Iron supplement for plastic biodegradation

Abstract

The present invention relates to methods for enhancing plastic bio-degradation by adding to at least one plastic article at least one strain of plastic-degrading bacteria and at least one additive including iron to form a plastic-degrading composition, and to plastic-degrading compositions.

Description

[0001] IRON SUPPLEMENT FOR PLASTIC BIODEGRADATION

[0002] FIELD OF THE INVENTION

[0003] The present invention is generally directed to biodegradation of plastic materials. More specifically, the invention relates to providing improved conditions for biodegradation of certain polymers including polyolefins by bacteria.

[0004] BACKGROUND OF THE INVENTION

[0005] In our modem lifestyle, plastic is one of the most widely used polymers around the globe. Every year a billion tons of plastic waste accumulates in the environment as a result of its massive consumption and extremely slow degradation rate, a process that may take up to 1000 years in a natural environment.

[0006] Polyethylene (PE) is the most commonly produced polyolefin plastic. It is a polymer, primarily used for packaging. Many kinds of polyethylene are known, with most having the chemical formula (C2H4)n. PE is usually a mixture of similar polymers of ethylene, with various values of n. It can be low-density or high-density and many variations thereof. Its properties can be modified further by crosslinking or copolymerization. All forms are nontoxic as well as chemically resilient, contributing to polyethylene's popularity as a multi-use plastic. However, this chemical resilience also makes it a long-lived and decomposition-resistant pollutant when disposed of improperly.

[0007] The process of degrading materials using enzymatic action of microorganisms is known as biodegradation. This process is considered one of the most eco-friendly methods to deal with plastic waste. Several works have identified laccase and manganese peroxidase as the enzymes responsible for PE degradation (Sowmya et al., 2015, International Journal of Environmental Research 9.3: 823-830; liyoshi et al., 1998, J Wood Sci 44:222). Additional studies showed that a laccase-like enzyme is involved in plastic biodegradation by the bacterium Rhodococcus ruber (Santo et al., 2013, International Biodeterioration & Biodegradation 84 (2013): 204-210) and by the bacterium Brevibacillus agri (WO2014167562A1). In recent years, the use of bacterial laccases is increasing rapidly due to their many remarkable features. For example, bacterial laccases can work under a broad range of temperatures and pH levels and have substantial robustness against various inhibitory agents.

[0008] The production of the plastic-degrading enzymes is dependent on the growth conditions employed, i.e., the presence or absence of inducers, induction time, nature and composition of culture medium, and surrounding conditions. Indeed, different bio -stimulants increase laccase production by microorganisms.

[0009] Metals such as Cu+2, Cd2+, Mn2+, Ca2+, and Li2+have been shown to contribute to laccase production. Among all metals tested, copper was reported to be the most significant additive for improving laccase induction in fungi and bacteria (Mongkolthanaruk et al. 2012, African Journal of biotechnology, 11(39), 9391-9398; Santo et al., 2013, International Biodeterioration & Biodegradation 84: 204-210). In addition, phenolic and aromatic compounds structurally related to lignin or lignin derivatives (e.g., Veratryl alcohol, Guaiacol, 2,5-Xylidine, ferulic acid, and ABTS) were also used for laccase induction in fungi, and it was found that induction by phenolic substances may be caused by a response developed by fungi against toxic aromatic compounds. Several alcohols such as Methanol, 1 -Propanol, 2-Propanol, 2-Methyl-l -propanol, Ethanol, and 1- Butanol, were also tested for improving laccase induction. While all of them were reported to increase laccase activity and specific activity, ethanol exhibited the best effect (Lee et al. 1999, Biotechnology Letters, 21(11), 965-968).

[0010] Accordingly, it appears that finding additives and conditions which increase production and / or activity of plastic-degrading enzymes may improve biodegradation of plastic materials.

[0011] SUMMARY OF INVENTION

[0012] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0013] In some embodiments, there is provided a method for enhancing plastic bio-degradation, the method including adding to at least one plastic article at least one strain of plastic-degrading bacteria and at least one additive including iron which together form a plastic -degrading composition.

[0014] In some embodiments, the plastic article is the only carbon source in the composition for the bacterial strain. In some embodiments, the plastic article includes a polymer selected from polyethylene (PE), polyethylene terephthalate (PET), polyamide (PA), polypropylene (PP); polyurethane (PU), polyvinyl chloride (PVC), polystyrene (PS), polyester, ethylene vinyl acetate (EVA), and a combination thereof.

[0015] In some embodiments, the iron is in the form of an iron salt. In some embodiments, the iron salt is selected from iron sulfate and iron chloride. In some embodiments, the iron salt is iron sulfate.

[0016] In some embodiments, the iron is added to a concentration of about 0.01 mM-10 mM in the composition. In some embodiments, the iron is added to a concentration of about 0.05 mM-5 mM in the composition.

[0017] In some embodiments, at least one further additive is added to the plastic article, the further additive being selected from mineral oil, ethanol, xylan, veratryl alcohol, copper, copper sulfate, polyvinyl alcohol, manganese sulfate, and ammonium nitrate.

[0018] In some embodiments, the at least one strain of plastic-degrading bacteria expresses at least one enzyme selected from a dioxygenase, a monooxygenase, a laccase, a manganese peroxidase, and a combination thereof. In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus, Acinetobactor, Bacillus, Klebsiella, Ralstonia, Rhodococcus, Staphylococcus, Stenotrophomonas, Streptococcus, and a combination thereof. In some embodiments, the at least one strain of the plastic -degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, Rhodococcus ruber, Bacillus subtilis, Ideonella sakaiensis, and a combination thereof. In some embodiments, the bacterial strain is added to a concentration of about 102-108CFU / ml in the composition.

[0019] In some embodiments, the method includes incubating the plastic-degrading composition for at least a week, a month, 3 months at a temperature suitable for the bacterial strain.

[0020] In some embodiments, the at least one additive including iron is added to the plastic at the same time as, or after, addition of the at least one bacterial strain.

[0021] In some embodiments, the method further includes at least one pretreatment step prior to adding the at least one bacterial strain and / or the at least one additive. In some embodiments, the at least one pretreatment step includes cutting, shredding or grinding the plastic article. In some embodiments, the pretreatment step includes sterilizing the plastic article. In some embodiments, the pretreatment step includes pretreating the plastic article by a QUV treatment and / or by an oxidizing agent or catalyst.

[0022] In some embodiments, the method further includes a step of adding to the composition an additional dose of the at least one additive.

[0023] In some embodiments, the method further includes measuring the level of biodegradation.

[0024] In some embodiments, measuring the level of biodegradation includes: a) measuring or obtaining a first intensity of at least one Fourier Transform Infra-Red Spectroscopy (FTIR) peak corresponding to at least one chemical group in the plastic article or in a part thereof prior to adding the at least one bacterial strain and / or the at least one additive; b) measuring a second intensity of the at least one FTIR peak corresponding to the at least one chemical group in the plastic article or in a part thereof at a certain time point after adding the at least one bacterial strain and the at least one additive; and c) comparing the first intensity to the second intensity, wherein the second intensity being higher than the first intensity indicates an increase in the level of the chemical group; and the second intensity being lower than the first intensity indicates a decrease in the level of the chemical group.

[0025] In some embodiments, the second time point is at least about 30, 60, or 90 days after adding the at least one bacterial strain and the at least one additive.

[0026] In some embodiments, the increase or decrease in intensity is by at least 5%, 10%, 20%, 30%, 40%, or 50%.

[0027] In some embodiments, the certain chemical groups are selected from CH2 groups, -C-O-C- groups, C-C-0 groups, O-C-C groups, and / or C=O groups.

[0028] In some embodiments, the bio-degradation of the plastic article is manifested by an increase in levels of -C-O-C- groups in the plastic article or in a part thereof; a decrease in levels of CH2 groups, aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups in the plastic article or in a part thereof; and / or visually detectable newly -appearing cracks, tears, and / or holes in the plastic or in a part thereof.

[0029] In some embodiments, the increase or decrease is by at least 5%, 10%, 20%, 30%, 40%, or 50%. In some embodiments, the increase or decrease is measured after at least about 30, 60, or 90 days after adding the at least one bacterial strain and the at least one additive.

[0030] In some embodiments, there is provided a composition including at least one strain of plasticdegrading bacteria, at least one additive including an iron salt, and at least one plastic article.

[0031] In some embodiments, there is provided a composition including at least one strain of plasticdegrading bacteria expressing a dioxygenase and / or a monooxygenase, at least one additive including iron sulfate, and at least one plastic article.

[0032] In some embodiments, there is provided a composition including at least one strain of plasticdegrading bacteria belonging to a species selected from Brevibacillus agri, Brevibacillus borstelensis, and Rhodococcus ruber, at least one additive including iron sulfate, and at least one plastic article.

[0033] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0036] Figs. 1A-1B show two images demonstrating a change in polyethylene (PE) morphology in LDPE after month and a half of incubation with B. agri bacteria and ImM iron sulfate as an additive.

[0037] Figs. 2A-2B show confocal laser scanning microscope images of biofilm formed by B. agri on LDPE PE after month of incubation in minimal synthetic medium (SM) and ImM iron sulfate. Fig. 2A. Light field. Fig. 2B Fluorescence and light fields image. Viable cells stained green and dead cells stained red with the BacLight® Dead / Live Kit. Image processing to obtain 3-D images was done using IMARIS software (Bitplane, Zurich, Switzerland). Bar: 30p.

[0038] Figs. 3A-3H show high-resolution scanning electron microscope (HR-SEM) photographs of old biofilm on the surface of LDPE PE after incubation during a month (second experiment). Fig. 3A-3B. untreated LDPE. Fig. 3C-3D. LDPE treated with B. agri. Fig. 3E-3F. LDPE treated with B. agri and ImM iron sulfate. Fig. 3G-3H. LDPE treated with B. agri and 0.05mM iron sulfate. Magnification: Figs. 3A, 3C, 3E, 3G: XI, 000; Figs. 3B, 3D, 3F, 3H: X5,000.

[0039] Figs. 4A-4D show four high-resolution scanning electron microscope (HR-SEM) images of LDPE PE surface after B. agri and ImM iron sulfate treatment followed by biofilm removal, at different magnifications. Bar = 40 pm (Fig. 4A), 5 pm (Fig. 4B), 5 pm (Fig. 4C), 2 pm (Fig. 4D).

[0040] Figs. 5A-5D show high-resolution scanning electron microscope (HR-SEM) images of SM media after incubation of B. agri with ImM iron sulfate and LDPE and EVA 18% mix, at different magnifications. Bar = 500 pm (Fig. 5A), 50 pm (Fig. 5B), 30 pm (Fig. 5C), 10 pm (Fig. 5D).

[0041] Figs. 6A-6D show high-resolution scanning electron microscope (HR-SEM) images of SM media after incubation of PE with B. agri and with different additives. Fig. 6A. ImM Iron sulfate and 1% ethanol. Fig. 6B. ImM iron sulfate and 0.01% xylan. Fig. 6C. 0.05mM iron sulfate and 0.05mM copper sulfate. Fig. 6D. ImM iron chloride. bar= 40 pm.

[0042] Figs. 7A-7D show high-resolution scanning electron microscope (HR-SEM) photographs of LDPE after one-month treatment. Fig. 7A. R. ruber alone. Fig. 7B. R. ruber and 0.05mM iron sulfate. Fig. 7C. B. borstelensis alone. Fig. 7D. B. borstelensis with 0.05mM iron sulfate. Bar = 100 pm.

[0043] Figs. 8A-8B show ATR-FTIR analysis of PE after different treatments, with and without B. agri bacteria. Fig. 8A. At a wavenumber range of 500-4000 cm1. The thick arrow indicates the - C-O-C- group peak around 1047 cm1. Fig. 8B. Focus at a wavenumbers range of 800-1300 cm1. Samples tested were: LPDE alone, B. agri alone, ImM iron sulfate, ImM copper sulfate, 1% ethanol, ImM iron sulfate + 1% ethanol, and samples including B. agri + [ImM iron sulfate (first exp and second exp, see arrows), ImM copper sulfate, ImM iron sulfate + 1% ethanol, or 1% ethanol]. The different treatment are shown in different colors. Since most samples behaved in a similar way, the outstanding peaks are marked with arrows.

[0044] Fig. 9 shows ATR-FTIR- microscope analysis of LDPE PE with and without treatment.

[0045] Fig. 10 shows FTIR spectra (600-3600 1 / cm) of PE after 30 days of incubation (upper panel) and untreated PE (lower panel).

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0048] A variety of plastic-degrading enzymes from various bacteria have been used in order to eliminate the environmental problem of plastic accumulation. However, many factors and conditions affect the level and activity of these enzymes.

[0049] The inventors have surprisingly found that addition of iron to a bacteria-plastic mix enhances plastic degradation for polyethylene (PE).

[0050] Three bacterial species, namely Brevibacillus agri, Brevibacillus borstelensis and Rhodococcus ruber, were used for PE degradation experiments (Examples 1-5). The experiments were carried out for a period of over one month, and adding iron sulfate clearly enhanced degradation, as can be seen from Figs. 3 and 7, The plastic degradation experiments in the presence of iron sulfate showed visible holes in the PE sheets, and the missing polyethylene fragments were found to be covered with bacterial biofilm and having pits and cracks all over the surfaces, as observed by high-resolution scanning electron microscope (HR-SEM).

[0051] Fourier Transform Infra-Red Spectroscopy (FTIR) indicated that a new molecular group of C-O-C was detected on the surface of iron sulfate and bacteria treated PE pieces (Fig 8). This finding also indicates a novel mechanism for plastic degradation, involving oxygenation of the carbon chain possibly via iron dependent dioxygenases or other related enzymes.

[0052] Additionally, degradation could also be seen from a comparison of the spectrum of PE following treatment by bacteria with iron sulfate to untreated PE spectrum, which revealed a significant decrease (21%) in the concentration of the methylene (CH2) group in the plastic. Methods for plastic degradation

[0053] Accordingly, in some embodiments, the present invention provides a method for enhancing plastic bio-degradation, the method including adding to at least one plastic article at least one strain of plastic-degrading bacteria and at least one additive including iron, which together form a plasticdegrading composition.

[0054] The term “enhancing” means that plastic degradation with the addition of iron is faster or more complete compared to plastic degradation at the same conditions, but without including iron in the composition. The enhancement may be measured by comparing the rate at which the same level of degradation is obtained, or by comparing the level of degradation obtained after a certain time of incubation, between two compositions, e.g. one including no additive and another including the tested additive, or between compositions including two different additives.

[0055] In some embodiments, the enhancing is compared between a composition including bacteria only and a composition including bacteria and iron containing additive. In some embodiments, the enhancing is compared between a composition not including bacteria or iron (e.g., natural degradation of the plastic article, or degradation by other means) and adding the bacteria and the iron containing additive.

[0056] The level of degradation may be measured by any suitable method, including measuring the weight of the plastic article, measuring the amount of small plastic pieces (microplastic) in solution, measuring the amount of methylene groups remaining in the plastic article, and / or measuring the level of certain chemical bond indicating plastic degradation, as further explained herein. The measurement may be by any suitable method, including weighing, imaging, image analysis, microscopy, FTIR, etc.

[0057] In some embodiments, the enhancement is by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, the enhancement may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in the rate of reduction in plastic size, or the enhancement may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in the rate of reduction in the concentration of the methylene (CFh) group in the plastic, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase or decrease in the level of a certain chemical bond, compared to a reference, as further explained below.

[0058] The at least one plastic article may be any type of article, or multiple articles or parts / pieces of plastic, made from plastic in any shape or form, such as but not limited to a plastic package, a plastic bottle, a plastic sheet, a plastic film, a plastic flake, a piece of plastic, etc.

[0059] In some embodiments, the plastic article is the only carbon source for the plastic-degrading bacteria in the composition. In some embodiments, an additional carbon source may be added.

[0060] In some embodiments, the plastic article includes a polymer. In some embodiments, the plastic article includes a polymer which is a polyolefin.

[0061] In some embodiments, the plastic article includes a polymer selected from polyethylene (PE), polyethylene terephthalate (PET), polyamide (PA), polypropylene (PP); polyurethane (PU), polyvinyl chloride (PVC), polystyrene (PS), polyester, ethylene vinyl acetate (EVA), and a combination thereof.

[0062] In some embodiments, the polymer is a PE. In some embodiments, the polymer is a PE selected from low-density polyethylene (LDPE), high-density polyethylene (HDPE), Linear low- density polyethylene (LLDPE), and Branched low-density polyethylene (BLDPE).

[0063] In some embodiments, the iron is in the form of an iron salt.

[0064] In some embodiments, the iron salt is selected from iron sulfate and iron chloride.

[0065] In some embodiments, the iron salt is iron sulfate.

[0066] In some embodiments, the iron salt is not iron stearate.

[0067] In some embodiments, the iron is added to a concentration of about 0.01 mM-10 mM in the composition. In some embodiments, the iron is added to a concentration of about 0.05 mM-5 mM in the composition. In some embodiments, the iron is added to a concentration of about 0.01-1, 0.05-1, or 0.01-0.5 mM in the composition.

[0068] In some embodiments, the iron is added to a concentration of at least about 0.01, 0.05, or 1 mM in the composition.

[0069] In some embodiments, the iron is added to a concentration of not more than about 1, 2, 3, 4, 5, or 10 mM in the composition.

[0070] In some embodiments, the at least one additive including iron is added to the plastic before addition of the at least one bacterial strain. In some embodiments, the at least one additive including iron is not added to the plastic before addition of the at least one bacterial strain.

[0071] In some embodiments, the at least one additive including iron is added to the plastic after addition of the at least one bacterial strain. In some embodiments, the at least one additive including iron is added to the plastic at the same time as, or after, addition of the at least one bacterial strain.

[0072] It is appreciated that the statements “the at least one additive including iron is not added to the plastic before addition of the at least one bacterial strain” and “the at least one additive including iron is added to the plastic at the same time as addition of the at least one bacterial strain” mean that nothing significant is intended to happen (such as plastic degradation, or an additional treatment) between the addition of iron and the addition of the bacteria, and therefore they may be added together. In other words, adding the at least one additive including iron before adding the bacteria is equivalent to adding it together with or after adding the bacteria, provided that no plastic degradation and no plastic treatment step occur between the addition of the at least one additive including iron and the addition of the bacteria. This is due to the fact that according to the present invention, the iron is intended as a cofactor of bacterial enzymes.

[0073] Accordingly, in some embodiments, no plastic degradation and no treatment step occurs between the addition of the at least one additive including iron and the addition of the bacteria.

[0074] In some embodiments, at least one further additive is added to the plastic article.

[0075] In some embodiments, at least one the further additive is selected from mineral oil, ethanol, xylan, veratryl alcohol, copper, copper sulfate, polyvinyl alcohol, manganese sulfate, and ammonium nitrate.

[0076] In some embodiments, the further additive is mineral oil.

[0077] In some embodiments, the mineral oil is at a concentration of about 0.01%-l%.

[0078] In some embodiments, the mineral oil is at a concentration of about 0.05%-0.5%.

[0079] As mentioned above, disintegration of the plastic is carried out by various enzyme from different bacterial species. In general, microorganisms colonize the plastic surface, and then secrete enzymes which degrade the plastic into its monomers, which can be taken up by the microbial cells. These monomers are further enzymatically degraded in the cells, which use the monomers as carbon source for bacterial growth. The mechanism of degradation and byproducts depends on the type of bacterial enzymes participating in this process.

[0080] Many bacterial genera have been shown to degrade different types of PE, including Gramnegative and Gram-positives species belonging to the genera Ralstonia, Stenotrophomonas, Klebsiella, Acinetobactor, Rhodococcus, Staphylococcus, Streptococcus, Streptomyces, Bacillus, etc. These bacteria include enzymes such as laccase, manganese peroxidase. As explained herein, the present inventors propose a novel mechanism which involves oxygenases, such as monooxygenases and dioxygenases.

[0081] Accordingly, in some embodiments, the at least one strain of plastic-degrading bacteria expresses at least one enzyme selected from a dioxygenase, a monooxygenase, a laccase, a manganese peroxidase, and a combination thereof.

[0082] Accordingly, in some embodiments, the at least one strain of plastic-degrading bacteria expresses at least one enzyme selected from a dioxygenase and a monooxygenase.

[0083] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus, Rhodococcus, Acinetobactor, Bacillus, Klebsiella, Ralstonia, Staphylococcus, Stenotrophomonas, Streptococcus, and a combination thereof. In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus and Rhodococcus.

[0084] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, Rhodococcus ruber Bacillus subtilis, Ideonella sakaiensis, and a combination thereof.

[0085] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, and Rhodococcus ruber.

[0086] In some embodiments, the bacterial strain is added to a concentration of about 102-108or 104-108CFU / ml in the composition. In some embodiments, the bacterial strain is added to a concentration of at least about 102or 104CFU / ml in the composition.

[0087] In some embodiments, the method includes incubating the plastic-degrading composition for at least about a week, a month, or 3 months at a temperature suitable for the bacterial strain.

[0088] In some embodiments, the incubating is at a temperature selected from room temperature, ambient temperature, 30°C, 37°C, 42°C, and combinations thereof.

[0089] In some embodiments, the method does not include photo-degradation treatment of the plastic and the iron prior to adding the bacteria. In some embodiments, the method does not include irradiating the plastic and the iron prior to adding the bacteria. In some embodiments, the method does not include treating the plastic and the iron by UV irradiation prior to adding the bacteria.

[0090] The method may further include pretreatment steps, which help remove certain chemicals embedded in the plastic, and prepare the plastic for degradation. Nonlimiting examples for such pretreatments include Thermo-UV pretreatment such as in a QUV™ accelerated weathering tester(QLAB), pro-oxidant additives, and photo-catalysis (e.g. using titanium dioxide (TiO2)). QUV pretreatment includes cycles of exposure to UV at certain temperatures with intervals at lower temperatures.

[0091] In some embodiments, the method further includes at least one pretreatment step prior to adding the at least one bacterial strain and / or the at least one additive to the plastic.

[0092] In some embodiments, the pretreatment step is conducted prior to adding the at least one bacterial strain. In some embodiments, the pretreatment step is conducted prior to adding the at least one additive.

[0093] In some embodiments, the pretreatment step includes a QUV treatment.

[0094] In some embodiments, the QUV treatment includes cycles of UV (312 nm) exposure for 3- 4 hours at 60°C-70°C followed by intervals of 1-4 hours at 50°C.

[0095] In some embodiments, the total QUV treatment is conducted for about 20-500, 50-500, or 50-200 hours. In some embodiments, the pretreatment step includes sterilizing the plastic article. In some embodiments, the sterilizing includes treatment by a ultrasonic and / or by ethanol.

[0096] In some embodiments, the pretreatment step includes cutting, shredding, or grinding the plastic article, to increase the surface area and accessibility of chemical groups in the plastic article. Following cutting, shredding, or grinding, the plastic article turns into smaller plastic pieces such as plastic flakes or plastic power.

[0097] The efficacy of the treatment may be improved by adding further doses of the at least one additive at later time points.

[0098] Accordingly, in some embodiments, the method further includes a step of adding to the composition an additional dose of the at least one additive. In some embodiments, the additional dose includes an amount of the at least one additive that is about the same as the amount of the at least one additive initially added to the plastic article. In some embodiments, the additional dose includes an amount of the at least one additive that is lower than the amount of the at least one additive initially added to the plastic article. In some embodiments, the additional dose includes an amount of the at least one additive that is about half the amount of the at least one additive initially added to the plastic article.

[0099] In some embodiments, the additional dose is added to the composition about a day, 2 days, 3 days, 4 days, 6 days, a week, or a month after adding the initial dose of the at least one additive.

[0100] In some embodiments, the method further includes adding additional doses of the at least one additive. Such additional doses may be added at a certain frequency such as daily, weekly, or monthly.

[0101] In some embodiments, the additional dose includes about 0.5mM or about 0.05mM iron sulfate.

[0102] Several methods are known for assessing the level of biodegradation. Some nonlimiting examples include weighing the plastic and comparing to the initial weight of the plastic, assessing the plastic morphology by microscopy and / or imaging analysis, and following the formation or removal of certain bonds or groups, e.g., by FTIR. It is noted that some methods (e.g. weighing) are problematic and inaccurate as they may reflect decomposition of other ingredients in the mixture, or since slow changes are difficult to measure. Therefore, measurement of changes in chemical bonds / groups by FTIR advantageously reflect changes in the plastic itself.

[0103] Some relevant groups in which changes are known to be associated with plastic degradation include groups such as carbonyl, carboxyl, methylene, etc. Additionally, the present inventors have found changes in FTIR patterns for additional groups which were formed, or presented with increased or decreased levels, including -C-O-C- groups, aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups.

[0104] In some embodiments, the method includes a further step of measuring the level of biodegradation.

[0105] In some embodiments, measuring the level of biodegradation includes measuring the intensity of specific FTIR peaks corresponding to certain chemical bonds / groups.

[0106] Accordingly, in some embodiments, measuring the level of biodegradation includes: a) measuring or obtaining a first intensity of at least one FTIR peak corresponding to at least one chemical group in the plastic article or in a part thereof prior to adding the at least one bacterial strain and / or the at least one additive; b) measuring a second intensity of the at least one FTIR peak corresponding to the at least one chemical group in the plastic article or in a part thereof at a certain time point after adding the at least one bacterial strain and the at least one additive; and c) comparing the first intensity to the second intensity, wherein the second intensity being higher than the first intensity indicates an increase in the level of the chemical group; and the second intensity being lower than the first intensity indicates a decrease in the level of the chemical group.

[0107] In some embodiments, the first intensity of at least one FTIR peak is obtained from an FTIR measurement of a reference article. The term “reference article” encompasses an article made of plastic, having the same or similar composition as the plastic article, such that FTIR peaks measured for the reference article can be used for inferring levels of chemical groups in the plastic article under the same conditions.

[0108] In some embodiments, the first intensity is measured prior to adding both the at least one bacterial strain and the at least one additive.

[0109] In some embodiments, the at least one chemical group is selected from: -C-O-C- groups, CH2 groups, aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups.

[0110] In some embodiments, the second time point is at least about 30, 60, or 90 days after adding the at least one bacterial strain and the at least one additive.

[0111] In some embodiments, the increase or decrease in intensity is by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70%.

[0112] In some embodiments, bio-degradation of the plastic article is manifested by an increase or a decrease in levels of CH2 groups, -C-O-C- groups, C-C-0 groups, O-C-C groups, and / or C=O groups in the plastic article or in a part thereof.

[0113] In some embodiments, bio-degradation of the plastic article is manifested by an increase in levels of -C-O-C- groups in the plastic article or in a part thereof.

[0114] In some embodiments, bio-degradation of the plastic article is manifested by a decrease in levels of CH2 groups in the plastic article or in a part thereof.

[0115] In some embodiments, bio-degradation of the plastic article is manifested by a decrease in levels of aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups in the plastic article or in a part thereof.

[0116] In some embodiments, bio-degradation of the plastic article is manifested by visually detectable newly-appearing cracks, tears, and / or holes in the plastic article or in a part thereof.

[0117] In some embodiments, the increase in the levels of -C-O-C- groups in the plastic article or in a part thereof is by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0118] In some embodiments, the decrease in the levels of CH2 groups, aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups in the plastic article or in a part thereof is by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0119] In some embodiments, the decrease in the levels of CH2 groups in the plastic article or in a part thereof is by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0120] The time point at which the measurement is conducted may be flexible, depending on needs, and may be days, weeks, months, and even years, after adding the bacterial strain and the additive. In some embodiments, the increase and / or decrease is measured after at least about a week, 2 weeks, 3 weeks, one month, 2 months, 3 months, etc. after adding the at least one bacterial strain and the at least one additive.

[0121] The measurement may be repeated at several time points to follow up on degradation of the plastic article.

[0122] In some embodiments, the method of the invention is preceded by another method of plastic degradation. In some embodiments, the method of the invention is followed by another method for plastic degradation.

[0123] Plastic-degrading compositions

[0124] In some embodiments, the present invention provides a composition including at least one strain of plastic-degrading bacteria, at least one additive including iron, and at least one plastic article.

[0125] Definitions and embodiments mentioned above and which may be relevant to the composition embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0126] In some embodiments, the composition includes iron salt. In some embodiments, the composition includes iron sulfate. In some embodiments, the composition does not include iron stearate.

[0127] In some embodiments, the iron is at a concentration of about O.OlmM-lOmM in the composition. In some embodiments, the iron is at a concentration of about 0.05mM-5mM in the composition.

[0128] In some embodiments, the iron is at a concentration of at least about O.OlmM, 0.05mM, or ImM in the composition. In some embodiments, the iron is at a concentration of not more than about 5mM or lOmM in the composition.

[0129] In some embodiments, the at least one strain of plastic-degrading bacteria expresses at least one enzyme selected from a dioxygenase and a monooxygenase.

[0130] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus, Rhodococcus, Acinetobactor, Bacillus, Klebsiella, Ralstonia, Staphylococcus, Stenotrophomonas, Streptococcus, and a combination thereof.

[0131] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus and Rhodococcus.

[0132] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, Rhodococcus ruber, Bacillus mobilis, Bacillus subtilis, Ideonella sakaiensis, and a combination thereof.

[0133] In some embodiments, the at least one strain of the plastic-degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, and Rhodococcus ruber.

[0134] In some embodiments, the plastic article includes -C-O-C- groups.

[0135] In some embodiments, the present invention provides a composition including at least one strain of plastic-degrading bacteria expressing a dioxygenase and / or a monooxygenase, at least one additive including iron sulfate, and at least one plastic article.

[0136] In some embodiments, the present invention provides a composition including at least one strain of plastic-degrading bacteria belonging to a species selected from Brevibacillus agri, Brevibacillus borstelensis, and Rhodococcus ruber, at least one additive including iron sulfate, and at least one plastic article.

[0137] In some embodiments, the present invention provides the composition disclosed herein for use in enhancing plastic bio-degradation.

[0138] In some embodiments, the present invention provides the use of the composition disclosed herein for enhancing plastic bio-degradation.

[0139] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0140] The term "about" when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0141] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0142] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0143] EXAMPLES

[0144] Example 1: Polyethylene (PE) Biodegradation experiments

[0145] In the biodegradation experiment, B. agri bacteria were cultured in 250 ml flasks, each containing 100 ml minimal synthetic medium (SM) supplemented with 0.1 g films of ethylene vinyl acetate (EVA) 18% and Low-density polyethylene (LDPE) (referred to below as “PE”) as the only carbon source, and with different ingredients having the potential to act as biodegradation inducers. The flasks were incubated on a rotary shaker (150 rpm) at 42°C for a period of a month and a half. At the end of the incubation time, the turbidity of the media and colony-forming unit (CFU) was tested. Based on these results PE samples from each flask were tested. The biofilm was separated from the PE by soaking the pieces in 2% SDS overnight and then washing them with double-distilled water (DDW) and finally drying them under a laminar hood overnight. These PE pieces were weighed, analyzed by Fourier Transform Infra-Red Spectroscopy (FTIR) spectroscopy and the prominent sample was analyzed by differential scanning calorimetry (DSC). PE pieces that had not undergone biofilm removal were observed under high resolution scanning electron microscopy (HR-SEM). All tests and results of the first biodegradation experiment are summarized in Table 1.

[0146] Table 1. Summary of the analysis of the first PE biodegradation experiment

[0147] CFU / ml: number of bacteria (colony forming units) per 1ml media after month and a half of incubation. ATR-FTIR: attenuated total reflection (ATR)-FTIR.

[0148] The experiment was repeated with the additives that had shown the best biodegradation results, selected according to bacterial growth parameters such as high CFU, turbidity, and / or biofilm. In addition, the combined effect of two materials was tested, including the combined effect of iron sulfate with ethanol, iron sulfate with xylan, and copper sulfate with xylan. Since the results presented in Table 1 showed that iron sulfate was a leading candidate as a biodegradation - additives, iron chloride was also as a possible candidate additive. To better understand the bacterial viability in the media during the experiment, the CFU and turbidity were tested weekly. As a control for the effect of the additive material, the second experiment included flasks with the additives alone without bacteria. In that experiment the only carbon source was LDPE. To reduce initial PE contamination, the PE were sterilized with 30 minutes treatment in ultrasonic bath sonicator then soaked in 70% ethanol for 1 hr. followed by two washes with DDW, and final drying in laminar or flow hood. All tests and results of the second biodegradation experiment are summarized in Table 2.

[0149] Table 2. Summary of the analysis of the second PE biodegradation experiment

[0150] CFU / ml: number of bacteria (colony forming units) per 1ml media after a month and a half of incubation. ATR-FTIR: attenuated total reflection-FTIR.

[0151] The third biodegradation experiment is conducted to estimate several new aspects including the influence of weekly addition of additives and the effect of pre-treatment by UV photooxidation on the PE biodegradation efficiency. In this experiment all three PE types are added to flasks and two additives are chosen: ImM iron sulfate and 1% ethanol. Once a week, half of the initial concentration of the additives is added. The pre-treatment by UV photo-oxidation is carried out in a QUV™ Accelerated Weathering Tester (Q-Panel, Cleveland, OH, USA). The polyethylene is subjected to a program of alternating exposure of 4 hours UV (312 nm) at 60°C and 4 hours condensation at 50°C. LDPE is treated for 200 hours, 100 hours on each side. EVA 18% and LDPE are treated for half of the time, totally 100 hours, and the films are turned over every few hours. The shorter treatment is determined due to the EVA 18% melting temperature and LDPE ’s tendency to break under UV. The PE spectrum after the QUV™ treatment is analyzed by ATR-FTIR and indicates the initiation of degradation. After the QUV™ treatment, the PE pieces are sterilized as detailed above, for the second experiment.

[0152] Example 2: Analysis of bacterial growth and PE morphology

[0153] In order to identify the additives that enabled and enhanced polymer biodegradation, the tested treatments were compared by assessment of the media turbidity, changes in plastic morphology, and CFU (colony forming units) in the solution. The turbidity and CFU results are summarized in Tables 1 and 2 above. It should be noted that there were no significant changes in the plastic mass according to gravimetric weight.

[0154] Turbid medium indicates that the bacterial growth was induced by the additives using material from the plastic and floating micro-plastic. On the other hand, a clear medium is likely the result of bacterial inhibition. This indicates that the additive does not allow or does not encourage the polyethylene biodegradation by the bacteria. The higher number of live bacteria in the samples with certain additives at the end of the experiment implies that these additive materials allows better bacterial growth for a long period of time. For example, a CFU of -1.8-107bacteria in 1 ml of media with Veratryl alcohol, compared to no bacteria in the Erlenmeyer with ImM copper chloride (first biodegradation experiment). In should be mentioned that low CFU does not eliminate the possibility that bacteria present in the solution actually attached to the PE surface.

[0155] The texture and morphology of PE were tested visually, and the amount of non-uniform textures, holes, or cracks on the surfaces was recorded. All of the LDPE pieces that were incubated with B. agri together with ImM iron sulfate appeared with holes at the end of the first experiment (see Figs. 1A-1B). No holes were observed in the second biodegradation experiment, probably since the incubation time was shorter.

[0156] Based on these initial results it was found that addition of iron sulfate produced promising results with high medium turbidity and visible holes in PE in multiple films. In the first biodegradation experiment the CFU was checked only in the end and showed that no bacteria survived in the medium with ImM iron sulfate. Therefore, in the second biodegradation experiment the CFU was checked weekly. This time the results showed that the bacteria were not present in the media after two weeks. To understand this, additional tests were used. First, to understand if ImM iron sulfate is too high a concentration for the bacteria for such a long period, the effect of lower concentration of 0.05mM iron sulfate was tested as well in a different flask during the second experiment. Throughout all the experiment the number of bacteria in this flask was extremely low and at the end of the experiment there were 8-103bacteria / ml. Next, the PE pieces from the second experiment were stained with BacEight® Dead / Live Kit (Invitrogen, Eugene, OR, USA) and observed at the end of the experiment under confocal laser scanning microscope (Olympus, Tokyo, Japan). The results indicated that the surface of LDPE was covered with mostly live bacteria that mainly aggregate in the cracks in the plastic, likely inducing the creation of these cracks (see Fig. 2). Finally, in the third biodegradation experiment (described above but not completed due to COVID closures), 0.5mM iron sulfate was added to the suitable flasks weekly and the bacteria thrived in the medium during the month as well as at the end of the experiment. At the end of the month, there were 4-106bacteria / ml. The conclusion from all these tests is that low initial concentration of iron sulfate enables low amounts of bacteria to live in the medium. Bacteria then continue to live and thrive on the polyethylene surface, even when it is not in the medium, and finally, an addition of iron sulfate weekly encourages bacterial growth also in the medium.

[0157] Additional materials showed biodegradation potential (based on the above Tables 1 and 2). These included: 0.1% veratryl alcohol, 1% ethanol, 0.1 mM copper sulfate, 4g / L ammonium nitrate, ImM iron sulfate, 0.01% xylan, 0.5% polyvinyl alcohol, and O.lmM manganese sulfate. The biodegradation activity of the bacteria in the presence of these materials was tested twice, in the first and second biodegradation experiments. The PE films that were incubated with these materials and bacteria were further analyzed using HR-SEM and ATR-FTIR.

[0158] Example 3: Analysis by Scanning Electron Microscopy (SEM) imaging of PE samples

[0159] Polyethylene samples (1x1 cm) colonized with bacteria were removed from the medium and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde. After fixation, the samples were metal- coated under a deep vacuum and examined using a high-resolution scanning electron microscope (HR-SEM) (DualBeam Microscope Helios G4 UC, Thermo Scientific). PE samples that were incubated in SM without any additives were considered controls. The surfaces of LDPE or EVA 18% samples without treatment were all similar, showing clean surfaces with few defects and thin microscopic cracks up to 1 pm long. Treated PE films with B. agri and without additional additives exhibited the natural attachment of the bacteria to the surface. The results display thriving bacterial colonies growing on LDPE, while on EVA 18% the B. agri bacteria attached uniformly on the surface with no biofilm development. HR-SEM summarization is presented in Table 1 (first biodegradation experiment) and Table 2 (second biodegradation experiment).

[0160] Analysis of biofilm

[0161] In both biodegradation experiments, the most significant results were achieved with any of the three bacteria tested and an addition of iron sulfate. This included the development of a massive and vast biofilm of B. agri that completely covered the PE surfaces and included an excessive amount of extracellular polymeric substance (EPS). Fig. 3 displays HR-SEM images from the second biodegradation experiment of control of LDPE that wasn’t treated with any additives, control of LDPE that was treated only with B. agri, LDPE after treatment with B. agri and ImM iron sulfate, and LDPE after treatment with B. agri and 0.05mM iron sulfate. LDPE without treatment was characterized by long natural cracks, while the control of LDPE that was treated only with B. agri exhibited some bacterial colonies. The treatment with ImM iron sulfate led to massive EPS and bacteria all over the surface, and the lower concentration gave some moderate results without dramatic change. In order to measure the thickness of the LDPE biofilm a focused ion beam (FIB) operation in the Dual-Beam (FIB-SEM) system was used. The biofilm thickness was measured as -350 nm in the scanned area.

[0162] Analysis of surface under the biofilm

[0163] With the aim of observing the impact of the bacteria together with the iron sulfate on the PE morphology, the biofilm was removed following overnight shaking culture of the PE pieces in 2% SDS and followed by a washing with DDW and drying overnight. In the first biodegradation experiment, under the biofilm (following treatment with iron sulfate), the LDPE surface began to peel and presented remarkable cracking of up to 10 pm in length appearing all over the surface (see Fig. 4). The LDPE surface changed according to the treatment and presented different coarse structures with long stripes (not shown). The EVA 18% surface appeared with -lOOnm holes and a wavy surface (not shown).

[0164] Analysis of the medium

[0165] In order to observe and characterize the PE fragments and the bacterial spreading in the medium with iron sulfate, a drop from the medium was scanned under HR-SEM without fixation. In the first experiment the SEM revealed that the medium was filled with micro-plastic up to 100 pm in length and covered with bacterial biofilm (see Fig. 5) In the second experiment the result was similar with enormous amount of microplastic (not shown).

[0166] To enhance the bacterial polyethylene biodegradation, combinations of iron sulfate with promising additives from the first experiment were tested (see Fig. 6). Among the synergic effects of iron sulfate with ethanol, xylan or copper sulfate, the best outcome resulted from addition of iron sulfate with ethanol. In this case a layer of bacteria and EPS cover almost all the surface. The combination of iron sulfate and xylan led to an organize biofilm up to the size of 50 pm. The synergistic effect of iron sulfate with copper sulfate encouraged bacterial thriving, but with less organized biofilm structures. Iron chloride was tested as alternative source of iron but its impact was found to be less effective than iron sulfate.

[0167] In addition to testing the impact of different additives with B. agri bacteria, the impact of R. ruber and B. borstelensis bacteria alone or with the addition of 0.05mM iron sulfate was also tested (see Fig. 7). The iron addition to B. borstelensis bacteria enhanced bacteria growth and attachment on the surface. It should be mentioned that R. ruber alone attached well to the surface as was observed in both biodegradation experiments due to its hydrophobic character. The low effect of the iron sulfate on the bacteria was ascribed to the low concentration that was tested, 0.05mM iron sulfate, and assume that higher concentration may lead to better results.

[0168] The initial results together with the microscopic analyses suggest effective biodegradation of LDPE when exposed to B. agri with iron sulfate as obtained in both biodegradation experiments. The molecular changes occurring in the polymers using analytical equipment including ATR- FTIR, FTIR-microscope, and DSC were assessed next.

[0169] Example 4: Fourier Transform Infra-Red Spectroscopy (FTIR) of PE samples

[0170] In the first biodegradation experiment the ATR-FTIR analysis indicated that ImM iron sulfate or ImM copper sulfate added to PE following B. agri treatment result in a new molecular bond that include oxygen to the PE surface. In the second biodegradation experiment the possible effector that induces of the newly detected bond was sought, i.e., whether the additives solely induce the creation of these bonds or whether the combination with B. agri affected the PE. Fig. 8 presents the most interesting results from both experiments. In both unrelated experiments the combination of ImM iron sulfate with B. agri gave remarkable result of new peaks that represent C-O-C bonds in the PE. In the second experiment, PE was treated only by ImM iron sulfate without bacteria as a control and the results indicate that no new peaks were present. Consequently, the conclusion is that B. agri itself uses the iron in the process of the PE biodegradation. The flask containing ImM copper sulfate with B. agri was contaminated at the beginning of the second experiment and therefore no spectrum change was observed. Yet, the control sample containing only ImM copper sulfate without bacteria exhibited three peaks around 1070 cm1. Therefore, it was concluded that the molecular change of the PE is an outcome of the copper sulfate alone and is not involved in biodegradation.

[0171] The combination of B.agri with ImM iron sulfate and 1% ethanol presented the same peaks as those from the bacteria with ImM iron sulfate, however, the former peaks (with ethanol) were lower. The bacteria with 1% ethanol alone did not change the spectrum. Thus, the peaks that are shown in the combination treatment are probably the outcome of the iron sulfate alone. The treatment with a lower concentration of iron sulfate (0.05mM) with B. agri did not result in the same effects as the higher concentration of ImM. It is possible that the effect was minor and wasn’t detectable in FTIR analysis. Treatment with ImM iron chloride with B.agri ended with high concentration of biofilm, as shown with HR-SEM imaging, however, no molecular change was detected by ATR-FTIR (not shown). The effect of all other additives was analyzed as well, and no detectable change of the PE was observed.

[0172] Due to spectrum changes of the LDPE surface following iron sulfate treatment, the correlated sample was analyzed with FTIR-microscope as well (Thermo Scientific, Nicolet iN10 MX IR Spectrometer). The IR spectra were recorded at the range of 4000-500 cm1, with a resolution of 8 cm1and 16 scans. The scanned area was 6x6 microns using a transmittance mode that enables quantitative analysis. Fig. 9 presents the average spectra of hundreds of scans for each sample. A comparison of the LDPE spectrum after iron sulfate treatment, to the control spectrum, reveals a significant decrease in the intensity of the methylene (CH2) group. According to Mayerhofer et al 2019 (Chemphyschem 20(21):2748-2753), integrated absorbance depends linearly on concentration. The combined peak area between 2773 cm1to 3101 cm1is 344.5 and 270.4 for both the control and treated samples, indicating a decrease of 21.5% in methylene group concentration after treatment. These intensity reductions indicate PE biodegradation. The spectral differences received from ATR-FTIR and FTIR-microscope techniques are due to different analytic modes, where the first one is analyzing the reflection of the surface, while the second analyzing direct absorption. These results also correlate to the HR-SEM images of LDPE after the B. agri and iron sulfate treatment that exhibits erosion of the surface of the PE.

[0173] The data so far suggest the involvement of oxidation enzymes in the degradation process, and that the effect of iron sulfate may perhaps be connected to dioxygenase group of enzymes, which use iron as a cofactor. Example 5: PE biodegradation by Rhodococcus Ruber in beta small scale mini membrane bioreactor (MBR) 100ml system

[0174] The experiment was held for one month under constant conditions of 25°C (room temperature), constantly stirred and air-diffused by small aquarium air pump. Two replicates were prepared, each with 100 ml SM minimal medium with 6 grams of PE as sole carbon source, and with the addition of 5mM FeSCE. Because of the slow reaction rate, the bioreactor ran in batch mode. PE samples were taken after 30 days of incubation and analyzed by FTIR to detect molecular changes compared to untreated control (Fig. 10). Spectra changes presented in 1000-1300 1 / cm region, which imply the addition of -C-O-C- functions of ethers and esters in treated PE, compared to untreated samples.

Claims

CLAIMSWhat is claimed is:

1. A method for enhancing plastic bio-degradation, the method comprising adding to at least one plastic article at least one strain of plastic-degrading bacteria and at least one additive comprising iron which together form a plastic-degrading composition.

2. The method of claim 1, wherein the plastic article is the only carbon source in the composition for the bacterial strain.

3. The method of claim lor 2, wherein the plastic article comprises a polymer selected from polyethylene (PE), polyethylene terephthalate (PET), polyamide (PA), polypropylene (PP); polyurethane (PU), polyvinyl chloride (PVC), polystyrene (PS), polyester, ethylene vinyl acetate (EVA), and a combination thereof.

4. The method of any one of claims 1-3, wherein the iron is in the form of an iron salt.

5. The method of claim 4, wherein the iron salt is selected from iron sulfate and iron chloride.

6. The method of claim 5, wherein the iron salt is iron sulfate.

7. The method of any one of claims 1-6, wherein the iron is added to a concentration of about 0.01-10 mM in the composition.

8. The method of claim 7, wherein the iron is added to a concentration of about 0.05-5 mM in the composition.

9. The method of any one of claims 1-8, wherein at least one further additive is added to the plastic article, the further additive being selected from mineral oil, ethanol, xylan, veratryl alcohol, copper, copper sulfate, polyvinyl alcohol, manganese sulfate, and ammonium nitrate.

10. The method of any one of claims 1-9, wherein the at least one strain of plastic-degrading bacteria expresses at least one enzyme selected from a dioxygenase, a monooxygenase, a laccase, a manganese peroxidase, and a combination thereof.

11. The method of any one of claims 1-10, wherein the at least one strain of the plastic-degrading bacteria belongs to a genus selected from Brevibacillus, Acinetobactor, Bacillus, Klebsiella, Ralstonia, Rhodococcus, Staphylococcus, Stenotrophomonas, Streptococcus, and a combination thereof.

12. The method of any one of claims 1-11, wherein the at least one strain of the plastic-degrading bacteria belongs to a species selected from Brevibacillus agri, Brevibacillus borstelensis, Rhodococcus ruber, Bacillus subtilis, Ideonella sakaiensis, and a combination thereof.

13. The method of any one of claims 1-12, wherein the bacterial strain is added to a concentration of about 102-108CFU / ml in the composition.

14. The method of any one of claims 1-13, the method comprises incubating the plastic-degrading composition for at least a week, a month, 3 months at a temperature suitable for the bacterial strain.

15. The method of any one of claims 1-14, further comprising at least one pretreatment step prior to adding the at least one bacterial strain and / or the at least one additive.

16. The method of claim 15, wherein the at least one pretreatment step comprises cutting, shredding or grinding the plastic article.

17. The method of claim 15 or 16, wherein the pretreatment step comprises sterilizing the plastic article.

18. The method of claim 15 or 16, wherein the pretreatment step comprises pretreating the plastic article by a QUV treatment and / or by an oxidizing agent or catalyst.

19. The method of any one of claims 1-18, further comprising a step of adding to the composition an additional dose of the at least one additive.

20. The method of any one of claims 1-19, further comprising measuring the level of biodegradation.

21. The method of claim 20, wherein measuring the level of biodegradation comprises: a) measuring or obtaining a first intensity of at least one Fourier Transform Infra-Red Spectroscopy (FTIR) peak corresponding to at least one chemical group in the plastic article or in a part thereof prior to adding the at least one bacterial strain and / or the at least one additive; b) measuring a second intensity of the at least one FTIR peak corresponding to the at least one chemical group in the plastic article or in a part thereof at a certain time point after adding the at least one bacterial strain and the at least one additive; and c) comparing the first intensity to the second intensity,wherein the second intensity being higher than the first intensity indicates an increase in the level of the chemical group; and the second intensity being lower than the first intensity indicates a decrease in the level of the chemical group.

22. The method of claim 21, wherein the second time point is at least about 30, 60, or 90 days after adding the at least one bacterial strain and the at least one additive.

23. The method of claim 21 or 22, wherein the increase or decrease in intensity is by at least 5%, 10%, 20%, 30%, 40%, or 50%.

24. The method of anyone of claims 21-23 wherein the certain chemical groups are selected from CH2 groups, -C-O-C- groups, C-C-0 groups, O-C-C groups, and / or C=O groups.

25. The method of any one of claims 1-23, wherein the bio-degradation of the plastic article is manifested by an increase in levels of -C-O-C- groups in the plastic article or in a part thereof; a decrease in levels of CH2 groups, aromatic groups, C-C-0 groups, O-C-C groups, and / or C=O groups in the plastic article or in a part thereof; and / or visually detectable newly- appearing cracks, tears, and / or holes in the plastic or in a part thereof.

26. The method of claim 25, wherein the increase or decrease is by at least 5%, 10%, 20%, 30%, 40%, or 50%.

27. The method of claim 25 or 26, wherein the increase or decrease is measured after at least about 30, 60, or 90 days after adding the at least one bacterial strain and the at least one additive.

28. A composition comprising at least one strain of plastic-degrading bacteria, at least one additive comprising an iron salt, and at least one plastic article.

29. A composition comprising at least one strain of plastic-degrading bacteria expressing a dioxygenase and / or a monooxygenase, at least one additive comprising iron sulfate, and at least one plastic article.

30. A composition comprising at least one strain of plastic-degrading bacteria belonging to a species selected from Brevibacillus agri, Brevibacillus borstelensis, and Rhodococcus ruber, at least one additive comprising iron sulfate, and at least one plastic article.

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