PROCESS FOR THE ENHANCEMENT OF THE BIODEGRADABILITY OF POLYOLEFINIC MATERIALS

Abstract

A process for enhancing biodegradability of polyolefinic materials is provided. The process includes providing a polyolefinic material, mixing the polyolefinic material with at least one fatty reagent, heating up the polyolefinic material mixed with the at least one fatty reagent to the melting temperature of the polyolefinic material to obtain a melted material, letting the melted material cool at room temperature for a time sufficient to obtain a solidified product, and incubating the solidified product with at least one fungal mycelium selected from fungal strains secreting Unspecific Peroxygenases (UPO), in presence of a fungal culture medium.

Claims

1. A process for enhancing biodegradability of polyolefinic materials, the process comprising the steps of: (a) providing a polyolefinic material; (b) mixing the polyolefinic material with at least one fatty reagent, with a ratio of fatty reagent/polyolefinic material ranging from 1/5 to 1/1 by weight; (c) heating up the polyolefinic material mixed with the at least one fatty reagent to the melting temperature of the polyolefinic material to obtain a melted material; (d) letting the melted material cool at room temperature for a time sufficient to obtain a solidified product; and (e) incubating the solidified product with at least one fungal mycelium selected from fungal strains secreting Unspecific Peroxygenases (UPO), in presence of at least a fungal culture medium.

2. The process of claim 1, wherein the polyolefinic material is selected form from: low-density polyethylene (LDPE), polypropylene (PP), high-density polyethylene (HDPE).

3. The process of claim 2, wherein the polyolefinic material is low-density polyethylene (LDPE).

4. The process of claim 1, wherein the at least one fatty reagent is selected from: fatty acid (FA), vegetable oil.

5. The process of claim 4, wherein the at least one fatty reagent is selected from: oleic acid (OA), olive oil (OO).

6. The process of claim 1, wherein the at least one fungal mycelium is Agrocybe aegerita mycelium.

7. The process of claim 1, wherein the fungal culture medium comprises sodium, potassium, magnesium, iron (II) cations, nitrate, hydrogenphosphate, sulfate, chloride anions, and further wherein the fungal culture medium is free from any carbon sources.

8. The process of claim 7, wherein the fungal culture medium is modified Czapek-Dox Broth comprising sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, iron(II) chloride.

9. The process of claim 1, wherein between steps (c) and (d), the process also further comprises the following step: once the melting temperature of the polyolefinic material is reached, maintaining the melting temperature of the polyolefinic material for a time comprised between 4 minutes and 6 minutes.

10. The process of claim 1, wherein, in step (d), the melted material is let to cool at room temperature for a time comprised between 4 minutes and 6 minutes.

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

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

13. The process of claim 1, wherein steps (c) and (d) are repeated in sequence from 1 to times.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0043] FIG. 1 shows: a) Ganoderma lucidum (Gl) mycelium growth conducted in presence of Potato Dextrose Broth (PDB). Mycelia did not grow on the sheet of pristine LDPE despite covering the available space in the Petri dish and consuming PDB (left), whereas they colonized the OA:LDPE mixed 1:1 and the space around it (right). b) Gl mycelium growth conducted in presence of a culture medium that does not contain any carbon source (modified Czapek Dox Broth, without sucrose). The mycelium could use exclusively the mixed material as carbon source: mycelia did not grow neither on the sheet of pristine LDPE nor in the Petri dish (left), whereas they colonized the OA:LDPE mixed 1:1 and the space around it (right).

[0044] FIG. 2 shows Gl mycelium grown on LDPE mixed with olive oil (ratio 1:1). a) Growth conducted in presence of PDB; mixed LDPE was colonized by the mycelium. b) Growth conducted a culture medium that does not contain any carbon source (modified Czapek Dox Broth, without sucrose); mycelium was able to use LDPE mixed with OO 1:1 as sole carbon source.

[0045] FIG. 3 shows Gl mycelium grown in PDB (a), in PDB supplemented with 20% of either oleic acid (b) or olive oil (c). Fatty acids and oils inhibit mycelium growth.

[0046] FIG. 4 shows the mycelium grown on the mixed polyolefinic material. a) Cross section of the material and the mycelium after the incubation. The mycelium grew in depth and consumed part of the treated material; (rectangular dotted line—the section of the material before the incubation; curved dotted line—the profile of the interface between the material and the mycelium, after the growth of the latter). b) Trace left on the material after the removal of the mycelium.

[0047] FIG. 5 shows SEM images of samples obtained by incubating fungal mycelium on LDPE mixed with oleic acid. Specimens were collected after the separation of the biological component from the polyolefinic material in order to highlight the deep interaction between mycelium and treated material. a) 2000× magnification of the surface of Gl mycelium, grown in direct contact with the polyolefinic material. Fragments of the polyolefinic material trapped within the hyphal net are clearly visible. b) and c) show 1000× and 500× magnification of the polyolefinic material after the incubation with, respectively, Gl and Aae mycelium and the removal of the biological material. Several mycelial hyphae penetrate in the material (tubular structures with circular section).

[0048] FIG. 6 shows a comparison of the ATR-FTIR spectra of oleic acid (OA), pristine LDPE (LDPE), OA-mixed LDPE (OA-mixed LDPE) and OA-mixed LDPE after the incubation with Aae (OA-mixed LDPE incubation with Aae).

[0049] FIG. 7 shows a zoom on the carbonyl region (1600-1800 cm.sup.−1) of FIG. 6; CH peak at 1465 is indicated as reference.

[0050] FIG. 8 shows a ATR-FTIR spectra of Agrocybe aegerita and Ganoderma lucidum mycelium, zoom on the carbonyl region 1600-1800 cm.sup.−1. The spectra present a broad peak with two maxima at about 1645 cm.sup.−1 and 1720 cm.sup.−1. No signal is present at 1750 cm.sup.−1.

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

EXAMPLES

Example 1

[0052] LDPE pellets were grinded into 3 mm size particles with a dry mill. 500 mg of grinded LDPE was mixed with a fatty reagent (either pure oleic acid or vegetable oil), the ratio fatty reagent/LDPE was 1/1 in the final material. Afterwards, the specimens were heated up to the melting temperature of LDPE (about 120° C.) for 5 minutes, using a hot plate, and let cool and solidify at room temperature for 5 minutes, repeating 5 cycles of melting/solidification.

[0053] Melting was easily detected because the polyolefinic material became transparent and undistinguishable 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 was finally solidified in sheets (thickness 1.0 mm). Control samples of pristine LDPE underwent the same temperature cycles. Sheets, kept in sterile environment after the thermal treatment to avoid contamination, were cut into squares having sides of 1 cm and placed on 5 cm diameter Petri dish.

[0054] Subsequently, 8 mm diameter disks were punched from mycelial mats of Agrocybe aegerita grown on Potato Dextrose Broth (PDB) (as well known by the skilled person in the art, PDB is a commercially known media for growth of fungi) and were put on the surface or in the vicinity of the polyolefinic samples. Incubation of the mycelium in presence of the polyolefinic material was conducted in either PDB or a modified Czapeck-Dox Broth, prepared by mixing only the inorganic salts (sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, iron(II) chloride) without any carbon source. Growth conditions of 26° C. and 70-80% RH were maintained within a plant growth chamber (Memmert). Mycelia were let grow for three months.

[0055] In parallel, Ganoderma lucidum (Gl), another white-rot basidiomycete was chosen as reference strain, which secretes lignin-modifying enzymes such as laccase, manganese-dependent peroxidase, and lignin peroxidase [D'souza et al. Appl. Environ. Microbiol. (1999) 65(12) 5307-5313]. The comparison between the action of Aae and Gl aimed at confirming that the capability of Agrocybe aegerita to oxidize LDPE chains is higher compared to that of other lignin degrading mycelia, as Gl.

[0056] Biodegradation of LDPE was assessed by monitoring the biofilm formation and the changes in surface morphology of the polyolefinic substrates using Scanning Electron Microscopy (SEM). Tested mycelia grew only on pretreated (with FA) LDPE, confirming the determinant effect of the mixing with fatty acids to overcome the well-known microbiological inertness of the polyolefins. FIGS. 1 and 2 demonstrates characteristic pictures of the Gl mycelia grown on LPDE and OA-mixed LPDE. In detail, mycelia did not grow on the control (pristine LDPE), while they grew intensively on the polyolefinic materials modified with the fat reagents, both in presence (FIG. 1a) and in absence (FIG. 1b) of alternative carbon sources (PDB). The fact that mycelia do not grow on pristine LDPE, despite the availability of all the nutrients that allow their growth on the free space of the Petri dish (FIG. 1a), confirms that the modifications induced by the treatment on LDPE with fatty reagent are determinant to render it feasible for fungal mycelial colonization. In addition, the results shown in FIG. 1b, demonstrate that mycelia are able to grow on mixed (with FA) LDPE as sole carbon source.

[0057] FIG. 2 shows Gl mycelia grown on LPDE and olive oil-mixed LPDE. The behavior is consistent with what observed mixing LDPE with OA instead of olive oil. The Aae mycelium showed identical behavior to the one demonstrated in FIGS. 1-2 for Gl mycelium.

[0058] In this context, it is noteworthy that the ability of fatty reagents to promote LDPE colonization, when intercalated in the material, is in contrast with the well-known antimicrobial action of these molecules (see FIG. 3). In fact, FIG. 3 shows Gl mycelium growing in PDB (a) or in PDB supplemented with 20% of either oleic acid (b) or olive oil (c). As expected, fatty acids and oils inhibit mycelium growth.

[0059] Mycelial colonization was not limited to the surface of the treated LDPE, since the hyphae penetrated into the material modifying its morphology macroscopically. FIG. 4a shows a picture of a cross section of a sample of OA-mixed LDPE (ratio 1:1) after the incubation with Gl. Before incubation, the sample had a rectangular section (rectangular dotted line) while the mycelium progressively thrusted inside the material, consuming it. The curved line highlights the interface between the mycelium and the material after the incubation. FIG. 4b shows another sample that underwent the same process (pretreatment with FA and incubation) but, afterwards, the mycelium was detached. It can be clearly seen that part of the OA-mixed LDPE material that was in direct contact with the mycelium is missing due to its consumption by the mycelium.

[0060] SEM magnification (see FIG. 5) allowed observing intimate integration between the fungal mycelium and the polyolefinic material, with hyphae penetrating in depth inside the material. Specimens were collected after the separation of the biological component from the polyolefinic substrate in order to investigate morphologically the interface between mycelium and polyolefinic material. FIG. 5a shows a piece of Gl mycelium mat grown on treated LDPE (OA:LDPE 1:1), after its removal; polyolefinic fragments trapped inside Gl hyphal network can be observed. The intimate interaction between mycelium and polyolefinic substrate is further highlighted in FIGS. 5b and 5c, where hyphae penetrating the surface of the polyolefinic material are clearly visible in specimens of substrate inspected after the incubation of either Gl (b) or Aae (c), even though the main volume of the mycelium mat is already removed.

Example 2

[0061] Furthermore, chemical analysis performed through Fourier-Transform Infrared (FTIR) spectroscopy highlighted an outstanding capability of Agrocybe aegerita to oxidize LDPE chains.

[0062] FIG. 6 compares the spectra of oleic acid, pristine LDPE, OA-mixed LDPE (1:1) and OA-mixed LDPE after the incubation with Aae. FIG. 7 is a zoom of the carbonyl region of the spectra in FIG. 6.

[0063] The spectrum of LDPE is characterized by four peaks: 2915, 2845, 1465 and 720 cm.sup.−1. The spectrum of OA-mixed LDPE presents the characteristic peaks of LDPE, moreover three main differences can be identified: [0064] a broad peak spreading between 2250 cm.sup.−1 and 3550 cm.sup.−1, due to the OH of the free carboxylic moiety of OA; [0065] the “finger-prints” of oleic acid in the region below 1500 cm.sup.−1; [0066] the sharp intense peak at 1710 cm.sup.−1 due to the carbonyl of the carboxylic moiety of OA.

[0067] By comparing the spectrum of OA-mixed LDPE with those of pure OA and pristine LDPE, all the additional signals listed above are clearly derived from the presence of OA. In the spectrum of LDPE, as expected, no peak is detected in the carbonyl region (1600-1800 cm.sup.1). On the other hand, it results uncontroversial that the new peak appearing at 1710 cm.sup.−1 in the spectrum of the OA-mixed LDPE corresponds to the same peak present in the spectrum of OA. For this reason it is undoubtedly assigned to the carbonyl derived from the OA present in the OA-mixed LDPE. It is worth highlighting that no additional peak indicating LDPE oxidation is detected. Indeed, the thermal treatment is a quick melting just aiming at mixing LDPE with oleic acid and it does not provoke any chemical modification of the polyolefin.

[0068] Finally, the spectrum of OA-mixed LDPE, after its incubation with Aae, clearly indicates the effects of the mycelial biotic action. In details, the peak at 1710 cm.sup.−1 has much lower intensity, suggesting that, as expected, the mycelium consumed oleic acid. On the other hand, a new peak is detected in the carbonyl region, at 1745 cm.sup.−1. It is noteworthy that, from the comparison with the spectra of mycelia alone (see FIG. 8), it can be excluded that it derives from the fungal material. This peak was therefore assigned to carbonyls formed as consequence of the oxidation of LDPE.

[0069] Since this peak is present only in samples incubated with Aae (and, with much lower intensity, in those incubated with Gl), while it is absent in OA-mixed LDPE, it demonstrates that oxidation did not occur during the mixing process, but was caused by the biotic action of Aae.

[0070] The little oxidation detected in samples of OA-mixed LDPE incubated with Gl is lower compared with the oxidation caused by Aae.

[0071] A direct comparison of these results with those disclosed in literature was performed through the calculation of the carbonyl index (CI=A.sub.C=O/A.sub.1460). Indeed, this ratio allows a semiquantitative estimation of the level of oxidation, by normalizing the absorbance of the carbonyl peak over the value of the reference peak of LDPE (C—H bending at 1460 cm.sup.−1), as described in literature [Kyaw, B. Metal., Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species. Indian J Microbiol 2012, 52 (3), 411-419]. The results obtained demonstrate an oxidative biodegradation potential of Agrocybe aegerita mycelium fourfold higher than the best so far reported results in the literature using microorganisms.

BIBLIOGRAPHY

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