the mycelium gradually inserted inside the material, consuming it. The curve emphasizes the interface between the mycelium and the material after incubation. Figure 4 b shows another sample that underwent the same process (pre-treatment with FA and incubation), but afterwards, the mycelium was isolated. It can be clearly seen that the portion of OA-mixed LDPE material in direct contact with the mycelium is missing due to its consumption by the mycelium.

[0060] SEM magnification (see Figure 5 ) allows to observe the intimate association of the fungal mycelium with the polyolefin material, with the hyphae penetrating deeply inside the material. The sample was collected after isolating the biological component from the polyolefin substrate to morphologically study the interface between the mycelium and the polyolefin material. Figure 5 a shows a piece of Gl mycelial cluster growing on treated LDPE (OA:LDPE 1 : 1) and after removal of the treated LDPE; it can be observed that polyolefin fragments are trapped inside the Gl mycelial network. In Figure 5 b and 5c further emphasize the intimate interaction between the mycelium and the polyolefin substrate, where the hyphae penetrating the surface of the polyolefin material are still clearly visible in the substrate sample examined after incubation of Gl (b) or Aae (c), although the main volume of the mycelium has been removed.

[0061] Example 2

[0062] Furthermore, chemical analysis by Fourier-transform infrared (FTIR) spectroscopy emphasizes the outstanding ability of the Shaggy mane fungus to oxidize LDPE chains.

[0063] ​Figure 6 The spectra of oleic acid, raw LDPE, OA-mixed LDPE (1 :1) and OA-mixed LDPE after incubation with Aae were compared. Figure 7 is Figure 6 Enlargement of the carbonyl region of the mid-IR spectrum.

[0064] The spectrum of LDPE has four characteristic peaks: 2915, 2845, 1465 and 720 cm -1 The spectrum of OA-mixed LDPE presents the characteristic peaks of LDPE, in addition, three main differences can be identified:

[0065] A broad peak extending between 2250 cm -1 and 3550 cm -1 due to the OH of the free carboxyl group of OA;

[0066] The "fingerprint" of oleic acid in the region below 1500 cm -1 ;

[0067] A sharp strong peak at 1710 cm -1 due to the carbonyl group of the carboxyl group of OA.

[0068] By comparing the spectrum of OA-mixed LDPE with the spectra of pure OA and raw LDPE, all the additional signals listed above are clearly due to the presence of OA. In the LDPE spectrum, as expected, no peak is detected in the carbonyl region (1600-1800 cm -1 ). On the other hand, it is undisputable that the new peak appearing in the spectrum of OA-mixed LDPE at 1710 cm -1 corresponds to the same peak present in the spectrum of OA. Therefore, this is undoubtedly due to the carbonyl group originating from the OA present in the OA-mixed LDPE. It is worth emphasizing that no additional peaks were detected indicative of oxidation of LDPE. In fact, the thermal treatment is a fast melting, only aimed at mixing LDPE with oleic acid, and it does not induce any chemical modification of the polyolefin.

[0069] Finally, after incubation of OA-mixed LDPE with Aae, the spectrum of OA-mixed LDPE clearly indicates the effect of the mycelial biotic action. In particular, the peak at 1710 cm1is much lower in intensity, indicating that, as expected, the mycelium consumed the oleic acid. On the other hand, a new peak is detected in the carbonyl region, at 1745 cm1. It is worth noting that from the comparison with the spectrum of the mycelium alone (see Figure 8 ), it can be excluded that this new peak comes from the fungal material. Therefore, this peak is due to the carbonyl group formed after oxidation of LDPE.

[0070] Since this peak is present only in the sample incubated with Aae (and much less intense in the sample incubated with Gl) and is absent in the OA-blended LDPE, it is suggested that oxidation is not occurring during the blending process, but is caused by the biological action of Aae.

[0071] The small amount of oxidation detected in the OA-blended LDPE sample incubated with Gl is lower compared to the oxidation caused by Aae.

[0072] These results were directly compared with the results published in the literature by calculating the carbonyl index (CI = A C＝O / A 1460 ) of the peak at 1710 cm-1. In fact, as described in the literature [Kyaw, B. M., et al. Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species. Indian J Microbiol 2012, 52(3), 411-419], by normalizing the absorbance of the carbonyl peak with respect to the value of the reference peak of LDPE (C-H bending at 1460 cm -1 -1), this ratio allows a semi-quantitative estimation of the oxidation level. The results obtained show that the oxidative biodegradation potential of the Pleurotus ostreatus mycelium is 4 times higher than the best results reported in the literature so far using microorganisms.

[0073] Document Information Directory

[0074] Alshehrei F. 2017. Journal of Applied & Environmental Microbiology, Volume 5(1): 8.

[0075] Restrepo-Flórez et al. Int. Biodeterior. Biodegrad. 2014, 88, 83-90.

[0076] Sheik et al. International Biodeterioration & Biodegradation. 105 (2015): 21.

[0077] Yang et al. Environ. Sci. Technol. 2014, 48, 13776.

[0078] Bombelli et al. Current Biology 27, R283-R293, April 24, 2017.

[0079] Xiaoting Jin et al., 2016. Conditions Optimizing and Application of Laccase-Mediator System (LMS) for the Laccase catalyzed Pesticide Degradation. Scientific reports. 6:35787.

[0080] Laccase: new functions for an old enzyme. Laccase: new functions for an old enzyme.

[0081] Gupta et al., BMC Genomics (2018) 19:48; Liers et al., FEMS Microbiol Ecol (2011) 78:91.

[0082] D’souza et al., Appl. Environ. Microbiol. (1999) 65(12) 5307-5313.

[0083] Kyaw, B. M. et al. Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species. Indian J Microbiol 2012, 52(3), 411-419.

Claims

1. A method for enhancing the biodegradability of a polyolefin material comprising the following steps: a. providing a polyolefin material; b. mixing the polyolefin material with at least one fatty agent, the weight ratio of fatty agent / polyolefin material being comprised between 1 / 5 and 1 / 1; c. heating the polyolefin material mixed with at least one fatty agent to the melting temperature of the polyolefin material to obtain a molten material; d. allowing the molten material to cool at room temperature for a sufficient time to obtain a solidified product; e. incubating the solidified product with at least one fungal mycelium selected from fungal strains secreting non-specific peroxygenases (UPOs) in the presence of at least one fungal culture medium.

2. The method according to claim 1, wherein the polyolefin material is selected from the group consisting of: low-density polyethylene (LDPE), polypropylene (PP), high-density polyethylene (HDPE).

3. The method according to claim 2, wherein the polyolefin material is low-density polyethylene (LDPE).

4. The method according to any one of claims 1 to 3, wherein the at least one fatty agent is selected from the group consisting of: fatty acids (FA), vegetable oils.

5. The method according to claim 4, wherein the at least one fatty agent is selected from the group consisting of: oleic acid (OA), olive oil (OO).

6. The method according to claim 1, wherein the at least one fungal mycelium is a Shizophyllum commune mycelium.

7. The method according to claim 1, wherein the fungal culture medium comprises sodium, potassium, magnesium, divalent iron cations, nitrate, hydrogen phosphate, sulfate, chloride anions and further wherein the fungal culture medium is devoid of any carbon source.

8. The method according to claim 7, wherein the fungal culture medium is a modified Czapek-Dox broth medium comprising sodium nitrate, potassium hydrogen phosphate, magnesium sulfate, potassium chloride, ferrous (II) chloride.

9. The method according to claim 1, wherein between steps c and d, the method further comprises the following steps: - once the melting temperature of the polyolefin material is reached, maintaining said temperature for a time comprised between 4 minutes and 6 minutes.

10. The method of claim 1, wherein, - in step d of the method, the molten material is cooled at room temperature for a time comprised between 4 minutes and 6 minutes.

11. The method of claim 1, wherein, - in step e of the method, the incubation is carried out at a temperature comprised between 23°C and 29°C and a relative humidity (RH) comprised between 60% and 90% for a time comprised between 1 month and 5 months.

12. The method according to claim 11, wherein the temperature is 26°C, the RH is comprised between 70% and 80% and the time is 3 months.

13. The method according to claim 1, wherein steps c and d of the method are repeated from 1 to 5 times in succession.

Images