Microbes and enzymes for plastic degradation

Abstract

In some aspects, the present disclosure provides methods and compositions for degrading mixtures of plastics, such as mixed plastic waste, using Cupriavidus gilardii.

Description

[0001] MICROBES AND ENZYMES FOR PLASTIC DEGRADATION

[0002] RELATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63 / 631,244, filed April 8, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] The accumulation of plastic waste in the oceans, soil, and even in the human body is a major pollution issue. Over 5 billion tons of mixed plastic waste has been disposed of to date. Despite major efforts to recycle plastic products, actually making use of a mixture of plastic materials has remained a challenging issue. A key problem is that plastics come in so many different varieties, and chemical processes for breaking them down into a form that can be reused in some way tend to be very specific to each type of plastic. Sorting the mixture of plastic waste material, from soda bottles to detergent jugs to plastic toys, is impractical at large scale. Today, much of the plastic material gathered through recycling programs ends up in landfills. Management of plastic waste has become a global concern, and a solution is urgently needed.

[0006] SUMMARY

[0007] The present disclosure relates to a species of bacteria that is capable of degrading a variety of plastics, such as those commonly found in mixed plastic waste streams. The data provided herein shows that Cupriavidus gilardii (C. gilardii), an aerobic gram-negative bacterium isolated from the gut of waxworms (Galleria mellonellag is capable of metabolizing polypropylene (PP), polyethylene terephthalate (PET), and nylon within just a few hours and without chemical pre-treatment of the plastic materials.

[0008] Thus, some aspects of the disclosure relate to a biodegradation method, comprising: applying an effective amount of a composition comprising C. gilardii cells to a mixture of plastics, thereby degrading the mixture of plastics.

[0009] Other aspects of the disclosure relate to a biodegradation method, comprising: applying an effective amount of a composition comprising C. gilardii cells to a product comprising a mixture of plastics, thereby degrading the mixture of plastics.

[0010] In some embodiments, the C. gilardii cells are formulated as a solution, a powder or as granules. In some embodiments, the applying of the C. gilardii cells to a mixture or product comprises spraying the cells onto the mixture or product. Yet other aspects of the disclosure relate to a bioreactor comprising: a mixture of plastics; and C. gilardii cells in an effective amount to degrade the mixture of plastics.

[0011] Other aspects of the disclosure relate to a composition comprising granules of C. gilardii cells.

[0012] Still other aspects of the disclosure relate to an applicator comprising C. gilardii cells in an effective amount (e.g., at least IxlO3, IxlO4, or IxlO5C. gilardii cells / ml) to degrade a mixture of plastics.

[0013] Further aspects of the disclosure relate to a kit comprising: an applicator; and C. gilardii cells in an effective amount (e.g., at least IxlO3, IxlO4, or IxlO5C. gilardii cells / ml) to degrade a mixture of plastics. In some embodiments, the C. gilardii cells are formulated as a solution, a powder or as granules. In some embodiments, the kit comprises a composition comprising granules of the C. gilardii cells.

[0014] In some embodiments, the mixture of plastics comprises mixed plastic waste. In some embodiments, the mixture of plastics comprises two or more types of plastic. In some embodiments, the mixture of plastics comprises three or more types of plastic.

[0015] In some embodiments, the mixture of plastics is selected from polyolefin, polyester, and polyamide. In some embodiments, the mixture of plastics comprises polyolefin, for example, polypropylene. In some embodiments, the mixture of plastics comprises polyester, for example, polyethylene terephthalate. In some embodiments, the mixture of plastics comprises polyamide, for example, nylon.

[0016] In some embodiments, the mixture of plastics comprises: polypropylene and polyethylene terephthalate; polypropylene and nylon; polyethylene terephthalate and nylon; or polypropylene, polyethylene terephthalate, and nylon.

[0017] In some embodiments, at least 30% of the mixture of plastics comprises the polyolefin, optionally polypropylene. In some embodiments, at least 30% of the mixture of plastics comprises the polyester, optionally polyethylene terephthalate. In some embodiments, at least 30% of the mixture of plastics comprises the polyamide, optionally nylon.

[0018] In some embodiments, the composition comprises a carrier, for example, a carbon-free medium.

[0019] In some embodiments, the effective amount is at least IxlO3, IxlO4, or IxlO5C. gilardii cells per milliliter (ml) carrier. For example, an effective amount may be about or at least IxlO3, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, IxlO4, 2xl04, 3xl04, 4xl04, 5xl04, 6xl04, 7xl04, 8xl04, 9xl04, IxlO5, 2xl05, 3xl05, 4xl05, 5xl05, 6xl05, 7xl05, 8xl05, or 9xl05cells / ml. In some embodiments, the C. gilardii cells are in contact with the mixture of plastics for at least 200 minutes.

[0020] In some embodiments, the C. gilardii cells and the mixture of plastics is maintained for at least 200 minutes at a temperature of between about 25 °C and about 58 °C, for example between about 37 °C and about 50 °C, optionally about 45 °C.

[0021] In some embodiments, the mixture of plastics is untreated prior to the contacting.

[0022] In some embodiments, the method does not comprise treating the plastic with a cobalt or manganese catalyst prior to the contacting.

[0023] BRIEF DESCRIPTION OF DRAWINGS

[0024] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0025] FIG. 1 shows the growth curves of Cupriavidus gilardii and Staphylococcus hominis in different growth media. C. gilardii (CG) was grown in the presence of lysogeny broth (LB) media alone, carbon-free media (CFM) alone, or CFM and polypropylene (PP) nanoparticles (FIG. 1, left). C. gilardii (CG) was grown in the presence of LB media alone, CFM media alone, or CFM and polyethyleneterephthalate (PET) (FIG. 1, middle). S. hominis (SH) was grown in the presence of LB media alone, CFM media alone, or CFM and PET (FIG. 1, right). Growth was measured by optical density (OD) at 600 nm.

[0026] FIGs. 2A-2C shows bacterial biofilm growth in the presence of plastic nanoparticles. PET nanoparticles were plated without the presence of bacteria (FIG. 2A, magnified 100X). C. gilardii (CG) grown in the presence of PET nanoparticles formed a bacterial biofilm (FIG. 2B, magnified 100X). A 200 pL pipette tip (FIG. 2C, top) was dipped into a solution containing C. gilardii, at which point, a C. gilardii biofilm formed on the pipette tip (FIG. 2C, bottom).

[0027] FIG. 3 shows a plating protocol for measuring growth over time of C. gilardii (BS1) in the presence of plastic nanoparticles.

[0028] FIG. 4 shows growth over time of C. gilardii (BS1) grown in the presence of PP or PET. 24 hours after plating the cells (t=0 minutes), C. gilardii colonies were observed (~1000cell / ml) (FIG. 4, left). Growth of C. gilardii colonies grown in the presence of PP or PET was observed over a period of about 3,000 minutes (48 hours) (FIG. 4, middle). C. gilardii colony growth in the presence of PP or PET was observed after 24 hours and after 48 hours following acclimation in the CFM media at 37 °C (FIG. 4, right). Growth was measured as Colony Forming Units (CFU) count on a logarithmic scale (CFU count (log)).

[0029] FIGs. 5A-5B show production of carbon dioxide produced during the growth of C. gilardii (BS1) in CFM grown in the presence of PET (polyester) or PP (polyolefin). C. gilardii cells (e.g., an isolate C. gilardii) of were grown in the presence of plastic nanoparticles (PP or PET) in CFM (FIG. 5A). Carbon dioxide (CO2) (measured in parts per million (ppm)) produced from C. gilardii grown in the presence of PET (polyester) or PP (polyolefin) in CFM was measured over a period of around 180 hours. Ambient CO2 levels were determined to be 400 ppm.

[0030] FIG. 6 shows a liquid chromatography-mass spectrometry (LC-MS) analysis of byproducts produced during C. gilardii (BSl)-mediated plastic nanoparticle degradation. Samples from a blank solution (no bacteria or CFM), a solution of C. gilardii grown in CFM, a solution of C. gilardii grown in the presence of PP (polyolefin), and a solution of C. gilardii grown in the presence of PET (polyester) were collected and analyzed by LC-MS. Samples were collected after a 72 hour incubation period.

[0031] FIGs. 7A-7B show C. gilardii (BS1) grown in carbon-free media in the presence of nylon brush bristles after 0 days (FIG. 7A; 37°C, left and 45°C, right) and after 5 days (FIG. 7B, 37°C).

[0032] FIGs. 8A-8B show C. gilardii (BS1) grown in carbon-free media in the presence of nylon fishing wire after 0 days (FIG. 8A; 37°C) and after 6 days (FIG. 8B, 37°C).

[0033] FIGs. 9A-9C show C. gilardii (BS1) grown in carbon-free media in the presence of uncoated nylon dental floss after 0 days (FIG. 9A; 37°C), after 7 days (FIG. 9B, 37°C), and after 20 days (FIG. 9C, 37°C).

[0034] FIGs. 10A-10B show C. gilardii S ) grown in carbon-free media in the presence of a nylon membrane after 0 days (FIG. 10A; 37°C) and after 20 days (FIG. 10B, 37°C).

[0035] FIGs. 11A-11E show C. gilardii (BS1) plated on agar plates after being grown in carbon-free media without nylon (FIGs. 11A-11B) and in the presence of nylon (FIGs. 11C- 11E).

[0036] DETAILED DESCRIPTION

[0037] Despite efforts to recycle plastic, a significant portion of plastic waste ends up in landfills, incinerated or released into the environment. Because approximately 380 million metric tons of plastic are produced in a given year, the rapid accumulation of plastic in landfills is becoming a global health concern. Depending on the environmental conditions, some plastic waste can take several decades to degrade. Existing methods of plastic degradation often focus on single types of plastic; however, recycling schemes often do not distinguish between different types of plastic. Furthermore, consumer products include a complex array of different polymers resulting in co-mingling of different types of plastic in landfills. Other methods of plastic degradation require a chemical treatment step to breakdown the plastic, which can be costly and inefficient.

[0038] Accordingly, in some aspects, the present disclosure provides methods and compositions for efficient degradation of plastic waste, including mixed plastic waste, using Cupriavidus gilardii (C. gilardii). In some instances, the methods described herein result in the metabolism of plastic comprising polypropylene (PP), polyethylene terephthalate (PET), and / or nylon with just a few hours of applying the bacteria.

[0039] Cupriavidus gilardii

[0040] The present disclosure provides C. gilardii cells that are useful in the degradation of plastic. C. gilardii are aerobic gram-negative bacteria that are part of the Burkholderiaceae family. Subspecies of C. gilardii include Cupriavidus gilardii JI 1 (see, e.g., GenBank Accession No. GCA_007829435.1) and Cupriavidus gilardii CR3 (see, e.g., GenBank Accession No. GCA_001281465.1).

[0041] A C. gilardii isolate refers to one or more Cupriavidus gilardii cells that have been separated from their natural environment. In some embodiments, a C. gilardii isolate is a clonal population of cells. Non-limiting examples of C. gilardii isolates include the strain LMG 5886 [API 141-2-84, CCUG 38401, CIP 105966, Gilardii 4325, JCM 11283],

[0042] C. gilardii cells may be propagated under conditions well known in the art (e.g., temperature, culture and incubation times). For example, a population of C. gilardii cells may be expanded by propagating the C. gilardii cells in a nutrients-rich medium, such as agar medium or broth medium, to produce an effective amount of C. gilardii cells. For example, the medium may comprise tryptic soy agar and the broth medium may comprise tryptic soy broth. Tryptic soy agar may comprise tryptone, soytone, dextrose, NaCl, K2HPO4, and agar. In some embodiments, tryptic soy broth may comprise pancreatic digest of casein, pancreatic digest of soybean, dextrose, sodium chloride, dipotassium phosphate, and combinations thereof. In some embodiments, the medium comprises Euria Broth (EB). In some embodiments, cells are grown on LB agar plates. In some embodiments, the temperature is between 30°C and 50°C. In some embodiments, the temperature is 37°C. In some embodiments, the temperature is 45°C. In some embodiments, the incubation time is between 0 minutes and 20 days. In some embodiments, the incubation time is at least 0 minutes, at least 20 minutes, at least 60 minutes, at least 4 hours, at least 6 hours, at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 15 days, or at least 20 days. In some embodiments, the incubation time is 0 minutes. In some embodiments, the incubation time is 20 minutes. In some embodiments, the incubation time is 60 minutes. In some embodiments, the incubation time is 4 hours. In some embodiments, the incubation time is 6 hours. In some embodiments, the incubation time is 48 hours. In some embodiments, the incubation time is 3 days. In some embodiments, the incubation time is 4 days. In some embodiments, the incubation time is 5 days. In some embodiments, the incubation time is 6 days. In some embodiments, the incubation time is 7 days. In some embodiments, the incubation time is 10 days. In some embodiments, the incubation time is 15 days. In some embodiments, the incubation time is 20 days.

[0043] Plastic Waste

[0044] Aspects of the present disclosure relate to use of the bacterial strain e.g., C. gilardii to metabolize one or more types of plastic. As used herein, the term “plastic” refers to a material comprising one or more synthetic polymers and excludes breakdown byproducts of an oxidation reaction (e.g., byproduct of cobalt and / or manganese oxidation). The adaptability, durability, and flexibility of plastics have allowed for the production of a diverse array of consumer products and have resulted in numerous industrial uses. Many plastics can be manipulated (e.g., molded, extruded, and / or pressed) while others are rigid plastics with low elasticity. Non-limiting measures of elasticity include the modulus of elasticity. In some instances, a plastic is a thermoplastic, which is a material that becomes pliable or moldable at a certain temperature and then solidifies when cooled. For example, a thermoplastic may have a melting point between 130 °C to 350 °C.

[0045] Non-limiting examples of plastics include polyolefins, polyesters, and polyamides. Polyolefins are polymers comprising olefin monomer units. In some embodiments, a polyolefin comprises the chemical formula (CH2CHR)nwhere R is an alkyl group. Non-limiting examples of polyolefins include polyethylene (PE) and polypropylene (PP). Polyesters are polymers that comprise more than one ester functional group. In some instances, a polyester comprises an ester functional group in every repeating monomer. In some embodiments, a polyester is a polyethylene terephthalate (PET). Polyamides are polymers comprising repeating monomers linked by amide bonds. In some embodiments, a polyamide is nylon. In some embodiments, a plastic comprises PET, PP, and / or nylon. PET is the most common type of thermoplastic material and is an aliphatic polyester that comprises repeating CioHgCE units. In some instances, PET is as defined by the National Association for PET Container Resources (NAPCOR) and is derived from terephthalic acid (or dimethyl terephthalate) and mono ethylene glycol. In some instances, PET has a melting peak temperature of 225°C to 255°C. For example, the melting peak temperature may be determined during the second thermal scan in procedure 10.1 in ASTM D3418, when heating the sample at a rate of 10°C / minute. The American Society for Testing and Materials (ASTM) has set forth a biodegradation test standard for solid materials including plastics (ASTM D-6400). The ASTM standard tests for composting is about 58 °C. This lightweight material is present in numerous products, including but not limited to textiles, electronics, automotive parts, and packaging, and is often recycled.

[0046] PP is the second most common type of thermoplastic material and is a type of polyolefin. PP comprises the chemical formula (CaHe / n. and is considered a low density plastic. In some embodiments, PP has a density of 0.90-0.92 g / cm3. In some instances, PP has a melting temperature of about 170°C. PP may be found in a variety of products including consumer products, medical equipment, and automobiles. For example, many household appliances, caps and closures, packaging, luggage, bags, medical tools, and car dashboards comprise PP.

[0047] Nylon is a synthetic polymer that comprises polyamides. This thermoplastic is often made from petroleum. In some embodiments, nylon has a melting point of 190°C to 350°C. There are numerous uses for nylon including the production of textiles, automotive parts, electrical equipment, and films for food packaging.

[0048] The compositions and methods disclosed herein may be useful in degrading waste comprising plastic (z.e., plastic waste). Waste comprising plastic may be derived from any suitable source, including but not limited to one or more streams of domestic refuse. In some embodiments, plastic waste comprises PET, PP, and / or nylon.

[0049] A mixture of plastics, such as mixed plastic waste, comprises two or more types of plastic. For example, a mixture of plastics may comprise 2 to 10 different types of plastic (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different types of plastic). In some embodiments, in which the mixture of plastics includes mixed plastic waste, separate waste streams may be combined to produce mixed plastic waste or the mixed plastic waste may be derived from a single stream of comingled plastics.

[0050] In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyolefin, polyester, and / or polyamide (e.g., PET, PP, and / or nylon). In some embodiments, 5% to 100% of the mixture of plastics (e.g., mixed plastic waste) comprises polyolefin, polyester, and / or polyamide (e.g., PET, PP, and / or nylon). For example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the mixture of plastics (e.g., mixed plastic waste) may comprise polyolefin, polyester, and / or polyamide (e.g., PET, PP, and / or nylon). In some embodiments, at least 30% (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90%) of the mixture of plastics comprises polyolefin (e.g., PET). In some embodiments, about 10% to about 90% (e.g., about 20% to about 80%, about 30% to about 70%) of the mixture of plastics comprises polyolefin (e.g., PET). In some embodiments, at least 30% (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90%) of the mixture of plastics comprises polyester (e.g., PP). In some embodiments, about 10% to about 90% (e.g., about 20% to about 80%, about 30% to about 70%) of the mixture of plastics comprises polyester (e.g., PP). In some embodiments, at least 30% (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90%) of the mixture of plastics comprises polyamide (e.g., nylon). In some embodiments, about 10% to about 90% (e.g., about 20% to about 80%, about 30% to about 70%) of the mixture of plastics comprises polyamide (e.g., nylon).

[0051] In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyolefin and polyester. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyolefin and polyamide. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyester and polyamide. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyolefin, polyester, and polyamide.

[0052] In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polypropylene and polyethylene terephthalate. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polypropylene and nylon. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polyethylene terephthalate and nylon. In some embodiments, the mixture of plastics (e.g., mixed plastic waste) comprises polypropylene, polyethylene terephthalate, and nylon.

[0053] Methods of degrading plastic waste

[0054] Some aspects of the present disclosure provide methods for degrading plastic waste comprising contacting a Cupriavidus gilardii (C. gilardii) with a plastic. Surprisingly, the results disclosed herein show that one bacterial strain could be used to degrade three different types of plastics and a pre-treatment step was not required. The results disclosed herein also show that PP, PET, and / or nylon could be provided as the sole source of carbon for C. gilardii because each of these plastics increased C. gilardii growth in carbon-free media. Accordingly, in some embodiments, a method disclosed herein comprises contacting C. gilardii with a composition comprising two different types of plastics. In some embodiments, a method disclosed herein does not comprise one or more pre-treatment steps prior to contacting a plastic with C. gilardii. Chemical pretreatments are often used for degrading plastic waste, breaking it down into smaller, more biodegradable molecules. Non-limiting examples of pre-treatment steps that may be excluded from the methods provided herein include hydrolysis (process that uses water and heat to break down plastic polymers into smaller molecules), photodegradation (exposure to sunlight or other sources of UV radiation), chemical degradation (e.g., chemical oxidation, for example, with a cobalt or manganese-based catalyst, or ozone or peroxides), enzymatic degradation (e.g., lipases and esterases), and pyrolysis (heat). In some embodiments, a method disclosed herein comprises contacting a plastic with C. gilardii in carbon-free media.

[0055] In some embodiments, an effective amount of effective amount of C. gilardii cells is applied to or contacted with a plastic. As used herein, an effective amount of C. gilardii cells is the amount of C. gilardii cells that degrades a plastic. In some embodiments, an effective amount of C. gilardii cells is about IxlO3to about IxlO10cells. In some embodiments, an effective amount of C. gilardii cells is about IxlO4to about IxlO10cells. For example, an effective amount of C. gilardii cells may be about IxlO4to about IxlO9cells, about IxlO4to about IxlO8cells, about IxlO4to about IxlO7cells, about IxlO4to about IxlO6cells, about IxlO5to about IxlO10cells, about IxlO5to about IxlO9cells, about IxlO5to about IxlO8cells, about IxlO5to about IxlO7cells, about IxlO6to about IxlO10cells, about IxlO6to about IxlO9cells, or IxlO6to about IxlO8cells. In some embodiments, an effective amount of effective amount of C. gilardii cells is at least IxlO3, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10cells. In some embodiments, an effective amount is at least IxlO3C. gilardii cells. In some embodiments, an effective amount is at least IxlO4C. gilardii cells. In some embodiments, an effective amount is at least IxlO5C. gilardii cells.

[0056] In some embodiments, an effective amount of C. gilardii cells is about IxlO5to about IxlO10C. gilardii cells per ml (cells / ml). For example, an effective amount of C. gilardii cells may be about 100,000 to about IxlO9C. gilardii cells per ml, about 100,000 to about IxlO8C. gilardii cells per ml, about 100,000 to about IxlO7C. gilardii cells per ml, about 100,000 to about IxlO6C. gilardii cells per ml, about 100,000 to about 500,000 C. gilardii cells per ml, about 200,000 to about 2xl09C. gilardii cells per ml, about 200,000 to about 2xl08C. gilardii cells per ml, about 200,000 to about 2xl07C. gilardii cells per ml, about 200,000 to about 2xl06 C. gilardii cells per ml, about 300,000 to about 3xl09C. gilardii cells per ml, about 300,000 to about 3xl08C. gilardii cells per ml, about 300,000 to about 3xl07C. gilardii cells per ml, about 300,000 to about 3xl06C. gilardii cells per ml, about 400,000 to about 4xl09C. gilardii cells per ml, about 400,000 to about 4xl08C. gilardii cells per ml, about 400,000 to about 4xl07C. gilardii cells per ml, about 400,000 to about 4xl06C. gilardii cells per ml, about 500,000 to about 5xl09C. gilardii cells per ml, about 500,000 to about 5xl08C. gilardii cells per ml, about 500,000 to about 5xl07C. gilardii cells per ml, or about 500,000 to about 5xl06C. gilardii cells per ml.

[0057] In some embodiments, an effective amount of C. gilardii cells is about 100,000 cells per ml, about 200,000 cells per ml, about 300,000 cells per ml, about 400,000 cells per ml, about 500,000 cells per ml, about 1 x 106cells per ml, about IxlO7cells per ml, about IxlO8cells per ml, about IxlO9cells per ml, or about 1 xlO10cells per ml. In some embodiments, an effective amount of C. gilardii cells is at least 400,000 C. gilardii cells per ml. In some embodiments, the effective amount of C. gilardii cells is determined as the number of cells per ml of carbon-free media. In some embodiments, the effective amount of C. gilardii cells is determined as the number of cells per ml of carrier.

[0058] In some embodiments, a method disclosed herein comprises contacting C. gilardii with a plastic for a period of time of 5 minutes to 100 hours, or more. For example, C. gilardii may be cultured for a period of time of about 5 minutes to about 5,000 minutes (e.g., about 5 minutes to about 2,000 minutes, about 5 minutes to about 1,000 minutes, about 5 minutes to about 500 minutes, about 5 minutes to about 250 minutes, about 5 minutes to about 100 minutes, about 100 minutes to about 5,000 minutes, about 100 minutes to about 4,000 minutes, about 100 minutes to about 3,000 minutes, about 100 minutes to about 2,000 minutes, about 100 minutes to about 1,000 minutes, about 100 minutes to about 500 minutes, or about 150 minutes to about 450 minutes). In some embodiments, a method disclosed herein comprises contacting C. gilardii with a plastic for a period of time of at least 100 minutes, at least 200 minutes, at least 300 minutes, at least 400 minutes, at least 500 minutes, at least 1000 minutes, at least 1,500 minutes, at least 2,000 minutes, or at least 5,000 minutes. In some embodiments, a method disclosed herein comprises contacting C. gilardii with a plastic for a period of time of at least 200 minutes.

[0059] In some instances, a plastic is present in a composition and C. gilardii is contacted with the composition. As non-limiting example, one or more plastics may be present in waste (e.g., mixed plastic waste). In some embodiments, a method disclosed herein comprises contacting C. gilardii cells (e.g., a composition comprising an effective amount of C. gilardii cells) with mixed plastic waste. In some embodiments, a method disclosed herein comprises degrading mixed plastic waste using C. gilardii.

[0060] When used in reference to plastic or plastic waste, degradation refers to the loss of one or more properties of the plastic. For example, plastic degradation may refer the conversion of one or polymers in a plastic into one or more products, the breaking down of a piece of plastic into one or more pieces, or an increase in the pliability of the plastic. In some instances, plastic degradation may be measured as metabolism of the plastic by C. gilardii. C. gilardii metabolism may be detected by determining the rate of growth of the bacteria on the plastic, measuring the CO2 output by the bacteria following contact with the plastic (method suggested by ASTM D338 for aerobic biodegradation of plastic), and / or measuring the presence of one or more byproducts by mass spectrometry. See, also e.g., the Examples. In some instances, an effective amount of C. gilardii cells disclosed herein is used to degrade two or more types of plastic. The two or more types of plastic may be in the same composition (e.g., in waste).

[0061] In some embodiments, C. gilardii are contacted with a plastic at a temperature that is less than 58 °C. In some embodiments, C. gilardii are applied to a plastic at room temperature. In some embodiments, C. gilardii are applied to a plastic at a temperature of at least 21 °C. In some embodiments, C. gilardii are applied to a plastic at a temperature of at least 25 °C. In some embodiments, C. gilardii are applied to a plastic at a temperature of at least 37 °C. In some embodiments, C. gilardii are applied to a plastic at a temperature between about 21 °C and about 50 °C. In some embodiments, C. gilardii are applied to a plastic at a temperature between about 37 °C and about 50 °C, e.g., between about 37 °C and about 49 °C, between about 37 °C and about 48 °C, between about 37 °C and about 47 °C, between about 37 °C and about 46 °C, between about 37 °C and about 45 °C, between about 37 °C and about 44 °C, between about 37 °C and about 43 °C, between about 37 °C and about 42 °C, between about 37 °C and about 41 °C, between about 37 °C and about 40 °C, between about 40 °C and about 50 °C, between about 40 °C and about 49 °C, between about 40 °C and about 48 °C, between about 40 °C and about 47 °C, between about 40 °C and about 46 °C, between about 40 °C and about 45 °C, between about 40 °C and about 44 °C, between about 40 °C and about 43 °C, between about 43 °C and about 50 °C, between about 43 °C and about 49 °C, between about 43 °C and about 48 °C, between about 43 °C and about 47 °C, between about 43 °C and about 46 °C, between about 43 °C and about 45 °C, or between about 43 °C and about 44 °C. In some embodiments, C. gilardii is contacted with a plastic at about 45 °C.

[0062] Some methods disclosed herein increase the rate of plastic degradation. In some embodiments, contacting C. gilardii with plastic increases the rate of plastic degradation by about 1% to about 5,000% as compared to a control. For example, the rate of plastic degradation may be increased by about 1% to about 10%, about 1% to about 25%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 1% to about 200%, about 1% to about 300%, about 1% to about 400%, about 1% to about 500%, about 1% to about 600%, about 1% to about 700%, about 1% to about 800%, about 1% to about 900%, about 1% to about 1,000%, about 1% to about 2,000%, about 1% to about 3,000%, about 1% to about 4,000%, about 1% to about 5,000%, about 50% to about 75%, about 50% to about 100%, about 50% to about 200%, about 50% to about 300%, about 50% to about 400%, about 50% to about 500%, about 50% to about 600%, about 50% to about 700%, about 50% to about 800%, about 50% to about 900%, about 50% to about 1,000%, about 50% to about 2,000%, about 50% to about 3,000%, about 50% to about 4,000%, about 50% to about 5,000%, about 50% to about 3,000%, about 50% to about 4,000%, about 50% to about 5,000%, about 100% to about 200%, about 100% to about 300%, about 100% to about 400%, about 100% to about 500%, about 100% to about 600%, about 100% to about 700%, about 100% to about 800%, about 100% to about 900%, about 100% to about 1,000%, about 100% to about 2,000%, about 100% to about 3,000%, about 100% to about 4,000%, about 100% to about 5,000%, about 100% to about 4,000%, about 100% to about 5,000%, about 100% to about 3,000%, about 100% to about 4,000%, about 100% to about 5,000%, about 1,000% to about 5,000%, as compared to a control. In some embodiments, the control is the rate of plastic degradation in the absence of the effective amount of C. gilardii cells. In some instances, the rate of plastic degradation may be determined as the rate of metabolism of a plastic by C. gilardii.

[0063] In some instances, a method described herein decreases the amount of time needed to degrade a plastic as compared to when the plastic is not contacted with C. gilardii. For example, plastic degradation may be initiated in less than 100 days, less than 50 days, less than 25 days, less than 10 days, less than 5 days, less than 4 days, less than 3 days, less 2 days, or less than 1 day of contacting C. gilardii with a plastic. In some embodiments, plastic degradation is initiated in less than 100 hours, less than 50 hours, less than 40 hours, less than 30 hours, less than 20 hours, less than 10 hours, less than 5 hours, or less than 1 hour. In some embodiments, plastic degradation may be initiated in about 1 day to about 10 days, about 1 day to 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 2 days to about 10 days, about 2 days to about 9 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 5 days, about 3 days to about 10 days, about 3 days to about 9 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, or about 5 to about 10 days. In some embodiments, initiation of plastic degradation is determined as when an increase in C. gilardii metabolism is detected.

[0064] In some embodiments, contacting of an effective amount of C. gilardii cells with a plastic increases production of about 1 to about 100 byproducts as compared to a control. For example, the production of plastic may increase the production of about 1 to about 10 , about 1 to about 25 , about 1 to about 50 , about 1 to about 75 , about 1 to about 100 , about 5 to about 10, about 5 to about 25, about 5 to about 50, about 5 to about 100, about 10 to about 25, about 10 to about 50, about 10 to about 100, about 25 to about 50, about 25 to about 100, or about 50 to about 100 byproducts compared to a control. In some embodiments, the control is the C. gilardii grown in carbon-free media without the plastic.

[0065] In some embodiments, contacting C. gilardii with a plastic comprises culturing C. gilardii in the presence of a plastic. “Culturing” refers to the process by which cells are grown under controlled conditions, typically outside of their natural environment. For example, C. gilardii may be cultured in media with a plastic added to the media. In some embodiments, the media is carbon-free media. In some instances, C. gilardii is cultured directly on a plastic without media.

[0066] In some embodiments, C. gilardii cells are cultured in the presence of plastic (e.g., in liquid media comprising plastic) to an optical density, measured at a wavelength of 600 nm (OD600), of about 0.1 to about 2. In some embodiments, C. gilardii cells are cultured to an OD600 of about 0.1 to about 0.5, about 0.1 to about 0.6, about 0.1 to about 0.7, about 0.1 to about 0.8, about 0.1 to about 0.9, about 0.1 to about 1, about 0.1 to about 1.1, about 0.1 to about 1.2, about 0.1 to about 1.3, about 0.1 to about 1.4, about 0.1 to about 1.5, about 0.1 to about 1.6, about 0.1 to about 1.7, about 0.1 to about 1.8, about 0.1 to about 1.9, about 0.1 to about 2, about 0.5 to about 1, about 0.5 to about 1.1, about 0.5 to about 1.2, about 0.5 to about 1.3, about 0.5 to about 1.4, about 0.5 to about 1.5, about 0.5 to about 1.6, about 0.5 to about 1.7, about 0.5 to about 1.8, about 0.5 to about 1.9, about 0.5 to about 2, about 1 to about 1.5, about 1 to about 1.6, about 1 to about 1.7, about 1 to about 1.8, about 1 to about 1.9, about 1 to about 2, or about 1.5 to about 2.

[0067] C. gilardii cells may be formulated in solutions, wettable powders, dusting powders, soluble powders, emulsions or suspension concentrates, tablets, water-dispersible granules, membranes, sheets, aerosols, soft-gel, hard-gel; water soluble granules (slow or fast release), and microencapsulated granules or suspensions.

[0068] Any suitable method may be used to contact C. gilardii cells with plastic. For example, an effective amount of a composition comprising C. gilardii cells may be sprayed onto one or more plastics or spread onto one or more plastics. For example, C. gilardii cells may be aerially sprayed onto one or more plastics. In some embodiments, one or more plastics are coated with C. gilardii cells. In some embodiments, C. gilardii cell are only applied to one portion of a plastic and allowed to spread over the plastic. In some embodiments, C. gilardii cells are applied as granules. In some embodiments, C. gilardii cells are applied as particles. In some embodiments, C. gilardii cells are applied using an applicator. Non-limiting examples of applicators include syringes, spray devices, ampules, brushes, and sprinkler devices. For example, useful spray devices include a boom sprayer, a hand or backpack sprayer, crop dusters (z.e., aerial spraying).

[0069] Bioreactors

[0070] Aspects of the present disclosure provide bioreactors for degrading a plastic. For example, a bioreactor disclosed herein may be useful in degrading plastic waste, including mixed plastic waste, using any of the methods disclosed herein. A bioreactor refers to a container in which cells are cultured, including a culture flask, a dish, a bag, or any other vessel. In some instances, a bioreactor may be single-use (disposable), autoclavable, or sterilizable. The bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials. Other non-limiting examples of bioreactors include stirred- tank bioreactors, bubble column bioreactors, packed-bed bioreactors, membrane bioreactors, photo-bioreactors, and hollow fiber bioreactors.

[0071] The bioreactors disclosed herein allow for C. gilardii to contact a plastic and may comprise any of the C. gilardii cells, any of the plastics, and / or any of the plastic waste disclosed herein. The bioreactor may comprise one or more compartments that direct the flow of C. gilardii and / or plastic, including plastic waste. Non-limiting modes of operating the bioreactor include continuous, semi-continuous or non-continuous processes. For example, a bioreactor is continuous when the feed and product streams are continuously being fed and withdrawn from the system. In contrast, batch fermentation is a process where all the feed is added with the cells and the cells are cultured with the feed until the feed is consumed. It is also possible to intermittently withdraw cells and / or feed from the bioreactor.

[0072] A bioreactor may comprise a sensor and / or mechanism to control or measure a condition inside the bioreactor. Non-limiting examples of conditions include growth rate of cells, cell number, cell density, cell viability, concentration of a polymer, concentration of a by-product (e.g., metabolite), oxygen concentration, CO2 concentration, humidity, and temperature. In some embodiments, the temperature in a bioreactor is between 30°C and 50°C. In some embodiments, the temperature in a bioreactor is 37°C. In some embodiments, the temperature in a bioreactor is 45°C. In some embodiments, the cell number in a bioreactor is between 5.0xl07CFU / mL and 9.5xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 5.0xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 5.5xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 6.0xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 6.5xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 7.0xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 7.5xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 8.0xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 8.5xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 9.0xl07CFU / mL. In some embodiments, the cell number in a bioreactor is 9.5xl07CFU / mL. Bioreactor sensors and control mechanisms are known to one of ordinary skill in the art.

[0073] In some instances, a bioreactor comprises plastic as the sole source of carbon. In some instances, the only source of carbon is plastic waste. In some instances, the plastic waste is mixed plastic waste. For example, C. gilardii may be grown in a bioreactor that comprises plastic waste and no other source of carbon, including media comprising carbon, is added to the bioreactor. Other sources of carbon include atmosphere, soil, fossil fuels, water, organic waste, and living organisms, any one or more of which may be specifically excluded from the bioreactor.

[0074] Kits and applicators

[0075] Also encompassed by the disclosure are kits and applicators. The kits provided may comprise any of the bacterial cells disclosed herein and a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising an excipient for dilution or suspension a bacterial cell described herein. In some embodiments, the composition described herein provided in the first container and the second container are combined to form one unit dosage form, e.g., for contacting with plastic. Excipients for bacterial cell suspensions are nonactive ingredients that are added to the suspension to aid in stabilization, preservation, and administration of the bacterial cells.

[0076] Thus, in one aspect, provided are kits including a first container comprising a bacterial cell described herein. In certain embodiments, the kits are useful for degrading plastic.

[0077] In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the kits and instructions provide for degrading one or more types of plastic. A kit described herein may include one or more additional excipients described herein as a separate composition.

[0078] In some embodiments, a spray device is used as an applicator. A spray device is a tool used to atomize and distribute liquid substances in a fine mist or spray. These devices can be powered by compressed air, electricity, or by hand, and come in a variety of shapes and sizes depending on their intended use. Common examples of spray devices include spray bottles, aerosol cans, boom sprayers, hand or backpack sprayers, crop dusters (e.g., aerial sprayers), and airbrushes. Spray devices can be operated manually or automated, and may include features such as adjustable spray patterns, variable pressure, and interchangeable nozzles for different types of liquids and applications.

[0079] In some embodiments, a sprinkler device is used as an applicator. A sprinkler device is a mechanical device used for distributing a liquid in a controlled manner. Sprinkler devices include, in some embodiments, a series of pipes or hoses that are connected to a liquid source (e.g., a composition comprising C. gilardiig which is then distributed to a series of nozzles or sprinkler heads. The nozzles or sprinkler heads are typically adjustable, allowing for a variety of spray patterns and coverage areas. Sprinkler devices can be operated manually or automatically, and may include features such as timers, sensors, and flow regulators to ensure efficient and effective water distribution. There are several types of sprinkler devices, including rotary sprinklers, impact sprinklers, and micro -irrigation systems. Rotary sprinklers spin in a circular motion, covering a large area, while impact sprinklers use a spring-loaded mechanism to create a rotating spray pattern.

[0080] EXAMPLES

[0081] Improved methods for the removal or degradation of plastic wastes, including mixed plastic waste, is an important area for innovation and research development. The present disclosure is related to the identification and characterization of bacterial strains capable of utilizing plastic nanoparticles as a sole source of nutrients, thus resulting in efficient degradation and consequent removal of plastic waste.

[0082] Example 1. Identification of bacterial strains capable of degrading plastic nanoparticles

[0083] To identify bacterial strains for use in the degradation of plastic nanoparticles, wax worms (Mellonella galleria) were starved of all nutrients and fed plastic for 3 days. After 3 days, bacterial cultures from the wax worm gut were isolated and cultured in nutrient-rich LB (lysogeny broth) and BHI (brain heart infusion) media. Following initial growth, isolated strains were cultured and iteratively screened in carbon-free media (CFM: KH2PO4, K2HPO4, MgSCE, NH4NO3, NaCl, FeSO4*7H2O, ZnSCU’ FhO, and MnSCE^FFO) in the presence of polypropylene (PP) and polyethylene terephthalate (PET) nanoparticles for 7 days. After 7 days, strains of interest were isolated for further analyses.

[0084] Two strains, Cupriavidus gilardii and Staphylococcus hominis, were identified that grew in CFM with plastic nanoparticles as the sole source of nutrients. To confirm these findings, a C. gilardii culture was plated in the presence of LB media alone, CFM alone, and CFM with polypropylene nanoparticles (FIG. 1, left). As expected, growth, as measured by optical density (OD) at 600 nm, was observed in LB media alone and growth was not observed in CBM alone. Surprisingly, C. gilardii growth was observed after 1,500 minutes in CBM with PP nanoparticles, indicating that C. gilardii could be used to metabolize PP. PP could be used as a sole source of nutrients for C. gilardii cultures. Next, C. gilardii and .S'. hominis were cultured in LB media alone, CBM alone, and CBM with PET nanoparticles (FIG. 1, middle and right). Growth was observed in the C. gilardii culture and the .S', hominis culture after 500 minutes, indicating that C. gilardii and .S', hominis could be used to metabolize PET. PET could be provided as a sole source of nutrients to both C. gilardii and .S', hominis. Notably, we found that the plastic particles interfere with the optical reading of bacterial growth, so growth curves for C. gilardii in the presence of PP and PET were also obtained using an agar plating method in Example 2 below.

[0085] In addition to observed growth in culture, C. gilardii was observed to form biofilm in the presence of PET nanoparticles. PET nanoparticles were plated alone (FIG. 2A at 100X magnification) or in the presence of C. gilardii in CFM (FIG. 2B at 100X magnification). C. gilardii in the presence of CFM and PET nanoparticles formed a biofilm. This result was confirmed further by dipping a plastic pipette tip into a solution of C. gilardii, which led to formation of a C. gilardii biofilm on the pipette tip (FIG. 2C).

[0086] This Example demonstrates that an effective amount of C. gilardii cells metabolized two different types of plastic (PP and PET), while .S', hominis is only able to utilize PET as a sole source of nutrients.

[0087] Example 2. Further analysis of plastic degradation by C. gilardii

[0088] Following the identification of a bacterial strain that metabolized both PP and PET, C. gilardii growth was analyzed in greater detail. FIG. 3 shows a plating protocol that was used for generating a growth curve for C. gilardii (BS1) in the presence of plastic nanoparticles (PNP) and CFM. First, a solution of PNP and carbon-free media was inoculated with C. gilardii and incubated overnight at 37°C. Next, the BS1 sample was diluted into aliquots for sampling at various time points. The aliquots were then incubated at 45°C and plated at 0 minutes, 20 minutes, 40 minutes, 60 minutes, 4 hours, 6 hours, 24 hours, and 48 hours. The time point plates were then incubated at 37°C and analyzed. After 24 hours, C. gilardii growth was observed in the presence of PP and PET (FIG. 4). C. gilardii grown in the presence of polyolefin produced carbon dioxide as a by-product of plastic nanoparticle degradation (FIG. 5A-5B), which indicates that a single bacterial strain could be used to metabolize multiple types of plastic. The metabolites produced by C. gilardii following exposure to plastic was further analyzed by mass spectrometry. Total ion current of supernatants from bacteria grown with PP nanoparticles (shown in red) and PET nanoparticles (black) (FIG. 6). Compared to a blank (orange) and supernatant of carbon-free media (green), the supernatants from the nanoparticle assay display peaks at -1.75 min and -3.5 min, with a prominent peak at -2.75 min for the case of bacteria grown in PP nanoparticle media (FIG. 6). The unique peaks observed in the PP and PET samples compared to CFM alone are yet to be determined. Notably different products were detected following contacting of C. gilardii with PP (polyolefin) or PET (polyester) as compared to growth of C. gilardii in carbon-free media alone. Therefore, C. gilardii grown in PP and PET resulted into different byproducts.

[0089] Together, these results demonstrate that C. gilardii metabolized two different plastics as the sole source of nutrients, thus leading to plastic degradation. In addition, these results suggest that C. gilardii produces a plastic degradation by-product, in addition to degrading plastics.

[0090] Example 3. Testing C. gilardii growth in the presence of nylon

[0091] To test whether C. gilardii (BS1) is able to degrade nylon, C. gilardii was grown in the presence of carbon-free media and various forms of nylon.

[0092] First, C. gilardii (BS1) was grown in the presence of carbon-free media and nylon brush bristles. After 5 days at 37°C, BS1 degraded nearly all of the nylon brush bristles (FIG. 7B compared to FIG. 7A). BS1 also degraded nylon brush bristles at 45 °C after 7-8 days (data not shown).

[0093] Next, C. gilardii (BS1) was grown in the presence of carbon-free media and nylon fishing wire. After 6 days at 37°C in a shaking incubator, BS1 degraded the nylon fishing wire (FIG. 8B compared to FIG. 8A). C. gilardii (BS1) was also grown in the presence of carbon- free media and uncoated nylon dental floss. After 7 days at 37°C in a shaking incubator, BS1 partially degraded the nylon dental floss (FIG. 9B compared to FIG. 9A), and after 20 days at 37°C in a shaking incubator, BS1 degraded the nylon dental floss (FIG. 9C compared to FIG. 9B, FIG. 9C compared to FIG. 9A). Finally, C. gilardii (BS1) was grown in the presence of carbon-free media and a nylon membrane that is typically used in gel permeation chromatography. After 20 days at 37°C in a shaking incubator, BS1 degraded nearly all of the nylon membrane (FIG. 10B compared to FIG. 10A), although degradation began after just 5 days (data not shown).

[0094] Following growth in various types of nylon, C. gilardii (BS1) was plated on agar and cell concentration was measured (FIGs. 11A-11E). As a control, a sample of carbon-free media containing nylon brush bristles was plated. As expected, no bacteria were detected from this sample (FIGs. 11A-11B). A sample of BS1 grown in the presence of carbon-free media and nylon brush bristles for 5 days was plated and concentrations of 9xl07CFU / mL (FIG. 11C) and 6.2xl07CFU / mL (FIG. 11D) were observed. Finally, a sample of BS1 grown in the presence of carbon-free media and nylon membrane for 20 days was plated and a concentration of 8.4xl07CFU / mL (FIG. HE) was observed. These results demonstrate that C. gilardii degrades multiple plastics, including nylon.

[0095] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0096] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0097] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0098] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0099] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0100] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A biodegradation method, comprising: applying an effective amount of a composition comprising Cupriavidus gilardii (C. gilardii) cells to a mixture of plastics, thereby degrading the mixture of plastics.

2. A biodegradation method, comprising: applying an effective amount of a composition comprising Cupriavidus gilardii (C. gilardii) cells to a product comprising a mixture of plastics, thereby degrading the mixture of plastics.

3. The method of any one of the preceding claims, wherein the mixture of plastics comprises mixed plastic waste.

4. The method of any one of the preceding claims, wherein the mixture of plastics comprises two or more types of plastic.

5. The method of claim 4, wherein the mixture of plastics comprises three or more types of plastic.

6. The method of any one of the preceding claims, wherein the mixture of plastics is selected from polyolefin, polyester, and polyamide.

7. The method of claim 6, wherein the mixture of plastics comprises polyolefin, optionally wherein the polyolefin is polypropylene.

8. The method of claim 6 or 7, wherein the mixture of plastics comprises polyester, optionally wherein the polyester is polyethylene terephthalate.

9. The method of any one of claims 6-8, wherein the mixture of plastics comprises polyamide, optionally wherein the polyamide is nylon.

10. The method of any one of claims 6-9, wherein the mixture of plastics comprises: polypropylene and polyethylene terephthalate;polypropylene and nylon; polyethylene terephthalate and nylon; or polypropylene, polyethylene terephthalate, and nylon.

11. The method of any one of the preceding claims, wherein at least 30% of the mixture of plastics comprises the polyolefin, optionally polypropylene.

12. The method of any one of claims 6-11, wherein at least 30% of the mixture of plastics comprises the polyester, optionally polyethylene terephthalate.

13. The method of any one of claims 6-12, wherein at least 30% of the mixture of plastics comprises the polyamide, optionally nylon.

14. The method of any one of claims 6-13, wherein the composition comprises a carrier, optionally wherein the carrier is a carbon-free medium.

15. The method of claim 14, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5C. gilardii cells per milliliter (ml) carrier.

16. The method of any one of the preceding claims, wherein the C. gilardii cells are in contact with the mixture of plastics for at least 200 minutes.

17. The method of any one of the preceding claims, wherein the C. gilardii cells and the mixture of plastics is maintained for at least 200 minutes at a temperature of about 37 °C and about 50 °C, optionally about 45 °C.

18. The method of any one of the preceding claims, wherein the mixture of plastics is untreated prior to the contacting.

19. The method of claim 18, wherein the method does not comprise treating the plastic with a cobalt or manganese catalyst prior to the contacting.

20. The method of any one of the preceding claims, wherein the C. gilardii cells are formulated as a solution, a powder or as granules.

21. The method of any one of the preceding claims, wherein the applying of the C. gilardii cells to a mixture or product comprises spraying the cells onto the mixture or product.

22. A bioreactor comprising: a mixture of plastics; andCupriavidus gilardii (C. gilardii) cells in an effective amount to degrade the mixture of plastics.

23. The bioreactor of claim 22, wherein the mixture of plastics comprises mixed plastic waste.

24. The bioreactor of claim 22 or 23, wherein the mixture of plastics comprises two or more types of plastic.

25. The bioreactor of claim 24, wherein the mixture of plastics comprises three or more types of plastic.

26. The bioreactor of any one of the preceding claims, wherein the mixture of plastics is selected from polyolefin, polyester, and polyamide.

27. The bioreactor of claim 26, wherein the mixture of plastics comprises polyolefin, optionally wherein the polyolefin is polypropylene.

28. The bioreactor of claim 26 or 27, wherein the mixture of plastics comprises polyester, optionally wherein the polyester is polyethylene terephthalate.

29. The bioreactor of any one of claims 26-28, wherein the mixture of plastics comprises polyamide, optionally wherein the polyamide is nylon.

30. The bioreactor of any one of claims 26-29, wherein the mixture of plastics comprises: polypropylene and polyethylene terephthalate; polypropylene and nylon; polyethylene terephthalate and nylon; or polypropylene, polyethylene terephthalate, and nylon.

31. The bioreactor of any one of claims 26-30, wherein at least 30% of the mixture of plastics comprises the polyolefin, optionally polypropylene.

32. The bioreactor of any one of claims 26-31, wherein at least 30% of the mixture of plastics comprises the polyester, optionally polyethylene terephthalate.

33. The bioreactor of any one of claims 26-32, wherein at least 30% of the mixture of plastics comprises the polyamide, optionally nylon.

34. The bioreactor of any one of the preceding claims, further comprising a medium, optionally a carbon-free medium.

35. The bioreactor of claim 34, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5C. gilardii cells per milliliter (ml) medium.

36. The bioreactor of any one of the preceding claims, wherein the temperature inside the bioreactor is about 37 °C and about 50 °C, optionally about 45 °C.

37. The bioreactor of any one of the preceding claims, wherein the mixture of plastics is untreated, optionally free of exposure to a cobalt or manganese catalyst.

38. An applicator comprising Cupriavidus gilardii (C. gilardii) cells in an effective amount to degrade a mixture of plastics.

39. The applicator of claim 38, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5C. gilardii cells per milliliter (ml) medium.

40. The applicator of claim 38 or 39, wherein the mixture of plastics comprises mixed plastic waste.

41. The applicator of any one of claims 38-40, wherein the mixture of plastics comprises two or more types of plastic.

42. The applicator of claim 41, wherein the mixture of plastics comprises three or more types of plastic.

43. The applicator of any one of the preceding claims, wherein the mixture of plastics is selected from polyolefin, polyester, and polyamide.

44. The applicator of claim 43, wherein the mixture of plastics comprises polyolefin, optionally wherein the polyolefin is polypropylene.

45. The applicator of claim 43 or 44, wherein the mixture of plastics comprises polyester, optionally wherein the polyester is polyethylene terephthalate.

46. The applicator of claim 44 or 45, wherein the mixture of plastics comprises polyamide, optionally wherein the polyamide is nylon.

47. The applicator of any one of claims 44-46, wherein the mixture of plastics comprises: polypropylene and polyethylene terephthalate; polypropylene and nylon; polyethylene terephthalate and nylon; or polypropylene, polyethylene terephthalate, and nylon.

48. The applicator of any one of the preceding claims, wherein the C. gilardii cells are formulated as a solution, a powder or as granules.

49. A composition comprising granules of C. gilardii cells.

50. A kit comprising: an applicator; andCupriavidus gilardii (C. gilardii) cells in an effective amount to degrade a mixture of plastics.

51. The kit of claim 50, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5C. gilardii cells per milliliter (ml) medium.

52. The kit of claim 50 or 51 , wherein the mixture of plastics comprises mixed plastic waste.

53. The kit of any one of claims 50-52, wherein the mixture of plastics comprises two or more types of plastic.

54. The kit of claim 53, wherein the mixture of plastics comprises three or more types of plastic.

55. The kit of any one of the preceding claims, wherein the mixture of plastics is selected from polyolefin, polyester, and polyamide.

56. The kit of claim 55, wherein the mixture of plastics comprises polyolefin, optionally wherein the polyolefin is polypropylene.

57. The kit of claim 55 or 56, wherein the mixture of plastics comprises polyester, optionally wherein the polyester is polyethylene terephthalate.

58. The kit of claim 56 or 57, wherein the mixture of plastics comprises polyamide, optionally wherein the polyamide is nylon.

59. The kit of any one of claims 56-58, wherein the mixture of plastics comprises: polypropylene and polyethylene terephthalate; polypropylene and nylon; polyethylene terephthalate and nylon; or polypropylene, polyethylene terephthalate, and nylon.

60. The kit of any one of the preceding claims, wherein the C. gilardii cells are formulated as a solution, a powder or as granules.

61. The kit of any one of the preceding claims, comprises a composition comprising granules of the C. gilardii cells.

62. The kit of any one of the preceding claims, comprises a composition comprising a spray formula of the C. gilardii cells.

Images