Microbes and enzymes for plastic degradation

Abstract

In some aspects, the present disclosure provides methods and compositions for degrading one or more plastics, such as polyethylene terephthalate, using Staphylococcus hominis.

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 / 631,125, 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 Staphylococcus hominis (.S', hominis). an aerobic gram-positive bacterium isolated from the gut of waxworms (Galleria mellonella). is capable of metabolizing polyethylene terephthalate (PET) 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 S. hominis cells to one or more plastics, thereby degrading the one or more plastics.

[0009] Other aspects of the disclosure relate to a biodegradation method, comprising: applying an effective amount of a composition comprising S. hominis cells to a product comprising one or more plastics, thereby degrading the one or more plastics.

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

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

[0012] Still other aspects of the disclosure relate to an applicator comprising S. hominis cells in an effective amount (e.g., at least IxlO3, IxlO4, or IxlO5S. hominis cells / ml) to degrade one or more plastics.

[0013] Further aspects of the disclosure relate to a kit comprising: an applicator; and S. hominis cells in an effective amount (e.g., at least IxlO3, IxlO4, or l x IO5.S', hominis cells / ml) to degrade one or more plastics. In some embodiments, the S. hominis cells are formulated as a solution, a powder or as granules. In some embodiments, the kit comprises a composition comprising granules of the S. hominis cells.

[0014] In some embodiments, the one or more plastics comprises plastic waste. In some embodiments, the one or more plastics comprise PET.

[0015] In some embodiments, the composition comprises a carrier, for example, a carbon-free medium.

[0016] In some embodiments, the effective amount is at least IxlO3, IxlO4, or IxlO5S. hominis cells per milliliter (ml) carrier. For example, an effective amount may be about or at least IxlO3, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xlO3, 9xl03, IxlO4, 2xl04, 3xl04, 4xl04, 5xl04, 6xl04, 7xl04, 8xl04, 9xl04, IxlO5, 2xl05, 3xl05, 4xl05, 5xl05, 6xl05, 7xl05, 8xl05, or 9xl05cells / ml.

[0017] In some embodiments, the S. hominis cells are in contact with the one or more plastics for at least 200 minutes.

[0018] In some embodiments, the S. hominis cells and the one or more plastics are 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.

[0019] In some embodiments, the one or more plastics are untreated prior to the contacting.

[0020] In some embodiments, the method does not comprise treating the one or more plastics with a cobalt or manganese catalyst prior to the contacting.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] 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:

[0023] FIG. 1 shows the growth curves of Staphylococcus hominis (.S'. hominis) in different growth media. S. hominis (SH) was grown in the presence of lysogeny broth (LB) media alone, carbon free media (CFM) media alone, or CFM and polyethylene terephthalate (PET). Growth was measured by optical density (OD) at 600 nm.

[0024] DETAILED DESCRIPTION

[0025] 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.

[0026] Accordingly, in some aspects, the present disclosure provides methods and compositions for efficient degradation of plastic waste using Staphylococcus hominis (S. hominis') . In some instances, the methods described herein result in the metabolism of plastic comprising polyethylene terephthalate (PET) within just a few hours of applying the bacteria.

[0027] Staphylococcus hominis

[0028] The present disclosure provides S. hominis cells that are useful in the degradation of plastic. S. hominis (Kloos & Schleifer 1975) are aerobic, coagulase negative, gram-positive bacteria that are part of the Staphylococcaceae family and Staphylococcus genus. Subspecies of S. hominis include S. hominis hominis (see, e.g., GenBank Accession No. GCA_002850375.1) and S. hominis novobiosepticus (see, e.g., GenBank Accession No. GCA_002902465.1).

[0029] A S. hominis isolate refers to one or more Staphylococcus hominis cells that have been separated from their natural environment. In some embodiments, a S. hominis isolate is a clonal population of cells. A clonal population of bacterial cells refers to a group of bacterial cells that are genetically identical and derived from a single ancestor. This occurs when a single bacterial cell undergoes cell division to produce daughter cells, which in turn divide to produce a colony of cells that are all genetically identical to the original cell. This clonal population can then continue to divide and grow, forming a bacterial culture. Non-limiting examples of S. hominis isolates include the strain ES 1-134. Other non-examples of S. hominis isolates include clinical isolates (e.g., isolated from clinical samples such as blood, urine, and wound swabs), biofilm isolates, and environmental isolates (e.g., isolated from environmental sources such as soil, water, and dairy products).

[0030] S. hominis cells may be propagated under conditions well known in the art e.g., temperature, culture and incubation times). For example, a population of S. hominis cells may be expanded by propagating the S. hominis cells in a nutrients-rich medium, such as agar medium or broth medium, to produce an effective amount of S. hominis 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.

[0031] Plastic Waste

[0032] Plastic waste refers to any discarded or abandoned plastic material that is no longer useful or needed. This can include a wide range of plastic products, such as single-use packaging, bags, bottles, and electronic devices. Plastic waste is a significant environmental problem, as it is not biodegradable and can persist in the environment for hundreds of years, leading to a buildup of plastic pollution that can harm wildlife and ecosystems. Plastic waste can also contribute to climate change, as the production and disposal of plastic products require significant amounts of energy and can generate greenhouse gas emissions. Proper disposal and management of plastic waste is essential to reduce its environmental impact and promote a more sustainable future.

[0033] Aspects of the present disclosure relate to use of the bacterial strain e.g., S. hominis to metabolize one or more types of plastic, such as those found in plastic waste. 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.

[0034] 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.

[0035] In some embodiments, a plastic comprises PET. A product may also comprise additional plastics such as 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.

[0036] PP is the second most common type of thermoplastic material and is a type of polyolefin. PP comprises the chemical formula (C.^Hrdn. and is considered a low density plastic. In some embodiments, PP has a density of 0.90-0.92 g / cm’. 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. Nylon is a synthetic polymer that comprises polyamides. This thermoplastic is often made from petroleum. In some embodiments, nylon has Lt melting point of 190°C to 35O°C. There are numerous uses for nylon including the production of textiles, automotive parts, electrical equipment, and films for food packaging.

[0037] 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. In some embodiments, plastic waste comprises PET and one or more additional plastics, such as PP and / or nylon.

[0038] A mixture of plastics, such as mixed plastic waste, comprises one or more types of plastic. For example, one or more plastics may comprise 1 to 10 different types of plastic (e.g., at least 1, 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.

[0039] In some embodiments, one or more 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 one or more 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 one or more 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 one or more 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 one or more 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 one or more 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 one or more 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 one or more 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 one or more plastics comprises polyamide (e.g., nylon).

[0040] In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polyolefin and polyester. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polyolefin and polyamide. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polyester and polyamide. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polyolefin, polyester, and polyamide.

[0041] In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polypropylene and polyethylene terephthalate. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polypropylene and nylon. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polyethylene terephthalate and nylon. In some embodiments, the one or more plastics (e.g., mixed plastic waste) comprises polypropylene, polyethylene terephthalate, and nylon.

[0042] Methods of degrading plastic waste

[0043] Some aspects of the present disclosure provide methods for degrading plastic waste comprising contacting a Staphylococcus hominis (S. hominis) with a plastic. Surprisingly, the results disclosed herein show that one bacterial strain could be used to degrade PET and a pretreatment step was not required. The results disclosed herein also show that PET can be provided as the sole source of carbon for S. hominis because this plastic increased S. hominis growth in carbon-free media.

[0044] In some embodiments, a method disclosed herein does not comprise one or more pretreatment steps prior to contacting a plastic with S. hominis. 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 .S', hominis in carbon-free media.

[0045] In some embodiments, an effective amount of effective amount of S. hominis cells is applied to or contacted with a plastic. As used herein, an effective amount of S. hominis cells is the amount of S. hominis cells that degrades a plastic. In some embodiments, an effective amount of S. hominis cells is about IxlO3to about IxlO10cells. In some embodiments, an effective amount of S. hominis cells is about IxlO4to about IxlO10cells. For example, an effective amount of S. hominis 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 S. hominis 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 IxlO3S. hominis cells. In some embodiments, an effective amount is at least IxlO4S. hominis cells. In some embodiments, an effective amount is at least IxlO5S. hominis cells.

[0046] In some embodiments, an effective amount of S. hominis cells is about IxlO5to about IxlO10S. hominis cells per ml (cells / ml). For example, an effective amount of S. hominis cells may be about 100,000 to about IxlO9S. hominis cells per ml, about 100,000 to about IxlO8S. hominis cells per ml, about 100,000 to about IxlO7S. hominis cells per ml, about 100,000 to about IxlO6S. hominis cells per ml, about 100,000 to about 500,000 S. hominis cells per ml, about 200,000 to about 2xl09S. hominis cells per ml, about 200,000 to about 2xl08S. hominis cells per ml, about 200,000 to about 2xl07S. hominis cells per ml, about 200,000 to about 2xl06S. hominis cells per ml, about 300,000 to about 3xl09S. hominis cells per ml, about 300,000 to about 3xl08S. hominis cells per ml, about 300,000 to about 3xl07S. hominis cells per ml, about 300,000 to about 3xl06S. hominis cells per ml, about 400,000 to about 4xl09S. hominis cells per ml, about 400,000 to about 4xl08S. hominis cells per ml, about 400,000 to about 4xl07S. hominis cells per ml, about 400,000 to about 4xl06S. hominis cells per ml, about 500,000 to about 5xl09S. hominis cells per ml, about 500,000 to about 5xl08S. hominis cells per ml, about 500,000 to about 5xl07S. hominis cells per ml, or about 500,000 to about 5xl06S. hominis cells per ml. In some embodiments, an effective amount of S. hominis 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 S. hominis cells is at least 400,000 S. hominis cells per ml. In some embodiments, the effective amount of S. hominis cells is determined as the number of cells per ml of carbon-free media. In some embodiments, the effective amount of S. hominis cells is determined as the number of cells per ml of carrier.

[0047] In some embodiments, a method disclosed herein comprises contacting S. hominis with a plastic for a period of time of 5 minutes to 100 hours, or more. For example, S. hominis 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 S. hominis 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 S. hominis with a plastic for a period of time of at least 200 minutes.

[0048] In some instances, a plastic is present in a composition and S. hominis is contacted with the composition. As non-limiting example, one or more plastics may be present in waste. In some embodiments, a method disclosed herein comprises contacting S. hominis cells (e.g., a composition comprising an effective amount of S. hominis cells) with plastic waste. In some embodiments, a method disclosed herein comprises degrading plastic waste using S. hominis.

[0049] 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 S. hominis. S. hominis 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 S. hominis 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).

[0050] In some embodiments, S. hominis are contacted with a plastic at a temperature that is less than 58 °C. In some embodiments, S. hominis are applied to a plastic at room temperature. In some embodiments, S. hominis are applied to a plastic at a temperature of at least 21 °C. In some embodiments, S. hominis are applied to a plastic at a temperature of at least 25 °C. In some embodiments, S. hominis are applied to a plastic at a temperature of at least 37 °C. In some embodiments, S. hominis are applied to a plastic at a temperature between about 21 °C and about 50 °C. In some embodiments, S. hominis 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, S. hominis is contacted with a plastic at about 45 °C.

[0051] Some methods disclosed herein increase the rate of plastic degradation. In some embodiments, contacting S. hominis 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 .S'. hominis cells. In some instances, the rate of plastic degradation may be determined as the rate of metabolism of a plastic by .S'. hominis.

[0052] 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 .S', hominis. 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 .S', hominis 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 .S', hominis metabolism is detected.

[0053] In some embodiments, contacting of an effective amount of .S', hominis 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 .S'. hominis grown in carbon-free media without the plastic. In some embodiments, contacting .S'. hominis with a plastic comprises culturing .S'. hominis 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, .S'. hominis may be cultured in media with a plastic added to the media. In some embodiments, the media is carbon-free media. In some instances, .S', hominis is cultured directly on a plastic without media.

[0054] In some embodiments, .S', hominis 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, .S'. hominis 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.

[0055] .S', hominis 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.

[0056] Any suitable method may be used to contact .S', hominis cells with plastic. For example, an effective amount of a composition comprising .S', hominis cells may be sprayed onto one or more plastics or spread onto one or more plastics. For example, .S', hominis cells may be aerially sprayed onto one or more plastics. In some embodiments, one or more plastics are coated with .S'. hominis cells. In some embodiments, .S', hominis cell are only applied to one portion of a plastic and allowed to spread over the plastic. In some embodiments, .S', hominis cells are applied as granules. In some embodiments, .S', hominis cells are applied as particles. In some embodiments, .S', hominis 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).

[0057] Bioreactors Aspects of the present disclosure provide bioreactors for degrading a plastic. For example, a bioreactor disclosed herein may be useful in degrading 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.

[0058] The bioreactors disclosed herein allow for .S'. hominis to contact a plastic and may comprise any of the .S'. hominis 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 .S'. hominis 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.

[0059] 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. In some instances, a bioreactor comprises plastic as the sole source of carbon. In some instances, the only source of carbon is plastic waste. For example, 5. hominis 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.

[0060] Kits and applicators

[0061] 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. 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.

[0062] 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. 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.

[0063] 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.

[0064] 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. 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 5. hominis), 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.

[0065] EXAMPLES

[0066] 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.

[0067] Example 1. Identification of bacterial strains capable of degrading plastic nanoparticles

[0068] 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, MgSCU, NH4NO3, NaCl, FeSO4*7H2O, ZnSCL’ EhO, and MnSCU^FLO) in the presence of polyethylene terephthalate (PET) nanoparticles for 7 days. After 7 days, strains of interest were isolated for further analyses.

[0069] A bacterial strain, Staphylococcus hominis (S. hominis), was identified that grew in CFM with plastic nanoparticles as the sole source of nutrients. To confirm these findings, a .S'. hominis (SH) culture was plated in the presence of LB media alone or CFM with PET nanoparticles and further compared to PET in CFM without .S'. hominis (FIG. 1). As expected, .S'. hominis growth, as measured by optical density (OD) at 600 nm, was observed in LB media alone and growth was not observed in the condition containing PET in CFM without .S'. hominis. Surprisingly, .S'. hominis growth was observed after around 700 minutes in CFM with PET nanoparticles, indicating that .S'. hominis is able to metabolize PET. PET can be used as a sole source of nutrients for .S'. hominis cultures.

[0070] 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.

[0071] 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.”

[0072] 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.

[0073] 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.

[0074] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0075] 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 Staphylococcus hominis (S. hominis) cells to one or more plastics, thereby degrading the plastics.

2. A biodegradation method, comprising: applying an effective amount of a composition comprising Staphylococcus hominis (S. hominis) cells to a product comprising one or more plastics, thereby degrading the one or more plastics.

3. The method of claim 1 or 2, wherein the one or more plastics comprise polyester, optionally wherein the polyester is polyethylene terephthalate.

4. The method of any one of claims 1-3, wherein the composition comprises a carrier, optionally wherein the carrier is a carbon-free medium.

5. The method of claim 4, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5.S'. hominis cells per milliliter (ml) carrier.

6. The method of any one of the preceding claims, wherein the .S'. hominis cells are in contact with the one or more plastics for at least 200 minutes.

7. The method of any one of the preceding claims, wherein the .S', hominis cells and the one or more plastics are maintained for at least 200 minutes at a temperature of about 37 °C and about 50 °C, optionally about 45 °C.

8. The method of any one of the preceding claims, wherein the one or more plastics are untreated prior to the contacting.

9. The method of claim 8, wherein the method does not comprise treating the one or more plastics with a cobalt or manganese catalyst prior to the contacting.

10. The method of any one of the preceding claims, wherein the .S'. hominis cells are formulated as a solution, a powder or as granules.

11. The method of any one of the preceding claims, wherein the applying of the .S'. hominis cells to a product comprises spraying the cells onto the product.

12. A bioreactor comprising: one or more plastics; andStaphylococcus hominis (S. hominis) cells in an effective amount to degrade the one or more plastics.

13. The bioreactor of claim 12, wherein the one or more plastics comprise polyester, optionally wherein the polyester is polyethylene terephthalate.

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

15. The bioreactor of claim 14, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5.S', hominis cells per milliliter (ml) medium.

16. 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.

17. The bioreactor of any one of the preceding claims, wherein the one or more plastics are untreated, optionally free of exposure to a cobalt or manganese catalyst.

18. An applicator comprising Staphylococcus hominis (S. hominis) cells in an effective amount to degrade one or more plastics.

19. The applicator of claim 18, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5.S', hominis cells per milliliter (ml) medium.

20. The applicator of claim 18 or 19, wherein the one or more plastics comprise polyester, optionally wherein the polyester is polyethylene terephthalate.

21. The applicator of any one of the preceding claims, wherein the .S'. hominis cells are formulated as a solution, a powder or as granules.

22. A composition comprising granules of .S', hominis cells.

23. A kit comprising: an applicator; andStaphylococcus hominis (S. hominis) cells in an effective amount to degrade one or more plastics.

24. The kit of claim 23, wherein the effective amount is at least IxlO3, IxlO4, or IxlO5.S'. hominis cells per milliliter (ml) medium.

25. The kit of any one of the preceding claims, wherein the .S', hominis cells are formulated as a solution, a powder or as granules.

26. The kit of any one of the preceding claims, comprises a composition comprising granules of the .S', hominis cells.

27. The kit of any one of the preceding claims, comprises a composition comprising a spray formula of the .S', hominis cells.

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