Degradation of polyhydroxyalkanoate

Abstract

The present disclosure provides methods for degrading bioplastics. Also provided are articles suitable for degradation of bioplastics. The bioplastic may be a polyhydroxyalkanoate polymer and it may be degraded by an article comprising bacteria and one or more enzymes.

Description

CROSS REFERENCE TO RELATED APPLCIATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 513,856 filed July 14, 2023, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant no. 2137561 awarded by the National Science Foundation, and grant no. 2230641 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] Plastic pollution poses a significant threat to both marine life and coastal economies worldwide. By 2050, it is estimated that the oceans will contain more plastic than fish by weight. Marine animals that ingest plastic debris can suffer from entanglement, suffocation, and starvation, and the accumulation of plastic pollutants in the food chain could also have implications for human health. The biodegradation of plastic in the marine environment is a complex process that depends on various factors, including photo-exposure, temperature, and microbial activity. Photodegradation, the process by which plastic can be broken down into smaller pieces due to the energy from UV rays can take many years. The resulting microplastics from this process can be dangerous to marine life and can enter the food chain. In addition to plastic debris from consumer and industrial sources, discarded fishing gear from industrial fishing operations is a major contributor to ocean plastic pollution. Such debris can damage fishing gear, disrupt fishing activities, harm fish populations, and discourage tourists from visiting coastal areas, posing risks to the livelihoods of coastal communities that rely on fishing and tourism. Plastic bags and packaging, for instance, can persist in the environment for up to 450 years, further exacerbating the issue. Considering these challenges, it is crucial to develop effective solutions that address plastic pollution in the ocean, including monitoring technologies that cater to the needs of end-users, such as oceanographers who deploy equipment that is not recovered.

[0004] Despite efforts to reduce plastic waste through various initiatives, including recycling and the use of biodegradable plastics, the plastic pollution problem persists. Toaddress this issue, scientists have developed biodegradable plastics as a potential solution to the problem of plastic waste. One such type of biodegradable plastics are polyhydroxyalkanoates (PHAs), a class of polyesters produced by bacteria and stored as carbon and energy reserves. PHAs possess physical and mechanical properties similar to traditional plastics, making them suitable for marine applications that require rigid materials. Poly-3-hydroxybutyrate (P3HB or PHB) is a class of biopolymer that belongs to the category’ of polyhydroxyalkanoates (PHAs). PHB is a promising alternative to conventional plastics due to its environmentally-friendly properties (FIG. 1).

[0005] Bioplastics include plastics that are bio-based, biodegradable, or both, with some derived from biomass and others designed for biodegradability. Biodegradable plastics refer to conventional plastics that are designed to break down through natural processes, while bioplastics are derived from renewable sources and designed to be compostable or biodegradable, with differences in composition, degradation behavior, and potential environmental impacts. However, recent studies suggest that the bioplastic, such as Polylactic acid (PLA), Polyhydroxyalkanoates (PHA), Polybutylene succinate (PBS), Poly caprolactone (PCL). etc., may not be an effective way to solve the plastic pollution problem in marine environments. These plastics require industrial composting facilities with specific conditions, such as high temperatures and humidity7, to degrade effectively The conditions of the deep ocean, including low temperatures and darkness, may not allow for these plastics to biodegrade quickly, causing them to persist and cause harm to the marine environment. Furthermore, the natural degradation of biodegradable plastics can lead to the formation of microplastics, which pose a catastrophic hazard to marine life (FIG. 2).SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides methods for degrading bioplastics. Also provided are articles suitable for degradation of bioplastics. The articles may be used in a method of the present disclosure.

[0007] In an aspect, the present disclosure provides articles suitable for initiating the degradation of a plastic (e.g., a polymer). For example, the plastic is a bioplastic / polymer. In various examples, the bioplastic is a polyhydroxyalkonate. The bioplastic for degradation may be referred to as a target bioplastic or target polymer.

[0008] The article comprises one or more bacteria and / or one or more enzymes. The one or more bacteria may be capable of producing an enzyme that can initiate / catalyze thedegradation of the target bioplastic. The one or more enzymes may be capable of producing an enzyme that can initiate / catalyze the degradation of the target bioplastic.

[0009] In an aspect, the present disclosure provides a method of degrading plastics (e.g., polymers). For example, the plastic is a bioplastic / polymer. In various examples, the bioplastic is a polyhydroxyalkanoate.

[0010] In various examples, the method comprises contacting the bioplastic with an article that will trigger the degradation of the bioplastic (e.g., polyhydroxyalkanoate polymer). In various examples, the article is a “living article” comprising one or more bacteria that express enzymes capable of degrading the target bioplastic. In other examples, the article comprises one or more enzy mes capable of degrading the target bioplastic.BRIEF DESCRIPTION OF THE FIGURES

[0011] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0012] FIG. 1. Chemical structure of a bioplastic (e.g., polyhydroxyalkanoate polymer (PHA), specifically, polyhydroxybutyrate (PHB)). PHA (e.g., PHB) are biodegradable polymers produced by microorganisms as a form of energy' storage. It is a potential alternative to conventional plastics due to its degradation activity and compostability.

[0013] FIG. 2. Marine plastic pollution: persistent, hazardous, challenging. Marine plastic pollution is a pressing issue due to the persistent nature of traditional plastics and the resulting accumulation of hazardous microplastics. The cold and dark conditions of the deep sea hinder the degradation of bioplastics, and enrichment of microorganisms capable of degrading plastics in the marine environment is challenging.

[0014] FIG. 3. Bio-sticker for PHA Degradation via Bioprinting. The bio-sticker for the degradation of PHB through bioprinting technology . This bio-sticker involves the utilization of Bacillus NRRL B-14911, which produces PhaZ enzyme, to facilitate the degradation of PHB into smaller molecules.

[0015] FIG. 4. 3D Bioprinting for Marine Plastic Degradation. Bacteria are patterned in 3D via alginate chemistry' and immobilized within a hydrogel matrix post-bio-ink deposition. The incorporation of calcium ions facilitates the cross-linking of alginate chains, resulting in the formation of a robust, solidified structure. Conversely, the addition of sodiumions triggers the depolymerization of the cross-linked alginate, thereby enabling the transition of the solid matrix to a liquid state.

[0016] FIG. 5. PHB degradation capability of Bacillus NRRL B-14911, Comamonas testosteroni, Marinobacter sp. NK1 and Microbulbifer sp. SOL66 after one-week incubation.

[0017] FIG. 6. Growth curves of Bacillus NRRL B-14911, Comamonas testosteroni, Marinobacter sp. NK1 and Microbulbifer sp. SOL66 in marine broth.

[0018] FIG. 7 A. CFU profile.

[0019] FIG. 7B. Growth curves of Bacillus NRRL B-14911 growth in different media marine broth identifies optimal growth conditions for Bacillus NRRL B-14911.

[0020] FIG. 8. Viability assessment of Bacillus NRRL B-14911 growth in different concentration of NaCl in marine broth.

[0021] FIG. 9. Growth curve of Bacillus NRRL B-14911 in different temperature.

[0022] FIG. 10A. Viability' assessment of Bacillus NRRL B-14911 in marine broth,.

[0023] FIG. 10B. Viability assessment of Bacillus NRRL B-14911 in bio-ink (nonpolymerized).

[0024] FIG. 10C. Viability assessment of Bacillus NRRL B-14911 in bio-stickers (polymerized bio-ink).

[0025] FIG. 11. The growth curve of Bacillus NRRL B-14911 in the presence of different concentrations of CaCh.

[0026] FIG. 12. The CFU profile of Bacillus NRRL B-14911 bio-sticker culture on different concentrations of CaCk agar plate for 24 hours.

[0027] FIG. 13. Logistic Growth Curve fit of Bacillus NRRL B-14911 Bio-Stickers.

[0028] FIG. 14A. Schematic illustration of bio-ink and bio-sticker production process. Alginate and marine broth are combined to form a non-polymerized solution. Bacillus NRRL B-1491 1 is then mixed with the solution to create a non-polymerized bio-ink.

[0029] FIG. 14B. Schematic illustration of bio-ink and bio-sticker production process. Calcium salt is added to the bio-ink, inducing cross-linking of the hydrogel, which results in the formation of a polymerized bio-sticker. Sodium salt is added to the aggregated biosticker. causing de-polymerization and reverting it to a non-polymerized bio-ink state.

[0030] FIG. 15. Clear-zone analysis reveals time-dependent (0, 4, 6, 8, 10, 12, 14 days) PHB degradation by Bacillus NRRL B-14911 bio-sticker. The white color indicates the PHB pow der, while the orange-red color indicates the Bacillus NRRL B-14911 bio-stickers.

[0031] FIG. 16A. Bacillus NRRL B-14911 biosticker degradation of PHB for various PHB concentrations.

[0032] FIG. 16B. Clear zone radius of bio-stickers cultured for 22 days on Petri dishes with different PHB concentrations.

[0033] FIG. 16C. CFU assays of bio-stickers cultured on PHB dishes for various time durations.

[0034] FIG. 17A. Bacillus NRRL B- 14911 bio-sticker degradation of PHB in various incubation temperature.

[0035] FIG. 17B. Clear zone radius of bio-stickers cultured for 28 days at different incubation temperatures.

[0036] FIG. 17C. CFU assays of bio-stickers cultured on PHB dishes for various incubation temperature.

[0037] FIG. 18A. Bacillus NRRL B-14911 bio-sticker degradation of PHB with varied initial biomass.

[0038] FIG. 18B. Clear zone radius of bio-stickers cultured for 26 days with different initial cell densities.

[0039] FIG. 18C. CFU assays of bio-stickers cultured on PHB dishes for various initial biomass.

[0040] FIG. 19A. Bacillus NRRL B-14911 bio-sticker degradation of PHB with various alginate concentration.

[0041] FIG. 19B. Clear zone radius of bio-stickers cultured for 22 days with different alginate concentrations.

[0042] FIG. 20A. Bacillus NRRL B-1491 1 bio-stickers with various layer height degraded PHB.

[0043] FIG. 20B. Clear zone radius of bio-stickers cultured for 22 days with different 3D printing layer thicknesses.

[0044] FIG. 21 A. Bacillus NRRL B-14911 bio-stickers with various diameters degraded PHB.

[0045] FIG. 21B. Clear zone radius of bio-stickers cultured for 22 days with different 3D printing radii.

[0046] FIG. 21C. CFU assays of bio-stickers cultured on PHB dishes with various initial diameters.

[0047] FIG. 22A. Modeling of PHB Degradation via 3D Bio-sticker. Logistic grow th fit of mass of PHB degraded per unit time by Bacillus NRRL B-14911.

[0048] FIG. 22B. Modeling of PHB Degradation via 3D Bio-sticker. Correlation between viability7curve of marine bacteria and PHB degradation.

[0049] FIG. 23. Portrays the bio-stickers, shaped as UR logo, produced using bioprinting technology. Illustrates their effective PHB degradation on agar plates, visible by disappearing white PHB powder and clear UR logo-shaped zones. Emphasizes their robust adhesion, as exhibited by successful transfer to a fresh agar plate without detachment.

[0050] FIG. 24. Showcases bio-sticker mediated PHB degradation on agar plates, evidenced by vanishing white PHB powder and emerging clear UR logo-shaped zones. Underlines the repeatable degradation process, culminating in PHB decomposition during a 20-day culture at a constant 30 °C.

[0051] FIG. 25A. Bacillus NRRL B-14911 biosticker degrades PHB after transfer from original agar plate to fresh culture plates. Degradation data were collected after transfer onto fresh culture medium.

[0052] FIG. 25B. Clear zone radius of bio-stickers cultured for 10 days on Petri dishes after transfer to fresh culture plates.

[0053] FIG. 25C. CFU assay s of bio-stickers cultured on PHB dishes for various time durations.

[0054] FIG. 26. Processes and materials for mechanical testing.

[0055] FIG. 27. Bio-sticker tensile test. Samples of bio-sticker with Bacillus NRRL B-14911, incubated at 4 or 30 °C for three w eeks.

[0056] FIG. 28. Bio-sticker adhesion test. Samples of bio-sticker with Bacillus NRRL B-14911. incubated at 4 or 30 °C for three weeks.

[0057] FIG. 29. Freeze-drying w ith protectants enhances vi abil i ty of marine bacteria.

[0058] FIG. 30A. Bacillus NRRL B-14911 biosticker prepared using freeze-drying with protectant media, compared to the control group (freeze-dried without protectant media Bacillus NRRL B-14911), showing the formation of a clear zone and measuring the degradation efficiency.

[0059] FIG. 30B. Clear zone radius of freeze-dried Bacillus NRRL B-14911 biosticker with protectant media and the control group (freeze-dried without protectant media Bacillus NRRL B-14911) biosticker cultured for 16 days on Petri dishes containing PHB.

[0060] FIG. 31. Verification of Bacillus NRRL B-14911 spore production and removal of non-sporulating live cells via heat shock treatment.

[0061] FIG. 32A. Production of Bacillus NRRL B-14911 spores, indicated by removal of non-sporulating live cells via heat shock treatment.

[0062] FIG. 32B. Analysis of PHB degradation efficacy by Bacillus NRRL B-14911 spores in bio-sticker.

[0063] FIG. 33A. PCR gel of BL21 / pSBlC3-T7-phaZ::chlr.

[0064] FIG. 33B. Plasmid map of BL21 / pSBlC3-T7-phaZ::chlr.

[0065] FIG. 34. Induction of phaZ protein expression in BL21 / pSBlC3-T7-phaZ: :chlrby IPTG.

[0066] FIG. 35. Degradation of PHB by the constructed strain BL21 / pSBlC3-T7- phaZ::chlr.

[0067] FIG. 36. High-fidelity prototypes for producing living PHB-degrading materials, bioprinted stickers; enzyme / spore-embedded materials; bio-inserts; and co-printing PHB and bacteria (from left to right).

[0068] FIG. 37. Surface erosion of solid PHB discs incubated with Bacillus NRRL B- 14911 biostickers for 28 days, or negative control incubated without biostickers, assayed by scanning electron microscopy.

[0069] FIG. 38A. Low-cost additive manufacturing (3D printing) of ELMs. In a foundational, proof-of-concept study, bacteria were 3D printed using a low-cost 3D printer outfitted to extrude alginate-based hydrogels that contained living cells.

[0070] FIG. 38B. Low-cost additive manufacturing (3D printing) of ELMs. When the alginate ink contacts the Ca2+-rich substrate, chemical crosslinking occurs, encapsulating the living cells in a rigid, freestanding 3D structure. Our approach can be used to spatially pattern material architectures with a variety of bio-inks, living cells, and biological functionalities.

[0071] FIG. 39A. Prototypes used in the Phase I human-centered design (HCD) process. Mock ‘menu’ of materials with different degradation timescales and shelf lives.

[0072] FIG. 39B. Prototypes used in the Phase I human-centered design (HCD) process. Rapid clearance of PHB powder by a marine bacterium isolated at UCSB.

[0073] FIG. 39C. Prototypes used in the Phase I human-centered design (HCD) process. 3D-printed, reusable bacterial sticker demonstrating PHB clearance underneath; created in the Meyer Lab.

[0074] FIG. 39D. Prototypes used in the Phase I human-centered design (HCD) process. 3D-printed examples of rigid PHB structures. Based on the demand for rapid prototyping ability expressed in our interviews, Omand created the first printer filament from Mango Materials’ PHB.

[0075] FIG. 39E. Prototypes used in the Phase I human-centered design (HCD) process. Hollow, threaded PHB inserts that will be co-printed with PHB-degrading bacteria - an example of a scalable product that can be added to an ocean instrument by an end-user to adjust the degradation speed.

[0076] FIG. 40. Oxygen-based respiration of PLA and Mango Materials’ injection molding (IM) grade PHB in seawater measured using an ’ autoBOD " automated respirometry system. All treatments conducted in triplicate.

[0077] FIG. 41. One-page promotional graphic (1920xl080p) created by Gesso Designs illustrating our delivery method prototypes.

[0078] FIG. 42. Clear zone radius of Bacillus NRRL B- 14911 biosticker cultured for 16 days on Petri dishes containing PHB.

[0079] FIG. 43. Survival of Bacillus NRRL B-14911 lyophilized in liquid culture followed by storage for up to 28 days at 4 (bottom) or 22 °C (top). Cells were resuspended in PBS salt buffer, 10% sucrose, or protective media prior to lyophilization.

[0080] FIG. 44. Survival of Bacillus NRRL B-14911 lyophilized in bio-ink followed by storage for up to 28 days at 4 (bottom) or 22 °C (top). The lyophilized bio-ink was prepared in either PBS salt buffer, 10% sucrose, or protective media.

[0081] FIG. 45. Survival of Bacillus NRRL B-14911 ly ophilized in 3D-printed biostickers followed by storage for up to 28 days at 4 (bottom) or 22 °C (top). Bio-stickers were incubated in PBS salt buffer. 10% sucrose, or protective media prior to lyophilization.

[0082] FIG. 46. PHB degradation by Bacillus NRRL B-14911 lyophilized in 3D- printed bio-stickers followed by storage for up to 28 days at 4 (bottom) or 22 °C (top). Biostickers were incubated in PBS salt buffer, 10% sucrose, or protective media prior to lyophilization.

[0083] FIG. 47. Colony forming units of living Bacillus NRRL B-1491 1 bacteria isolated from sterile artificial seawater in which PHB discs containing embedded lyophilized vegetative cells or spores had been incubated for 5 weeks. CFU values represent bacteria that had been revived following injection molding embedding within the PHB discs.

[0084] FIG. 48. Images of PHB discs containing embedded lyophilized vegetative cells or spores that were incubated for 5 weeks in artificial seawater. The discs containing embedded bacteria all show visible degradation.

[0085] FIG. 49. Conceptual graphic describing the dual-head 3D printing approach developed in the Omand lab (left panel) and the direct heated injection / extrusion of living cells with PHA powder or pellets (right panel).

[0086] FIG. 50. Clear zone radius of PHB degradation by biostickers cultured at 15 °C on Petri dishes containing PHB. Bacteria strains were isolated from the Pacific Ocean.

[0087] FIG. 51. Images of a variety of 3D-printed hydrogel polymers after incubation in artificial seawater for 4 weeks. Alginate :GelM A blends maintain stable gelation upon incubation in artificial seawater.

[0088] FIG. 52A. Bio-sticker tensile test. Samples of bio-sticker with Bacillus NRRL B-14911, incubated at 30 °C for two or three weeks.

[0089] FIG. 52B. Bio-sticker tensile test. Samples of bio-sticker. Negative control group without bacteria, incubated at 30 °C for two or three weeks.

[0090] FIG. 52C. Bio-sticker tensile test. Samples of bio-sticker. Positive control group containing E. coli BL21 bacteria, incubated at 30 °C for two or three weeks.DETAILED DESCRIPTION OF THE DISCLOSURE

[0091] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0092] As used herein, unless otherwise indicated, ‘‘about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to. those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / -10% or less, + / -5% or less. + / -!% or less, and + / -0. 1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before aquantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0093] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to. all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of ‘'0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as ‘"about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0094] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0095] As used herein, unless otherwise stated or indicated, “s” refers to second(s), “min” refers to minute(s), and “h” refers to hour(s).

[0096] The present disclosure provides methods for degrading bioplastics. Also provided are articles suitable for degradation of bioplastics. The articles may be used in a method of the present disclosure.

[0097] In an aspect, the present disclosure provides articles suitable for initiating the degradation of a plastic (e.g., a polymer). For example, the plastic is a bioplastic / polymer. In various examples, the bioplastic is a polyhydroxyalkonate. The bioplastic for degradation may be referred to as a target bioplastic or target polymer.

[0098] The article comprises one or more bacteria and / or one or more enzymes. The one or more bacteria may be capable of producing an enzyme that can initiate / catalyze the degradation of the target bioplastic. The one or more enzymes may be capable of producing an enzyme that can initiate / catalyze the degradation of the target bioplastic.

[0099] The article may comprise bacteria of various genera. For example, at least a portion of the bacteria are of the genus Bacillus. Nocardioides, Comamonas , Marinobacter , Shew ane Ila, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia. Microbulbifer.Alteromonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas, Arenicella, or Marisediminitalea. In various embodiments, the genus is Bacillus. In various examples, at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6,la. Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp. , Ralstonia sp., Marinobacter maritimus, Microbulbifer sp. , Alteromonas sp. , Bowmanella sp. , Alcanivorax sp. , Vibrio diabolicus, Pseudoalteromonas sp. , Arenicella sp. , Marisediminitalea aggregata, andPseudoalteromonas Hpolytica A, or a combination thereof. In various examples, at least a portion of the bacteria are Bacillus NRRL-14911.

[0100] The bacteria may be lyophilized prior to incorporation with the article. The composition comprising the bacteria that is lyophilized prior to incorporation may comprise one or more lyoprotectants. Examples of lyoprotectants include, but are not limited to, saccharides (e.g., sucrose, trehalose, or the like, or combinations thereof), milk, and the like, and combinations thereof. The composition of lyophilized bacteria may still comprise one or more of these lyoprotectants. In various examples, the article comprises one or more lyoprotectants and the lyoprotectant is sucrose having a concentration of 10 wt% or less (e.g., 1 wt%, 2, wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt%).

[0101] The bacteria may be sporulated prior to incorporation into the article. The sporulation is performed under conditions conducive to spore formation and verified by a method such as light microscopy, electron microscopy, spore staining, heat shock survival, or any other suitable method for detecting bacterial spores. Methods for sporulation are described herein and in Example 1. Additional methods are known in the art.

[0102] Sporulation may be achieved by various methods. For example, sporulation may be achieved by nutrient depletion. For example, the bacteria may be placed in a buffered medium comprising one or more salts and no nutrients. For example, the buffer medium may comprise water, KC1 (e.g., about 0.1% KC1), MgCb (e.g., about 0.012 MgCh), CaCb (e.g..about 1 mM CaCb), MnCh (e.g., 10 pM MnCh), and, optionally. FeSO4 (e.g., 10 pM FeSO-i). Following nutrient depletion, the bacteria may be sporulated.

[0103] The article may comprise various enzymes. For example, the one or more enzymes may be polyhydroxyalkanoate hydrolase, PHB depolymerase, PHA depolymerase, or the like, or combinations thereof. In various examples, the enzyme is a polyhydroxy alkanoate hydrolase. In various examples, the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase, such as. for example. PhaZ, PhaZ2, PhaZ5, PhaZ7, or the like, or a combination thereof. In various examples, the PHB monomer hydrolase is PhaZ.

[0104] The article may be of various shapes and / or sizes and / or compositions. For example, the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, insert, or the like. The article may be 3D printed or made by injection molding. The article may have various textures. Examples of various textures are presented FIG. 39B. Fort example, additional manufacturing approached, include, but are not limited to, rotational molding, tubular extrusion, or by application a PHA coating to another article.

[0105] 3D printing may be used to manufacture the article. Various parameters of the 3D printing can be adjusted. For example, the printhead speed and extrusion can all be individually changed. For example, the resolution can be 10 pm to 10 cm, including all 0.1 pm values and ranges therebetween. For example, the printhead speed can be 100 to 500 mm / min. including all 0. 1 mm / min values and ranges therebetween. In various examples, the extrusion rate may be 10 to 50 pL / min. For example, the resolution may be around or be 0.3 mm.

[0106] In various examples, the article is manufactured by 3D printing with a bioink comprising one or more enzymes, where the bioink suitable for 3D printing. Such a configuration may allow for the one or more enzymes to diffuse throughout the article. Printing of the enzyme may use a similar alginate-based bioprinting (or with a different type of polymers or constituents making up the bio-ink), or with the enzyme cross-linked to the bio-ink polymers. For example, the enzyme may be covalently attached to the polymers or constituents of the hydrogel. For example, the enzyme may comprise a tag (e.g., His-tag. SpyTag, HA tag, or other known biochemical binding tab) that allows for attachment (e.g., covalently or non-covalently) to the polymers or constituents of the hydrogel.

[0107] In various examples, the article may comprise various additional components. For example, additional components include, but are not limited to. monitoring equipment, sensors, cameras, and the like, and combinations thereof.

[0108] In various examples, the article has adhesive properties and may be referred to as a sticker. The sticker may be a hydrogel or formed from a bioink. The sticker (or hydrogel or bioink) may further comprise water and other constituents or polymers, such as, for example, gelatin methacryloyl, pluronic F127, collagen, cellulose, gelatin, fibrinogen / fibrin, gellan gum, silk, hyaluronic acid, dextran, agarose, alginate, chitosan, polyvinyl alcohol, polyethylene glycol, poly caprolactone, poly(lactic-co-glycolic acid), or the like, or any combination thereof. In vanous examples, the article comprises alginate and calcium chloride. The concentration of the calcium chloride may be 0.05 to 0.5 M, including all 0.001 M values and ranges therebetween. In various examples, the calcium chloride may slowly release throughout the hydrogel.

[0109] Various methods may be used to prepare a hydrogel. For example, the constituents, water, and bacteria and / or enzy mes can be mixed and then printed or cast onto / into a region optionally comprising a crosslinking chemical. In various examples, the gelation may be triggered by temperature. For example, the constituents, water, and bacteria and / or enzymes can be mixed and then printed or cast onto / into a region having a temperature that will induce gelation. In various examples, the gelation may be triggered by other environmental triggers, such as UV light. In other examples, the constituents, water, and bacteria and / or enzymes can be mixed and then gelate shortly thereafter. For example, gelation may occur upon mixing constituents of the hydrogel (e.g., water, additional polymers or constituents, bacteria, and / or enzymes).

[0110] In various embodiments, the one or more enzymes may be chemically modified (e.g., conjugated) with one or more of the polymers or constituents of the hydrogel. Without intending to be bound by any particular theory, it is considered that crosslinking the enzyme to the polymers of the polymer network of the hydrogel will immobilize the enzyme, thus preventing the enzyme from being washed away or diffusing away from article. In various examples, the enzy me may be covalently or non-covalently attached (e.g., via electrostatic interactions). Various ty pes of conjugation chemistry' (e.g., click chemistry', acylation chemistry’, or other known conjugation methods) can be used to attach the enzyme to the polymer or constituents of the hydrogel.[OHl] The sticker may have various desirable characteristics. For example, the sticker may have a desirable elastic modulus. For example, the elastic modulus may be 1 kPa to 10 MPa, including all 0.1 Pa values and ranges therebetween. The sticker could also be able to be adhered to solid surfaces, such as. for example, solid PHA (e.g., PHB) bioplastic.

[0112] In various examples, the article may have a sacrificial layer. The sacrificial layer may comprise a polyhydroxyalkanoate polymer. After at least a portion of the sacrificial layer is removed, a portion of the article may contact the bioplastic or the enzyme from the article may contact the bioplastic and initiate degradation of the bioplastic. The sacrificial layer may be a copolymer for 3-hydroxybutyratc. The sacrificial layer may comprise poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx). In various examples, the sacrificial layer comprises, consists essentially of, or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3- hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydro\ybutyrate-co-3- hy dr oxy hexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or the like, or combinations thereof. In various embodiments, the sacrificial layer is poly-3-hydroxybutyrate (PHB). The sacrificial layer may be an exterior layer of the article that contacts the bioplastic. The sacrificial layer does not initiate degradation of the bioplastic.

[0113] In various examples, the article is a screw. The screw may be contacted (e.g., screwed into) with the target bioplastic. After contact with the article (e.g., screw), the target bioplastic will degrade or partially degrade. In various examples, the screw has an external layer (e.g., sacrificial layer as described herein), where at least a portion of the external layer degrades and degradation of the target bioplastic is initiated after the at least partial degradation of the external layer.

[0114] In various examples, the article may be contacted with the target bioplastic to initiate degradation of the bioplastic. The initiation of degradation of the target plastic may be instantaneously, delayed, or initiated by a trigger (e.g., an environmental trigger). The article may be disposed or otherwise contacted with the target bioplastic. The bioplastic may be a polyhydroxy alkanoate polymer. The polyhydroxyalkanoate polymer may be a copolymer for 3-hydroxybutyrate. Examples of such polymers include, but are not limited to, poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3- hy dr oxy hexanoate) (PHBHHx). In various examples, the polyhydroxyalkanoate polymer comprises, consists essentially of, or is poly-3-hydroxybutyrate (PHB), poly -4- hydroxybutyrate (P4HB), poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or the like, or combinations thereof. In various embodiments, the polyhydroxyalkanoate poly mer is poly-3-hydroxybutyrate (PHB).

[0115] In an aspect, the present disclosure provides a method of degrading plastics (e.g.. polymers). For example, the plastic is a bioplastic / polymer. In various examples, the bioplastic is a polyhydroxyalkanoate.

[0116] In various examples, the method comprises contacting the bioplastic with an article that will trigger the degradation of the bioplastic (e.g., polyhydroxyalkanoate polymer). In various examples, the article is a “living article” comprising one or more bacteria that express enzymes capable of degrading the target bioplastic. In other examples, the article comprises one or more enzy mes capable of degrading the target bioplastic.

[0117] In various examples, the bioplastic (e.g., polyhydroxyalkanoate polymer) is contacted with an article comprising bacteria. In other examples, the bioplastic (e.g., polyhydroxyalkanoate polymer) may be mixed as a powder and mixed with lyophilized or sporulated bacteria. In various other examples, the bacteria may be in a vessel (e.g., pouch), which is embedded in the bioplastic (e.g., polyhydroxyalkanoate polymer).

[0118] The bioplastic may be a polyhydroxyalkanoate polymer. The polyhydroxyalkanoate polymer may be a copolymer for 3-hydroxybutyrate. Examples of such polymers include, but are not limited to, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhe\anoate) (PHBHHx). In various examples, the polyhydroxyalkanoate polymer comprises, consists essentially of, or is poly-3- hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB). poly-3-hydroxybutyrate-co-4- hy dr oxy butyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3-hydroxyhcxanoate) (PHBHHx), Polyphydroxy octanoate) (PHO). poly(3-hydroxydecanoate) (PHD). poly(3-hydroxydodecanoate) (PHDD). poly(3-hydroxyoctanoate-co-3-hydroxydecanaote) (PHO-co-PHD). or the like, or combinations thereof. In various embodiments, the polyhydroxyalkanoate polymer is poly-3- hydroxybulyrale (PHB).

[0119] In various examples, an article suitable for a method of the present disclosure is an article as described herein. The article may comprise one or more bacteria and / or one or more enzymes. The one or more bacteria may be capable of producing an enzyme that caninitiate / catalyze the degradation of the target bioplastic. The one or more enzymes may be capable of producing an enzyme that can initiate / catalyze the degradation of the target bioplastic.

[0120] Various bacteria may be used. For example, at least a portion of the bacteria are of the genus Bacillus, Nocar dioides, Comamonas, Marinobacter, Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia, Microbulbifer, Alteromonas, Bowmanella. Alcanivorax. Vibrio. Pseudoalteromonas . Arenicella. or Marisediminitalea. In various embodiments, the genus is Bacillus. In various examples, at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6,la, Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei. Marinobacter algicola DG893, Cupriavidus sp. , Ralstonia sp. , Marinobacter maritimus, Microbulbifer sp. , Alteromonas sp. , Bowmanella sp. , Alcanivorax sp. , Vibrio diabolicus, Pseudoalteromonas sp. , Arenicella sp. , Marisediminitalea aggregate, and Pseudoalteromonas Upolytica A, or a combination thereof. In various examples, at least a portion of the bacteria are Bacillus NRRL-14911.

[0121] Various enzymes may be used. For example, the one or more enzymes may be polyhydroxyalkanoate hydrolase, PHB depolymerase, PHA depolymerase, or the like, or combinations thereof. In various examples, the enzyme is a polyhydroxyalkanoate hydrolase. In various examples, the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase, such as, for example, PhaZ. PhaZ2. PhaZ5. PhaZ7. or the like, or a combination thereof. In various examples, the PHB monomer hydrolase is PhaZ.

[0122] The article may be of various shapes and / or sizes and / or compositions. For example, the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, or the like. The article may be 3D printed or made by injection molding.

[0123] In various examples, the bioplastic (e.g., polyhydroxyalkanoate polymer) is contacted with water prior to, during, and / or after contacting the article. In various examples, water initiates the degradation of the bioplastic after the article has contacted the bioplastic. In various examples, the method is performed in a controlled environment (e.g., in a laboratory). In other examples, the method is performed in a natural environment, such as a body of water. Non-limiting examples of water include a lake, sea, bay, ocean, pond, river, stream, tributary, lagoon, creek, delta, estuary, drainage basin, reservoir, gulf, puddle, canal, wetland, fjord, strait, wastewater, soil, sludge, mud. or the like. In various examples, the body of water is a body of salt water.

[0124] In various examples, the rate of degradation is affected by one or more variables. For example, the temperature and salinity of the water may increase or decrease the rate of degradation. In various examples, the composition of the article may be modulated to tune the rate of degradation based on one or more of these variables.

[0125] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0126] The following Statements are various examples of the present disclosure.Statement 1. A method for degrading a polyhydroxyalkanoate polymer comprising contacting the polyhydroxyalkanoate polymer with an article, wherein after contacting the degradation of the polyhydroxy alkanoate polymer is initiated.Statement 2. A method according to Statement 1, wherein the polyhydroxy alkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutj rate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly (3 -hydroxy decanoate) (PHD). poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or combinations thereof.Statement 3. A method according to Statement 1, wherein the polyhydroxy alkanoate polymer comprises or is a copolymer of 3-hydroxybutyrate.Statement 4. A method according to any one of the preceding Statements, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).Statement 5. A method according to any one of the preceding Statements, wherein the article is 3D printed or injection molded.Statement 6. A method according to any one of the preceding Statements, wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, insert, or the like.Statement 7. A method according to any one of the preceding Statements, wherein the article comprises bacteria.Statement 8. A method according to Statement 7, wherein at least a portion of the bacteria are of the genus Bacillus, Nocar dioides. Comamonas. Marinobacter , Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia, Microbulbifer , Alteromonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas, Arenicella, or Maris ediminitcdea.Statement 9. A method according to Statement 7, wherein the bacteria are Bacillus NRRL- 14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6, la, Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp.. Ralstonia sp., Marinobacter maritimus, Microbulbifer sp. , Alteromonas sp., Bowmanella sp., Alcanivorax sp. , Vibrio diabolicus, Pseudoalteromonas sp. , Arenicella sp. , Marisediminitalea aggregata, Pseudoalteromonas Upolytica A, or a combination thereof.Statement 10. A method according to Statements 7 or 8, wherein the bacteria are of the genus Bacillus.Statement 11. A method according to any one of Statements 7 to 10, wherein the bacteria are Bacillus NRRL-14911.Statement 12. A method according to any one of Statements 7 to 11, wherein the bacteria are lyophilized prior to incorporation into the article.Statement 13. A method according to any one of Statements 7 to 12, wherein the bacteria are lyophilized from a composition comprising the bacteria and one or more lyoprotectants.Statement 14. A method according to Statement 13, wherein the one or more lyoprotectants are chosen from saccharides, milk, and the like, and combinations thereof.Statement 15. A method according to Statement 14, wherein the saccharides are sucrose, trehalose, or the like, or combinations thereof.Statement 16. A method according to any one of Statements 13 to 15 c. wherein the composition comprises sucrose at a concentration of 10 weight % relative to the weight of the entire solution.Statement 17. A method according to any one of Statements 7 to 1 , wherein the bacteria are sporulated prior to incorporation into the article.Statement 18. A method according to any one of the preceding Statements, wherein the article comprises one or more enzymes.Statement 19. A method according to Statement 18, wherein at least a portion of the one or more enzymes are a polyhydroxyalkanoate hydrolase. PHB depolymerase, or PHA depolymerase, or combinations thereof.Statement 20. A method according to Statement 19, wherein the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase.Statement 21. A method according to Statement 20, wherein the PHB monomer hydrolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.Statement 22. A method according to Statement 21, wherein the PHB monomer hydrolase is PhaZ.Statement 23. A method according to any one of the preceding Statements, wherein polyhydroxyalkanoate polymer is contacted with water prior to, during, and / or after contacting the article.Statement 24. A method according to Statement 23, wherein the water is salt water.Statement 25. A method according to Statements 23 or 24, wherein the water is a body of water.Statement 26. A method according to Statement 25, wherein the body of water is a lake, sea, bay, ocean, pond, river, stream, tributary’, lagoon, creek, delta, estuary’, drainage basin, reservoir, gulf, puddle, canal, wetland, fjord, strait, wastewater, soil, sludge, mud, or the hke.Statement 27. An article suitable for degradation of a polyhydroxy alkanoate polymer, wherein the article comprises one or more bacteria and / or one or more enzymes.Statement 28. An article according to Statement 27, wherein the article is disposed on a surface of or in contact with the polyhydroxyalkanoate polymer.Statement 29. An article according to Statement 28, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3-hydro\ybutyrate- o-4-hydroxybut rate (P(3-HB-co-4-HB)), poly(3-hydroxybuty rate- co-3-hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or combinations thereof.Statement 30. An article according to Statement 28, wherein the polyhydroxyalkanoate polymer comprises or is a copolymer of 3-hydroxybutyrate.Statement 31. An article according to Statement 29, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).Statement 32. An article according to any one of Statements 27 to 31, wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, insert, or the like.Statement 33. An article according to Statement 32, wherein the article is a sticker.Statement 34. An article according to Statement 33, wherein the sticker is a hydrogel.Statement 35. An article according to Statements 33 or 34, wherein the sticker further comprises gelatin methacryloyl, pluronic Fl 27, collagen, cellulose, gelatin, fibrinogen / fibrin, gellan gum, silk, hyaluronic acid, dextran, agarose, alginate, chitosan, polyvinyl alcohol, polyethylene glycol, polycaprolactone, poly(lactic-co-glycolic acid), or the like, or combinations thereof.Statement 36. An article according to any one of Statements 33 to 35, wherein the sticker further comprises alginate and calcium chloride.Statement 37. An article according to Statement 36, wherein calcium chloride has a concentration of 0.05 to 0.5 M, including all 0.001 M values and ranges therebetween.Statement 38. An article according to Statements 33 to 37, wherein the sticker has an elastic modulus of 1 kPa to 10 MPa, including all 0.1 Pa values and ranges therebetween.Statement 39. An article according to Statement 32, wherein the article is a screw.Statement 40. An article according to any one of Statements 27 to 39, wherein at least a portion of the bacteria of the one or more bacteria are of the genus Bacillus, Nocardioides, Comamonas,Marinobacter, Shewanella, Pseudomonas , Paucimonas, Cupriavidus, Ralstonia, Microbulbifer, Alteromonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas. Arenicella. or Marisediminitalea.Statement 41. An article according to any one of Statements 27 to 40, wherein at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6,la, Pseudomonasstutzeri YM1006, Pseudomonas sp. DSDY0501 , Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp., Ralstonia sp., Marinobacter maritimus, Microbulbifer sp., Alteromonas sp., Bowmanella sp., Alcanivorax sp., Vibrio diabolicus, Pseudoalteromonas sp., Arenicella sp., Marisediminitalea aggregata, Pseudoalteromonas lipolytica_A, or a combination thereof.Statement 42. An article according to Statement 41, wherein the bacteria are of the genus Bacillus.Statement 43. An article according to any one of Statements 40 to 42, wherein the bacteria are Bacillus NRRL-14911.Statement 44. An article according to any one of Statements 27 to 43, wherein the one or more bacteria are lyophilized prior to incorporation into the article.Statement 45. An article according to any one of Statements 27 to 44, wherein the article further comprises one or more lyoprotectants.Statement 46. An article according to Statement 45, wherein the one or more lyoprotectants are chosen from saccharides, milk, and the like, and combinations thereof.Statement 47. An article according to Statement 46, wherein the saccharides are sucrose, trehalose, or the like, or combinations thereof.Statement 48. An article according to any one of Statements 27 to 47, wherein the one or more bacteria are sporulated prior to incorporation into the article.Statement 49. An article according to any one of Statements 27 to 48, wherein the article is 3D printed.Statement 50. An article according to any one of Statements 27 to 49, wherein the one or more enzymes are a polyhydroxyalkanoate hydrolase, PHB depolymerase, or PHA depolymerase, or combinations thereof.Statement 51. An article according to Statement 50, wherein the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase.Statement 52. An article according to Statement 51, wherein the PHB monomer hydrolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.Statement 53. An article according to Statement 52, wherein the PHB monomer hydrolase is PhaZ.Statement 54. An article according to Statement 53, wherein the article further comprises a polyhydroxyalkanoate polymer.Statement 55. An article according to Statements 53 or 54, wherein the article further comprises an exterior layer comprising a polyhydroxyalkanoate polymer.Statement 56. An article according to Statements 54 or 55, wherein the polyhydroxyalkanoate polymer comprises or is poly-3 -hydroxybutyrate (PHB).Statement 57. An article comprising a polyhydroxyalkanoate polymer and one or more bacteria and / or one or more enzymes embedded therein.Statement 58. An article according to Statement 57, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P(3 -HB-co-4-HB)), poly(3 -hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or combinations thereof.Statement 59. An article according to Statement 57, wherein the polyhydroxyalkanoate polymer comprises or is a copolymer of 3 -hydroxybutyrate.Statement 60. An article according to Statements 57 or 58, wherein the polyhydroxyalkanoate polymer comprises or is poly-3 -hydroxy butyrate (PHB).Statement 61. An article according to any one of Statements 57 to 60, wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, insert, or the like.Statement 62. An article according to Statement 61, wherein the article is a screw.Statement 63. An article according to any one of Statements 57 to 61, wherein at least a portion of the bacteria of the one or more bacteria are of the genus Bacillus, Nocardioides, Comamonas, Marinobacter, Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia, Microbulbifer, Alteromonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas, Arenicella, or Maris ediminitalea.Statement 64. An article according to any one of Statements 57 to 63, wherein at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6,la, Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501 , Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp., Ralstonia sp., Marinobacter maritimus, Microbulbifer sp., Alteromonas sp., Bowmanella sp., Alcanivorax sp., Vibrio diabolicus, Pseudoalteromonas sp., Arenicella sp., Marisediminitalea aggregata, Pseudoalteromonas lipolytica_A, or a combination thereof.Statement 65. An article according to Statement 63, wherein the bacteria are of the genus Bacillus.Statement 66. An article according to any one of Statements 57 to 65, wherein the bacteria are Bacillus NRRL-14911.Statement 67. An article according to any one of Statements 57 to 66, wherein the one or more bacteria are lyophilized prior to incorporation into the article.Statement 68. An article according to any one of Statements 57 to 67, wherein the article further comprises one or more lyoprotectants.Statement 69. An article according to Statement 68, wherein the one or more lyoprotectants are chosen from saccharides, milk, and the like, and combinations thereof.Statement 70. An article according to Statement 69, wherein the saccharides are sucrose, trehalose, or the like, or combinations thereof.Statement 71. An article according to any one of Statements 57 to 70, wherein the one or more bacteria are sporulated prior to incorporation into the article.Statement 72. An article according to any one of Statements 57 to 71, wherein the article is 3D printed.Statement 73. An article according to any one of Statements 57 to 72, wherein the one or more enzymes are a polyhydroxyalkanoate hydrolase, PHB depolymerase, or PHA depolymerase, or combinations thereof.Statement 74. An article according to Statement 73, wherein the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase.Statement 75. An article according to Statement 74, wherein the PHB monomer hydrolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.Statement 76. An article according to Statement 75, wherein the PHB monomer hydrolase is PhaZ.

[0127] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.EXAMPLE 1

[0128] This example provides a description of methods and articles of the present disclosure.

[0129] Engineered Living Materials (ELMs) and 3D Bioprinting for Marine Plastic Degradation. To provide a better solution to the Great Pacific Garbage Patch and ingestion, suffocation and entanglement of marine species. The example herein utilizes 3D bioprinting to create ELMs containing bioplastic degrading bacteria, allowing for controlled degradation of bioplastics while retaining plastic’s durability, versatility, and affordability.

[0130] ELMs as a Solution to Plastic Waste in Marine Environments. Traditionally, plastics have been treated with chemical methods to enhance their biodegradation. However, these methods have shown limited efficacy and can have negative environmental impacts. ELMs offer a promising alternative to traditional chemical treatments or natural biodegradation methods. ELMs are a class of functional materials composed of engineered biological systems that can create, modify, or maintain their own material structure or properties. Described herein is how ELMs can be utilized to tackle the problem of plastic waste in marine environments. A new concept that ELMs bind to the surface of bioplastic debris, releasing PHB monomer hydrolase that break down long polymers into carbon dioxide and water is described. (FIG. 4).

[0131] This Example aims to apply ELMs to the problem of plastic waste in marine environments. ELMs provide a highly targeted and efficient approach to reducing plastic pollution in the ocean. Unlike traditional chemical treatments or natural biodegradation, ELMs can be designed to specifically target certain types of polymers found in plastics. They can be applied to the surface of plastic materials or inserted into plastic equipment, allowing for a highly effective and targeted approach to reducing plastic waste. One of the key advantages of ELMs is their ability to degrade plastic more quickly and without releasing microplastics. With their ability to target specific types of plastics and degrade them quicklyand safely, ELMs represent a highly effective and sustainable approach to reducing plastic waste and preserving the health of oceans.

[0132] Programmable degradation of bioplastics using 3D bio-printed bacteria. 3D bioprinting is a cutting-edge technology that combines additive manufacturing with ELMs to create three-dimensional structures containing living cells. A custom-built 3D bioprinter, a proven, robust platform, was used for constructing ELMs. This bioprinter, armed with features such as precise layer-by-layer deposition and controllable printing parameters, has been optimized to maintain cell viability and function throughout the printing process. The printer creates ELMs containing bioplastic degrading bacteria deposited in specific three- dimensional patterns through direct alginate chemistry to degrade their surrounding bioplastic structures at programmable and controllable rates.

[0133] Alginate serves as a scaffold to hold the bacterial cells in place as they degrade the bioplastic material. Alginate, a natural polysaccharide, is derived from seaweed and can support cell growth and biomaterial formation. Alginate is commonly used as a bio-ink (nonpolymerized) material for 3D bioprinting due to its biocompatibility and ability to gel (polymerized) in the presence of calcium ions. The principle of alginate-based bio-3D printing is based on an ionotropic gelation process, which involves the interaction between positively charged calcium ions and negatively charged alginate molecules (FIG.4).

[0134] The printer can deposit the alginate and bacterial mixture in precise patterns to create three-dimensional structures. In this process, a solution of sodium alginate and cells or nutrients is extruded through a nozzle and deposited layer by layer to create a 3D structure. The bio-ink is extruded onto a calcium-containing agar surface or into a calcium-containing slurry, causing the alginate to gel and form a stable 3D scaffold. This process can be further optimized by adjusting the concentration of alginate and calcium ions, as well as the printing parameters such as nozzle diameter and printing speed.

[0135] The utilization of 3D bioprinting constitutes a favorable approach towards plastic waste management by means of enhanced sustainability. The living bacteria will convert a bioplastic (e.g., PHA, such as, for example, PHB) into carbon dioxide and water when exposed to plastic debris, resulting in the mitigation of potential environmental damage. The controlled and sustained degradation of the plastic material also reduces the risk of leaching hazardous chemicals into the environment. Additionally, the use of a hydrogel with nutrient support promotes the long-term viability of microorganisms that degrade PHAs. And also serves to adhere the bacteria to the bioplastic, ensuring direct release of the PHB depolymerase. This results in more efficient degradation of plastic waste, making ourinnovation a more cost-effective and environmentally friendly solution to plastic waste management.

[0136] Triggered PHA degradation using spore utilization and PHB monomer hydrolase purification. The strategic design of a trigger for PHA degradation, as opposed to fostering direct degradation, holds potential promise for industries such as fisheries, environmental stewardship, and oceanic monitoring. One advantage of this method is the facilitation of controlled degradation. In circumstances where the timing of material degradation is crucial, this capability is invaluable. As an illustrative example, consider the field of fisheries, where biodegradable equipment could be engineered to maintain its robustness and functionality over a prescribed duration, only to degrade upon the activation of the designed trigger. This strategy could potentially diminish the volume of equipment waste that ends up in oceans.

[0137] Furthermore, the use of a degradation trigger could contribute to the mitigation of pollution. Direct degradation often results in accelerated material breakdown, which can inadvertently lead to environmental pollution. By contrast, a triggered degradation process offers a more predictable and regulated strategy for managing the lifecycle of materials, thereby minimizing their environmental impact.

[0138] The concept of triggered degradation also presents opportunities for advanced materials management. In the realm of ocean monitoring, materials and devices that are intended to function over a specific timeframe can be designed to degrade post-use, thus circumventing disturbances to marine life or habitats. This approach could prove particularly beneficial for the deployment of temporary sensors or monitoring equipment.

[0139] Moreover, the adoption of materials capable of triggered degradation in fisheries and broader marine management could help mitigate the detrimental effects of ‘ghost gear’ fishing equipment that is lost or discarded in the ocean and poses significant risks to marine wildlife. The versatility of this degradation trigger system also allows for customization to suit a multitude of environments or applications. This adaptability could significantly enhance the design flexibility for products spanning a vast range of industries and applications.

[0140] Nevertheless, the practical implementation of a trigger system for degradation demands meticulous consideration of the potential environmental implications, the risk of misuse or unforeseen triggering, and the pragmatics of manufacturing and utilization. As with any innovative approach, this system warrants additional research for development, testing, and optimization to ensure its suitability for real-world application.

[0141] Application for Ocean Monitoring Devices. Biostickers, biodegradable sensors applicable to various surfaces, signify an emerging technology with potential for environmental impact. Their integration into marine monitoring devices using the subject matter of the present disclosure is overcomes previous limitations of these devices. Despite their role in marine research, these devices often contribute to marine pollution due to their non-recoverability post-deployment. The application of biostickers could bolster these devices’ capabilities to detect and quantify plastic pollutants, thereby mitigating the environmental footprint of monitoring activities.

[0142] Aspects of the present disclosure describe a living material for degrading PHAs at a tunable and triggerable rate by utilizing the marine PHB-degrading strain Bacillus NRRL B-14911. The viability of this strain was tested in 3D-printed bio-ink for PHB degradation, and the optimal temperature and culture conditions were determined by evaluating rate and metabolic activity in bioplastic samples. Bioprinting methods and patterns were optimized for PHB degradation, and modeling of PHB degradation with 3D bio-stickers was carried out via MATLAB. To assess potential applications in the field of sustainable bioplastics, the degradation activities of lyophilized bacteria, spores, and embedded enzymes in bioprinting were investigated. Furthermore, mechanical testing of the bio-sticker and analysis of PHB degradation were conducted to determine the effectiveness of the biostickers. Initial high-fidelity prototypes of living materials were developed with tunable rates of bioplastic degradation. The results of this research provide insights into the development of ELMs for PHB degradation, which may have implications in the field of sustainable bioplastics.

[0143] Viability of 3D-Printed Bio-stickers. To assess the viability of microorganisms in 3D-printed bio-ink for functional bio-sticker production, colony forming unit (CFU) assays were used to determine survival rates. These results show robust viability of 3D-printed microorganisms with minimal loss for at least two weeks, indicating that the bio-ink formulation provides a favorable environment for microorganism growth and proliferation. These findings are desirable for the development of functional bio-stickers capable of biodegradation.

[0144] Tunable PHB Degradation via Bioprinting. To determine parameters that affect rates of degradation, a systematic investigation of the effects of alginate concentration, bacteria concentrations, and modifications in printing matrix polymer concentrations and chemistry was conducted. Clear-zone analysis revealed a direct correlation between microbial degradation rate and initial bio-sticker biomass, PHB concentration, and incubationtemperature, providing valuable insights into the interplay between microbial behavior and the bioprinting process. These findings can be leveraged to tune the degradation activity of PHB-based materials.

[0145] Viability and Degradation of PHB Post-Transfer and storage of lyophilized bacteria. To assess the reusability of biodegradable stickers during transportation and storage, transfer experiments were conducted to investigate their biodegradability under different environmental conditions, with potential implications for reducing waste through repeated use in diverse settings. The degradation efficiency of lyophilized Bacillus NRRL B-14911 was tested. The freeze-drying process can preserve the activity of bacteria while rendering them dormant until rehydrated, potentially enabling the strategic triggering of PHB degradation.

[0146] Mechanical properties of Bio-sticker. To assess the suitability of bio-stickers for degrading PHB under marine shear stress, mechanical tests were conducted to determine their tensile strength and modulus of elasticity. These tests provide important information about the mechanical performance of the stickers, including their ability to withstand routine handling and other external stresses. Based on the results of these tests, the mechanical properties of the stickers can be optimized, such as their strength and elasticity, to improve their adhesion to the PHB surface and enhance their effectiveness in degrading in seawater environments.

[0147] Triggers for PHB Degradation via Lyophilized Bacteria, Bio-printed Spores, and Purified PHB monomer hydrolase. To explore different modes of triggering degradation, three distinct methods were implemented: lyophilized bacteria, bioprinted spores, and purified PHB degrading enzymes. Each technique offers unique advantages for controlling the timing and location of PHB degradation in various applications, spanning from fisheries equipment to temporary oceanic monitoring devices. The degradation efficiency of lyophilized Bacillus NRRL B-14911 was evaluated. The lyophilization process preserves bacterial activity and induces dormancy until rehydration, which may strategically trigger PHB degradation. The degradation capacity of Bacillus NRRL B-14911 spores was further assessed. Their resilience and ability to maintain dormancy until conditions favor growth potentially make them efficient triggers for PHB degradation. Lastly, a strategy for PHB degradation was designed. The strategy used purified PHB monomeric hydrolase PhaZ, potentially embedded directly into PHB materials, that could initiate degradation upon exposure to seawater.

[0148] Material and Instruments.

[0149] Table 1. Reagents Used in the Protocols.

[0150] 4% alginate in Marine Broth. The procedure began with the necessary materials, which included Marine Broth, Sodium Alginate, Distilled Water, an Autoclave, a Stirring Hot Plate, a Magnetic Stir Bar, a pH Meter, and a Sterile Storage Container.

[0151] Initially, the Marine Broth was prepared as directed by the manufacturer’s instructions. This typically involved dissolving a specified amount of the powdered broth in distilled water.

[0152] Subsequently, the required amount of sodium alginate was measured out to create a 4% solution. To exemplify, for the preparation of 100ml of Marine Broth, 4 grams of sodium alginate would be required.

[0153] The sodium alginate was then introduced to the Marine Broth. During this step, the solution was stirred with a magnetic stir bar on a stirring hot plate. Considering sodium alginate’s well-known difficulty to dissolve, heating the solution to 60-80 °C and stirring for several hours may be required. Assurance was sought that the alginate was fully dissolved before proceeding.

[0154] Following this, the solution was allowed to cool before it was stored in a sterile container. The solution was deemed suitable for storage at room temperature.

[0155] Throughout the procedure, cleanliness and sanitation of the workspace were prioritized to avoid contamination, and all safety procedures for handling the materials and equipment were adhered to. It was also ensured that, if the media was to be used for culturing organisms, the media was appropriate for the organisms under consideration.

[0156] 50% Marine Broth-0.1% PHB-0.3M CaC12 agar plates. The procedure was initiated by preparing a 50% Marine Broth. Depending on whether the broth was a powder or a liquid, the manufacturer’s instructions were followed for its preparation, before it was diluted with distilled water to achieve a 50% concentration. For instance, in the preparation of 1 liter of media, 500 ml of Marine Broth and 500 ml of PBS buffer were combined.

[0157] Next, a 0.1% PHB solution was prepared. This was achieved by dissolving 1 gram of PHB in the already prepared 50% Marine Broth to reach the desired concentration.

[0158] Subsequently, a 0.3M calcium chloride (CaCk) Solution was prepared. This involved dissolving 33. 15 grams of CaC12 in 1 liter of the previously prepared 50% Marine Broth.

[0159] Upon combination of the solutions, agar was added to the mixture. Typically, 15 grams of agar per liter of media were included, with the quantity varying depending on the desired firmness of the plates. The goal was to achieve a final concentration of 1 .5% in the mixture.

[0160] The mixture was then sterilized by autoclaving at 121 °C for 15 minutes. This procedure not only melted the agar but also sterilized the media.

[0161] After sterilization, the media was allowed to cool to around 50-55 °C. At this temperature, the media remained liquid. It was then poured into sterile Petri dishes within a laminar flow hood or another sterile environment. A typical volume of about 20 ml per 100 mm Petri dish was used.

[0162] The plates were subsequently left to cool at room temperature, allowing the agar to solidify and create a solid surface conducive for microbial growth.

[0163] Finally, the solid plates were stored upside down (agar side up) to prevent condensation from dripping onto the agar. These plates were stored at 4 °C until they were ready to be used.

[0164] PHB-Agar Plates Protocol (500 mL). The experiment commenced with the preparation of a IL Erlenmeyer flask that contained a stir bar, 500 mL of Artificial Seawater (ASW), 3 g of PHB powder, 0.5 g of NH4C1, less than 0.1 g of yeast extract, and 7.5 g of bacto agar.

[0165] Next, the flask was covered with a piece of aluminum foil large enough for handling later. The flask was then stirred and heated on a stir plate until the PHB and agar formed aggregates. After the formation of aggregates, the stir bar was removed, and the flask was autoclaved for 40 minutes on slow (liquid) exhaust. A potential overflow of the media due to bubbling was noted.

[0166] During the autoclaving process, a water bath at 50-60 °C was prepared using tap water. The flask was then cooled in this water bath until it reached a temperature that allowed handling without gloves.

[0167] A phosphorus addition was sterilized using a Bunsen burner, a 3 mL Luer-Lok syringe, and a 0. 1 pm Luer-Lok filter. Approximately 2 mL of the KH2PO4 stock solution was drawn, after which the filter was attached to the syringe and the solution was injected into the media.

[0168] Working near the flame, the media was poured into plates, with each plate being filled halfway. The flask was covered with the tinfoil cap when not in the process of pouring. The plates were then left to solidify overnight on the lab bench.

[0169] The following day. the plates, positioned upside down, were stored in a labeled Ziploc® bag in a room with a stable temperature. It was noted that this protocol typically filled one sleeve of large plates (100x15 mm) or tw o sleeves of small plates (60x15 mm). Any spillage of media was allow ed to solidify before removal with paper tow els and disposal in the trash. It was also mentioned that the PHB concentration could be adjusted as needed and that if the media was not poured immediately after autoclaving, reheating would liquefy it again.

[0170] Marine Broth-Agar (250 mL). In a 500 mL bottle, 250 mL MB media and 3.75g bacto agar were combined. They were stirred, autoclaved for 35 minutes, and poured into plates maintaining sterility. The plates solidified overnight on lab bench. Next day, they w ere stored in the refrigerator.

[0171] PHB Liquid Media (250 mL). In a 500 mL media bottle, 250 mL ASW, 2 g PHB powder, 0.25 g NH4C1, and <0.1 g yeast extract were all mixed. It was stirred on a stir plate and dispensed as 10 mL aliquots into culture tubes using a modified 5 mL pipette. Tubes were loosely capped and autoclavde on slow exhaust for 30 minutes. They were allow ed to let cool, the caps w ere tightened, and stored in stable temperature room.

[0172] Marine Broth 2216 (500 mL). In a IL flask, 500 mL MQ water and 18.7g Marine Broth 2216 were combined. It was stirred and heated (40-50 °C) until dissolved. 10 mL aliquots were pipetted into 50 culture tubes. Tubes were loosely capped and autoclavedon slow exhaust for 30 minutes. It was cooled, the caps were tightened, and stored in the refrigerator. Notes: Marine Broth-Agar protocol fills half a sleeve of large plates or one sleeve of small plates. For PHB Liquid Media, modify pipette tip to avoid clogging by PHB aggregates.

[0173] Bacto TSB I Tryptic Soy Broth (500 mL). In a IL flask, 500 mL MQ water and 15.0 g Bacto TSB were combined. It was stirred and heated (40-50 °C) until dissolved.10 mL aliquots were pipetted into 50 culture tubes. Tubes were loosely capped and autoclaved on slow exhaust for 30 minutes. It was cooled, the caps were tightened, and stored in the refrigerator.

[0174] Glycerol Stock (lOOmL of 50% Glycerol Solution). 50 mL glycerol and 50 mL MQ water were mixed on a stir plate until well-mixed. The mixture was autoclaved.

[0175] DSM Sporulation medium. Sporulation medium was constituted by dissolving either nutrient broth or tryptic soy broth in distilled water, following the manufacturer’s guidelines. Additional components, including yeast extract, calcium chloride, manganese sulfate, and potassium phosphate monobasic, were integrated into the broth solution.

[0176] The pH of the resultant medium was modulated to an optimal range of 7.2 to 7.4, employing a pH meter or indicator strips. When requisite, pH was adjusted using hydrochloric acid (HC1) or sodium hydroxide (NaOH).

[0177] For the preparation of solid medium, agar was incorporated into the liquid medium, ensuring thorough mixing. Subsequent to the addition of agar, the medium underwent sterilization in an autoclave at 121 °C for a duration of 15-20 minutes, which simultaneously facilitated the dissolution of agar.

[0178] Post-autoclaving, the medium was allowed to cool to approximately 50-55 °C. In the case of solid medium, the cooled preparation was dispensed into either petri dishes or culture tubes and left to solidify.

[0179] Upon solidification, the sporulation medium was set for application and could be introduced to the bacterial culture, initiating the process of sporulation.

[0180] Bacillus NRRL B- 14911 spore germination medium. Spore Preparation: Isolate Bacillus NRRL B-1411] spores according to the standard method of isolation.

[0181] Spore Germination Assay: The isolated spores were resuspended in sterile distilled water. In a sterile test tube, a known quantify of spores (e.g., IxlO6spores / mL) was added to the germination medium. The mixture was incubated at 37 °C with shaking at 200 rpm. At predetermined time intervals (e.g., 0, 1. 2, 4, 6, 8. and 24 hours), an aliquot of the germination medium was withdrawn and measure the optical density at 600 nm wasmeasured (OD600) using a spectrophotometer. An increase in OD600 indicated germination and outgrowth of the spores.

[0182] Data Analysis: The OD600 was plotted as a function of time to analyze the germination profile of Bacillus NRRL B- 14911 spores.

[0183] Table 2: Strains, Genotypes, and Channels of Purchase. Strains

[0184] Viability in 3D-Printed Bio-sticker. Enumeration of Bacterial Colony Forming Units (CFU) by Plate Count Assay.

[0185] The CFU assay was employed to determine the bacterial concentration. This involved performing a plate count assay to enumerate bacterial CFUs in a sample.

[0186] A series of dilutions of the bacterial sample were prepared. This was achieved by adding 1 mL of the sample to 9 rnL of sterile diluent with 1 xPBS in a series of tubes, which were then vortexed or mixed thoroughly. A sterile pipette or spreader was used to apply 100 pL of each dilution onto a nutrient agar plate in triplicate. A sterile glass spreader was used to evenly distribute the bacterial suspension over the surface of the plate.

[0187] The plates were incubated upside down at 30 °C and to allow for the development of colonies of Bacillus NRRL B-14911. The number of colonies was counted on each plate that fell within the range of 30-300 colonies per plate. The CFU per mL of the original bacterial sample was calculated by multiplying the number of colonies by the dilution factor.

[0188] Colony-Forming Unit (CFU) Profiles in Liquid Culture of Bacillus NRRL B- 14911. A culture of Bacillus NRRL B-14911 was cultivated in an appropriate medium until it achieved an optical density (O.D.eoo) of 1. The culture was then harvested through centrifugation 2500g 10 min. The resultant bacterial pellet was resuspended in a variety of liquid media, such as LB, Seawater, Nutrient Broth, and Marine Broth. This resuspension process was carried out until a final O.D.eoo value of 0.5 was reached.

[0189] These resuspended aliquots were divided into test tubes. The test tubes were then incubated at 30 °C with shaking for a duration of 48 hours. At specific intervals (Oh, 24h, and 48h), 100 pl samples were drawn from each test tube for CFU testing.

[0190] CFU assays were conducted on these samples to identify the quantity of viable bacterial cells. The resultant data were analyzed, resulting in growth curves and CFU profiles. This provided valuable insights into the growth dynamics of Bacillus NRRL B-14911 in different liquid media.

[0191] Growth Curves and CFU Profiles - Viability of Media Broth and Bio-ink. A bacterial culture with an initial optical density (O.D.eoo) of 0.5 was prepared, from which 50 mL was collected. This culture was then resuspended with an equal volume of either bio-ink or Marine broth.

[0192] The resuspended bacterial culture was incubated at 30 °C with shaking. Every' two days, samples were drawn from the culture for CFU analysis.

[0193] CFU assays were conducted on these samples to determine the number of viable bacterial cells. The resulting data was analyzed, producing growth curves and CFU profiles. These analyses provided insights into the viability of both Marine broth and bio-ink as media for supporting bacterial growth.

[0194] Growth Curves and Colony-Forming Unit (CFU) Profiles - Viability of Biosticker. Petri dishes were prepared with PHB for bacterial culture. On these, 1 mL of Bacillus NRRL B-14911 bio-ink (composed of 0.04 Alginate and Marine broth) w'as cultured.

[0195] The Petri dishes w ere then incubated at a suitable temperature for bacterial growth. After a period of four days, one Petri dish containing the bio-ink was removed.

[0196] The bio-ink from this dish was resuspended in 1 mL of IM sodium citrate solution. Serial dilutions of this resuspended bio-ink were prepared. CFU tests were performed on these serial dilutions using PBS solution. The data obtained from these tests were analyzed, resulting in growth curves and CFU profiles.

[0197] Optimized PHB Degradation via Bioprinting. Optimized PHB Degradation via Bioprinting with Biomass. A bacterial culture of Bacillus NRRL B-14911 was prepared in a suitable liquid medium and grown until the optical density (O.D.eoo) reached 1. This culture was then diluted to the desired cell densities by either enrichment or dilution.

[0198] Following this, a bio-ink mixture w as prepared by adding 4% alginate to Marine Broth (MB). Bacterial cells were added to the bio-ink mixture at varying concentrations (O.D.6oo=0.1, L 5, 10, 20). This bio-ink mixture was then bioprinted onto MB- CaCb-0.01 PHB agar plates using suitable equipment.

[0199] These plates were incubated at an appropriate temperature (30 °C) for four days. The radius of PHB degradation was measured every four days using appropriateimaging software, and the results were recorded. A graph was plotted to visualize the results at the end of the fourth day.

[0200] At the conclusion of the experiment, the bacterial cells and PHB degradation products were scraped from the agar plates. The biomass was collected, and the wet or dry weight was measured, as appropriate. The results were recorded, and data analysis was carried out. with statistical analysis applied if necessary.

[0201] Optimized PHB Degradation via Bioprinting with PHB concentration. The Bacillus NRRL B-14911 culture was harvested by centrifugation 2500g lOmin and resuspended in 10 mL of 4% Alginate-Marine broth to obtain a final optical density (O.D.eoo) value of 0.5. This sample was then bioprinted onto MB-CaCh Petri dishes, with three bioreplicates for each. The PHB concentration of the Petri dishes was controlled, varying between 0.1%, 0.5%, 1% 1.5%, and 2%.

[0202] Over a period of 24 days, the halo size of the bioprinting samples was measured daily. A graph was then plotted, displaying the halo size against time.

[0203] For data analysis, the mean and standard deviation of the hole size for each day were calculated. One-way ANOVA was used to ascertain if significant differences existed in hole size among the three different Alginate-MB concentrations. Following this, a post-hoc test was performed to determine which concentrations were significantly different from each other.

[0204] Optimized PHB Degradation via Bioprinting with Temperature. The Bacillus NRRL B-14911 culture was harvested through centrifugation 2500g, 10 min and was subsequently resuspended in 10 mL of 4% Alginate-Marine broth, resulting in a final O.D.eoo value of 0.5. This resuspended culture was bioprinted onto MB-CaCh Petri dishes, with three bio-replicates created for each sample. The Petri dishes were then incubated at one of four different temperatures: 10 °C, room temperature (measured), 30 °C, and 37 °C.

[0205] The halo size of the bioprinting samples was measured daily over the course of 24 days. A graph was plotted to present the halo size against time.

[0206] For the data analysis, the mean and standard deviation of the halo size were calculated for each day. A one-way ANOVA was performed to determine if there were significant differences in halo size among the different temperatures. A post-hoc test was carried out to identify which temperatures showed significantly different results from each other.

[0207] Optimized PHB Degradation via Bioprinting with Alginate concentration. The Bacillus NRRL B-14911 culture was harvested through a process of centrifugation 2500g, 10min, following which it was resuspended in 10 mL of 4% Alginate-Marine broth, resulting in a final O.D.eoo value of 0.45. This prepared bacterial culture was then bioprinted onto MB- CaCh Petri dishes, w ith three replicates created for each of the three different Alginate- Marine Broth concentrations: 2%, 4%, and 6%.

[0208] Over the course of 24 days, the halo size of the bioprinting samples was measured daily. This data was then graphically represented, plotting halo size against time.

[0209] For the data analysis, the mean and standard deviation of the halo size were calculated for each day. A one-way ANOVA was conducted to determine if there were significant differences in halo size across the three different Alginate-Marine Broth concentrations. Lastly, a post-hoc test was performed to determine which concentrations exhibited significantly different results from one another.

[0210] Viability and Degradation Rate Post-Transfer. The Bacillus NRRL B-14911 culture was harvested by centrifugation 2500g lOmin, then resuspended in 10 mL of 4% Alginate-Marine broth to achieve a final O.D.eoo value of 0.5. This prepared culture was bioprinted onto Marine Broth-PHB-CaCb Petri dishes, with three biological replicates created for each sample.

[0211] After an incubation period of 24 and 72 hours, the biological stickers were transferred to fresh culture plates. Throughout a period of 24 days, the halo size in the bioprinting samples was measured on a daily basis.

[0212] For the purpose of data representation, a graph was plotted showcasing the halo size against time.

[0213] In the analysis stage, the mean and standard deviation of the halo size were calculated for each day. A one-way ANOVA was conducted to identify any significant differences in halo size across the three different Alginate-Marine Broth concentrations. Lastly, a post-hoc test was performed to determine which concentrations had significantly different results from the others.

[0214] Mechanical properties of Bio-sticker. The experiment commenced with the preparation of 3D-printed dogbone structures in agarose slurry to test the stiffness and Young’s modulus of biological stickers or bio-gels. Three experimental groups were defined. The first group was incubated at 30 °C, then transferred to 4 °C for storage. A negative control group was established, consisting of a biological sticker without Bacillus NRRL B- 14911. A positive control group consisted of a biological sticker containing E. coli BL21.

[0215] Subsequently, the samples were mounted on a tensile testing machine using clamps or grips suitable for the sample size and shape. A uniaxial load was applied to eachsample at a constant rate of deformation using the tensile testing machine until the sample reached its maximum deformation or broke.

[0216] During the testing process, the load and deformation of each sample were recorded. This data was later utilized to calculate the stiffness and Young’s modulus of the bio-gel.

[0217] Following the tests, the data was analyzed by calculating the stress and strain of the sample. The stress was derived by dividing the load by the cross-sectional area of the sample, and the strain was computed by dividing the deformation by the original length of the sample.

[0218] Finally, the stress-strain data was used to calculate the stiffness and Young's modulus of the bio-gel. The stiffness was determined by dividing the change in stress by the change in strain, and Young’s modulus was calculated by dividing the stress by the strain.

[0219] The entire test was repeated with multiple samples to ensure consistency and reliability of the data.

[0220] Viability and Degradation Rate of Lyophilized Storage. The protectant medium was made up of sucrose, skimmed milk, trehalose. CaCh, MgCh, KOH, and three antioxidants (ascorbic acid, uric acid, and glutathione), with the pH of the medium adjusted to 7.3 ± 0.2.

[0221] In preparing the protectant medium, specific quantities of various elements per gram / liter were combined in phosphate-buffered saline (PBS). This included sucrose (10 g), skimmed milk (10 g), trehalose (5 g), CaCL (0.1 g), MgC12 (0.1 g), and KOH (0.3 / 0.6 g), along with the three antioxidants: ascorbic acid (1 g), uric acid (0.4 g), and glutathione (0.1 g)-

[0222] The sterilization process involved treating the skimmed milk at 121 °C for 15 minutes in an autoclave. The rest of the solution, including the antioxidants and sugar, was sterilized by filtration through 0.22 pm filters.

[0223] For the preparation of the bacterial suspension, fresh Bacillus NRRL B-14911 bacteria were harvested directly from agar plates after 24 hours. The harvested bacteria were mixed with two solutions: a normal saline solution (NaCl 0.9%) as a control and the protectant medium (milk 10% + sucrose 10% + trehalose 5% + antioxidants).

[0224] The overnight Bacillus NRRL B-14911 culture was divided into three equal parts, and the supernatant was removed by centrifugation 2500g lOmin. The first sample involved resuspending the Bacillus NRRL B-14911 pellet in a protective agent. For the second sample, the Bacillus NRRL B-14911 pellet was resuspended in a protective agentcontaining 4% Alginate, forming a bio-ink. Lastly, for the third sample, the Bacillus NRRL B- 14911 pellet was resuspended in a protective agent containing 4% Alginate, and a 0.1M calcium chloride solution was added to form a biosticker.

[0225] The bacterial suspensions, prepared with either the protectant medium or saline solution, were stored under different conditions for 48 hours or 30 days. These conditions encompassed freezing the suspensions at -80°C for a duration of 5 hours, followed by freeze-drying the samples.

[0226] Post freeze-drying, the samples were rehydrated and submerged in PBS to return to their original volume of 500 pL. This process took place at a temperature of 25°C. Subsequently, the samples were incubated at room temperature.

[0227] To evaluate the efficacy of the protectant medium in preserving Bacillus NRRL B-1491L a colony-forming units (CFUs) test was performed on the stored samples.

[0228] Bacillus NRRL B- 14911 sporulation. During the injection molding or 3D printing processes, it was observed that PHB bonded with Bacillus NRRL B-14911, which led to the direct incorporation of the spores into the bioplastic material. The embedded spores proved resilient and remained dormant for extended periods until the PHB began to exhibit physical cracks or fissures. This allowed seawater to penetrate and rehydrate the spores, triggering the degradation of the PHB and effectively preventing the release of microplastics into the marine environment.

[0229] To obtain a substantial quantity of Bacillus NRRL B-14911 spores, a CFU assay was used to approximate the spore yield and to confirm the removal of non-sporulating vegetative cells through heat shock treatment. Observations revealed that under heat shock at 95 °C, vegetative cells reduced to 0 CFU / mL, and the spore CFU decreased by 2 log, remaining at 102CFU / mL.

[0230] For testing the degradation efficacy of the spores, spore bio-ink was prepared by combining the spores with 4% alginate. The clear-zone method was then utilized to measure the size of the degradation clear zone of the spores bio-sticker (polymeric) on plates containing 1% PHB and 0.3M CaCh medium. A degradation trend line was plotted to quantify the amount of PHB degraded per unit time. The trend line indicated that the spore recovery and PHB degradation were active but exhibited a lag phase compared to vegetative cells.

[0231] Construction of Plasmids for the Purification of PHB-Degrading Enzy mes. The use of inorganic protective carriers or dispersion with heterogeneous polymers would enhance the stability and activity of the enzyme within the PHB. To test this, a plasmid wasdesigned containing the hydrolytic enzyme phaZ (bio-sourced from B. megaterium N- 18-25- 9), known for its PHB degradation properties. This plasmid, pSBlC3-T7-phaZ::chlr, was introduced into E. coli BL21(DE3) Competent Cells, creating the strain BL21 / pSBlC3-T7- phaZ::chlr.

[0232] Confirmation of the successful construction of the BL21 / pSBlC3-T7- phaZ::chlrstrain was achieved through PCR of the plasmid and full plasmid sequencing. When 0.5mM IPTG was added to the overnight culture of BL21 / pSBlC3-T7-phaZ::chlras well as to the bio-ink made by adding 4% alginate, and fermented for 3 hours, clear zones were observed on LB agar plates containing PHB and chloramphenicol after 5 days of incubation at 37 °C. This indicated that the constructed strain BL21 / pSBlC3-T7-phaZ::chlrwas successfully able to degrade PHB.

[0233] Viability of 3D-Printed Bio-stickers. The viability of Bacillus NRRL B-14911 in bioprinted stickers is essential for maintaining product effectiveness and identifying optimal storage and handling conditions to prevent bacterial degradation. Determining survival rate also aids in establishing an appropriate shelf life. To maximize / promote bacterial viability, investigated the survival ability of Bacillus NRRL B-14911 in bioprinting stickers has significant implications for the development and use of bioprinting products, including the potential use of this marine bacterium in biotechnology7.

[0234] Efficient PHB Powder Degradation by Bacillus NRRL B-14911. For our study, we selected four different bacterial strains to test their ability to degrade organic compounds. Bacillus NRRL B-14911 is a marine bacterium that has been shown to have potential in degrading polyhydroxy alkanoate (PHA), which is a type of biopolymer commonly used in bioplastics. Marinobacter sp. NK1 is another marine bacterium that has been shown to have the ability to degrade petroleum compounds, making it a promising candidate for bioremediation applications. Microbulbifer sp. SOL66 is a marine bacterium that has been isolated from a marine sponge and has shown potential in degrading lignin, which is a complex organic polymer found in plants. Finally, Comamonas testosteroni is known to be capable of degrading a wide range of organic compounds, including xenobiotic compounds commonly found in industrial effluents. By testing these four bacterial strains, we aimed to identify their potential for biodegradation applications.

[0235] The clear zone assay measures microorganisms’ ability7to degrade PHB powder by growing them on an agar plate with PHB powder and observing the appearance of a clear zone around the microbial colony as an indicator of efficiency. The clear-zone assay revealed that Bacillus NRRL B-14911 formed larger clear zones than other marine bacterialstrains (Comamonas testosteroni , Marinobacter sp. NK1, Microbulbifer sp. SOL66) per unit time, indicating its efficiency in degrading PHB powder compared to other marine bacteria (Figure 5). Therefore, Bacillus NRRL B-14911 was selected for subsequent experiments. This result highlights the potential of Bacillus NRRL B-14911 as a promising candidate for biodegradation of PHB powder and its potential application in biotechnology .

[0236] To evaluate the growth of marine bacteria, Growth curves of Bacillus NRRL B-14911. Comamonas testosteroni, Marinobacter sp. NK1, and Microbulbifer sp. SOL66 were generated to assess their proliferation in marine broth (Figure 6). Upon incubation in marine broth at 30°C for 24 hours, all four strains - Bacillus NRRL B-14911, Comamonas testosteroni, Marinobacter sp. NK1, and Microbulbifer sp. SOL66 - demonstrated growth, underscoring their ability’ to thrive under these conditions.

[0237] Marine Broth suitable for Bacillus NRRL B-14911 growth and bioprinting. To determine the optimal liquid culture medium for Bacillus NRRL B-14911, NQ cultured Bacillus NRRL B-14911 with agitation using a plate reader to generate a growth curve. The growth curve was generated using Marine Broth, Luria-Bertani broth, nutrient broth, or sea water to determine growth under different media types. These media were selected because they represent vary ing nutrient compositions that may impact bacterial growth. The growth curve of Bacillus NRRL B-14911 showed higher and sustained O.D.eoo values under Marine Broth conditions (FIG. 7B). The CFU results analysis similarly demonstrated a bacterial concentration of 109CFU / mL for Bacillus NRRL B-14911 cultured in Marine Broth (FIG. 7A).

[0238] Therefore, Marine Broth w as selected to use in subsequent experiments for Bacillus NRRL B-14911, such as Marine Broth was used to prepare bio-ink (non-polymeric) and bio-stickers. The use of Marine Broth as a culture medium can contribute to the production of high-quality bioprinting products and the effective biodegradation of these products in marine environments.

[0239] To assess the performance stability and viability of Bacillus NRRL B-14911 in bio-stickers deployed in actual marine environments, the effect of salt concentration and temperature on Bacillus NRRL B-14911 growth was tested. The success of bio-stickers in biodegradation applications relies on the survival and growth of Bacillus NRRL B-14911 in the bio-sticker. The optimal growth conditions to ensure the stability and viability’ of Bacillus NRRL B-14911, given that salt concentration and temperature are critical factors that can affect their survival and growth. In the Pacific Ocean Current Report 2018. the average salinity of the Pacific Ocean is approximately 35 ppt, with a range of 32 to 37 ppt, dependingon the location and depth. The 5% salt concentration mentioned in the experiment is equivalent to approximately 50 ppt, which is higher than the average salinity of the Pacific Ocean. Despite this, the growth curve of Bacillus NRRL B- 14911 in Marine Broth with varying salt concentrations showed that the strain could tolerate salt concentrations of around 5% (FIG. 8). Furthermore, in the Pacific Ocean Current Report 2018, the average surface temperature of the Pacific Ocean is approximately 17 °C. The strain could grow at temperatures ranging from 22 °C to 37 °C (FIG. 9). with the highest O.D.eoo value observed at 30 °C, indicating that 30 °C was the optimal temperature for the growth of Bacillus NRRL B-14911. These findings provide important information for the development of protocols for the growth and cultivation of Bacillus NRRL B-14911 in bioprinting and biodegradation applications.

[0240] Viability of Bacillus NRRL B-14911 in Marine Broth, Bio-ink, and Biostickers. To determine the viability of Bacillus NRRL B-14911 in liquid culture, bio-ink (nonpolymerized), and bio-stickers (polymerized). To create a bio-sticker, equal volumes of bioink (non-polymerized) were printed onto an agar plate containing calcium ions and PHB powder, allowed to incubate for several days, and then removed the bio-sticker from the plate and dissolve it in equal volumes of sodium salt solution before performing CFU testing. After incubating Bacillus NRRL B-14911 in liquid culture and non-polymerized bio-ink for several days, CFU testing indicated that the bio-stickers had the highest viability of Bacillus NRRL B-14911 compared to liquid culture and bio-ink (non-polymerized), with the maximum survival rate observed in the bio-stickers (polymerized) (FIG. 10). The viability of Bacillus NRRL B-14911 in liquid culture decreased by 4.29 log within a week (FIG. 10A), indicating a potential shelf life of less than one week. In contrast, the viability of Bacillus NRRL B-14911 in bio-ink (non-polymerized) staying fairly constant over the first 21 days, with a 2.31 log decrease in the fourth week (FIG. 10B), while the viability of Bacillus NRRL B-14911 in biostickers remained stable for three weeks. This suggests that bio-stickers can maintain their vitality for up to three weeks or more, with minimal loss in viability (FIG. 10C).

[0241] Based on these findings, it was hypothesized that the viability of Bacillus NRRL B-14911 may be affected by CaCb and alginate present in the bio-ink. as well as by the PHB powder itself, which could potentially impact the growth of the bacterium. It was postulated that alginate present in the bio-sticker may provide nutrients to Bacillus NRRL B- 14911, and that the biodegradation of PHB powder in the bio-sticker may provide additional nutrition for the bacteria.

[0242] Next, additional testing was performed to investigate the effect of CaCh on the viability of Bacillus NRRL B-14911(FLG. 11). The addition of calcium ions, such as CaCh, to the bio-ink (non-polymerized) solution is critical for the success of alginate-based 3D-bioprinting as it provides the necessary calcium ions for ion gelation. However, an excessive amount of calcium ions can lead to the formation of rigid gels, which may hinder cell proliferation and differentiation. Therefore, careful control of the concentration of calcium ions is necessary to ensure optimal gelation and maintain cell viability. Moreover, calcium ions play a crucial role as a cofactor in PhaZ -mediated PHB degradation, in marine biodegrading bacteria, where they bind to the enzyme’s active site, stabilizing the enzy mesubstrate complex and enhancing ester bond hydrolysis. To identify the effect of CaCh on Bacillus NRRL B-14911 viability, we generated growth curves of Bacillus NRRL B-14911 by varying calcium chloride concentrations in liquid culture. The grow th curve of Bacillus NRRL B-14911 in the presence of different concentrations of CaCh showed that more than 0.05M CaCh delayed Bacillus NRRL B-14911 growth (FIG. 11).

[0243] To determine the effect of CaCh on bacterial viability, bio-stickers using polymerized bio-ink on agar plates were prepared with different CaCh concentrations, followed by 24-hour incubation and CFU measurements. The CFU results indicated that there was no significant decrease in CFU when the CaCh concentration in agar plates was less than 0.3M. However, when the concentration was 0.3M, the CFU of Bacillus NRRL B-14911 in the bio-stickers decreased by 2.23 log. while a concentration of 0.7M resulted in a decrease of 3.68 log. (FIG. 12).

[0244] Logistic Growth Curve Predicts Viability of Bacillus NRRL B-14911 Biostickers. To determine the viability of Bacillus NRRL B-14911 bio-stickers with the CFU data described in FIG. 10, the logistic growth curve was fit using Matlab. f(x) = (N *k). / (N +((k N) *exp( r *x)))Coefficients (with 0.95 confidence bounds)'.N = 0.2002(- 1.607, 2.007) k - 6.685 (-1.092, 14.46) r = 1.31(- 2.635, 5.255)Goodnessoffit :SSE '. 8.868R - square 0.778R - squareAdjusted : 0.6299RMSE : 1.719

[0245] The aim was to predict the Bacillus NRRL B- 14911 content of the bio-stickers over a period of 2-6 months and estimate the shelf-life of the product. The model follows an exponential growth phase followed by a stable plateau phase, as described by the Logistic growth curve, where r represents the initial growth rate, K represents the maximum grow th rate, and N represents the initial biomass (FIG. 13). Although the model predicts long-term stability of biomass in the bio-stickers, it is worth considering the time frame over which this stability is expected to be maintained, as most liquid growth curves eventually exhibit a death phase after a period of stabi 1 ity. Therefore, it is important to assess whether it is reasonable to expect indefinite stability, but if confirmed, the stable state of bio-stickers could facilitate their deployment as an effective and reliable product for PHB degradation.

[0246] Tunable PHB Degradation via Bioprinting. To optimize PHB degradation, an investigation into the relationship between degradation rates and environmental and bioprinting parameters such as nutrient concentration, temperature, initial biomass, alginate concentration, layer structure, and printing radii was conducted. The ultimate goal of the experiments was to determine the optimal conditions for using Bacillus NRRL B- 14911 to degrade PHB through bioprinting, which may have potential applications in biodegradable materials and environmental remediation.

[0247] Evaluating PHB Degradation by Bacillus NRRL B-14911 bio-stickers with Clear-zone Assay. As previously discussed, it was found that the degradation efficiency of Bacillus NRRL B-14911 was superior to that of other marine bacteria. Additionally, the bacterium exhibited good growth performance in both bio-ink (non-polymerized) and biosticker (polymerized) formulations (FIG. 14). As a result of these favorable attributes, Bacillus NRRL B-14911 was selected for the testing procedures outlined herein (FIG. 5). A non-polymerized hydrogel created from alginate and marine broth forms a bio-ink when mixed with Bacillus NRRL B-14911. The addition of calcium salt to the non-polymerized bioink induces cross-linking of the hydrogel, resulting in the formation of a polymerized biosticker. Subsequently, the addition of sodium salt to the aggregated bio-sticker induces depolymerization, reverting it back to the non-polymerized state of bio-ink. To quantitatively characterize the PHB-degradation efficiency in a culture medium plate by a bio-sticker, the clear-zone method was employed. Bio-stickers of the same size are 3D printed onto culture medium plates containing PHB and cultured under specified conditions. At the end of thegrowth period, the extent of PHB degradation by the bio-sticker is quantitatively characterized by measuring the size of the transparent area around the PHB on the plate (FIG. 15). In this method, a smaller transparent area size indicates a lower degradation efficiency of PHB by the bio-sticker, while a larger transparent area size indicates a higher degradation efficiency of PHB by the bio-sticker.

[0248] During the first four days of cultivation, it was observed that the bottom of the culture dish remained covered with white PHB powder, without the formation of any distinct degradation zones or the presence of bio-stickers. On the fourth day, complete degradation of PHB powder underneath the bio-stickers was observed, resulting in the formation of clear zones. After four days, the bio-sticker continued to degrade the PHB powder, resulting in the gradual expansion of a transparent circle around it. This finding indicates that the bio-stickers effectively degraded PHB and provide valuable information for the optimization of bioprinting protocols and the development of eco-friendly materials.

[0249] PHB Concentration Impacts Biodegradation Efficiency of Bio-sticker. To investigate the influence of different concentrations of PHB (ranging from 0.1% to 2%) on the efficiency of Bacillus NRRL B-14911 bio-stickers in degrading PHB. the bio-stickers were grown in the presence of 0.1% to 2% concentrations of PHB, and the extent of PHB degradation was quantitatively assessed using the clear-zone method. The size of the clear zones in the Petri dishes was continuously measured for 24 days to determine the rate and extent of PHB degradation with all parameters of bio-stickers being kept constant.

[0250] Panel A (FIG. 16A) demonstrated that the bio-sticker exhibits a higher efficacy in degrading PHB at lower concentrations, while Panel B (FIG. 16B) revealed a correlation between the concentration of PHB and the clear zone radius, indicating that the bio-sticker is more efficient in degrading lower concentrations of PHB. The time required for degradation of PHB by bio-sticker per unit area varied with different PHB concentrations.These results collectively suggest that the efficient degradation of PHB at a low concentration can be attributed to the consistent efficacy of the hy drolytic enzy me PhaZ in cataly zing PHB degradation.

[0251] Bio-stickers fabricated through 3D panting were cultured on dishes containing PHB for 7, 14, 21, and 28 days, respectively, and subsequently subjected to CFU assays (FIG. 16C). The results of the CFU profiling revealed that the grow th of the bio-stickers was decrease by 1.97 log or more from w eek 3 to week 4 in culture plates containing a concentration of 1% or higher of PHB. The lower PHB concentrations (e.g., 0. 1%) may result in faster PHB degradation due to the higher efficacy of PHB-degrading enzymes, whilehigher PHB concentrations (1% or higher) may need more than 22 days to completely- degraded (FIG. 16 A).

[0252] Temperature Impacts Biodegradation Efficiency of bio-sticker. To determine the tunable incubation temperature of bio-sticker for PHB -degradation and assess how temperature affects enzyme activity- and bacterial grow th, tests at incubation temperatures of 4 °C, 20 °C, 30 °C and 37 °C for bio-stickers on agar plates containing PHB powder were conducted, and the extent of PHB degradation was quantitatively assessed using the clearzone method (FIG. 17A and FIG. 17B). Bio-stickers fabricated through 3D printing were cultured on dishes containing PHB for 7, 14, 21, and 28 days, respectively, and subsequently- subjected to CFU assays (FIG. 17C).

[0253] The clear-zone assay shows that the bio-sticker degradation efficiency was higher at 30 °C compared to 37 °C from day 4 to day 28 (FIG. 17A). The bio-stickers cultured at 37 °C show ed a delayed onset of degradation until day 6, followed by a higher degradation rate compared to bio-stickers cultured at 20 °C on day 28 (FIG. 17B). The clearzone result revealed that the degradation rate of incubation at 37 °C tended to increase (compared to 20 °C) from day 6 to 28. indicating that the temperature may simultaneously affect the growth rate and optimal enzyme activity', which may account for the observed impact of temperature on degradation efficiency.

[0254] The growth of Bacillus NRRL B- 14911 determined from analyzing the liquid culture growth curve (FIG. 9) demonstrated that the bacteria exhibited optimal growth within a temperature range of 22 °C to 30 °C, while no growth occurred at a temperature of 15 °C. In addition, it w as observed that the bio-stickers cultured at 4 °C did not show' any measurable degradation. Moreover, the CFU profile demonstrated that the bio-stickers cultured at 4 °C still had viable cells 107log CFU / mL after 4 weeks of culture (FIG. 17C). Therefore, it is possible that Bacillus NRRL B- 14911 formed spores under the low- temperature conditions.

[0255] It is proposed that below the optimum temperature, the efficiency or production of PHB-degrading enzymes decreases with decreasing temperature. Above the optimum temperature, the efficiency of PHB-degrading enzymes decreases with increasing temperature (FIG. 17A-B). Based on the general behavior of enzy mes and their activity under different temperature conditions. Enzy mes typically have an optimal temperature range for their activity, which can vary- depending on the specific enzyme. Below- this optimal range, enzyme activity decreases due to slower molecular motion and lower collision frequency, while above this range, enzyme activity decreases due to denaturation and loss of enzymestructure. However, the specific temperature range and behavior of PHB-degrading enzymes would need to be experimentally determined to confirm this proposition.

[0256] Initial Biomass Impacts Biodegradation Efficiency of bio-sticker. To determine the tunable initial biomass of PHB-degrading bio-sticker and evaluate its effect on the degradation rate of PHB powder, bio-stickers were cultured with initial cell densities ranging from O.D.eoo 0.1 to O.D.eoo 20 on Petri dishes containing PHB powder and 0.3M CaCh (FIG. 18 A). The degradation of PHB was quantitatively evaluated using the clear zone method. While keeping all parameters of the bio-stickers constant, we measured the clear zone radius in the Petri dishes for 24 consecutive days to determine the rate and extent of PHB degradation.

[0257] Panel A illustrates a consistent degradation trend over time (from 12 to 28 days) for bio-stickers with higher initial biomass (e.g., O.D.600 5, 10, 20) (FIG. 18A). Panel B (FIG. 18B) shows the clear zone radius of bio-stickers with an initial cell density of O.D.eoo 10 after 28 days of incubation, which was about 65% higher than that of bio-stickers with an initial cell density of O.D.eoo 0.1, indicating that bio-stickers with a higher initial cell density exhibit higher degradation efficiency.

[0258] CFU detection on bio-stickers was conducted with different initial cell densities (FIG. 18C). After being cultured in Petri dishes containing PHB powder for 7, 14, 21, and 28 days, the CFU results showed that bio-stickers with different initial cell densities exhibited a similar decreasing trend of 1.32-1.91 log from one to four weeks. The CFU method showed that different bio-ink printing volumes did not affect the final biomass content of the bio-stickers (FIG. 18C). One possible explanation for the observed trend is that the availability of nutrients in the culture medium and bio-ink directly impacts the final biomass of the bio-stickers. Therefore, higher initial biomass of the bio-stickers may have resulted in a greater amount of nutrient consumption, leading to an enhanced degradation of PHB over time.

[0259] It was hypothesized that the reason for the impact of high cell density on PHB degradation efficiency is the accumulation of PHB-degrading enzymes. The accumulation of PHB-degrading enzymes occurs in parallel with the increase of bacterial biomass, resulting in enhanced PHB degradation efficiency. This phenomenon can be attributed to the higher production of enzy mes as more bacterial cells are present, facilitating the enzymatic degradation of the PHB polymer through cooperative actions.

[0260] Alginate Concentration Does Not Impact Biodegradation Efficiency of biostickers. To investigate the influence of different concentrations of alginate (ranging from 2%to 6%) on the efficiency of Bacillus NRRL B- 14911 in degrading PHB, bio-stickers were prepared using varying concentrations of alginate and cultured them on dishes containing PHB powder and 0.3M CaCb. The extent of PHB degradation was quantitatively assessed using the clear-zone method. The size of the clear zones in the Petri dishes was continuously measured for 22 days to determine the rate and extent of PHB degradation with all parameters of bio-stickers being kept constant.

[0261] Panel A (FIG. 19A) demonstrates the change in degradation of PHB over 22 days of culture of bio-sticker, while Panel B (FIG. 19B) reveals the clear zone radius of biostickers prepared with different concentrations of alginate on day 22, indicating no significant effect of the concentration of alginate ranging from 2% to 6% on the rate of PHB degradation.

[0262] Printing Layers Does Not Impact Biodegradation Efficiency of bio-stickers. To investigate the effect of different 3D printing layer thicknesses (sample thicknesses of 1 mm, 5 mm, 10 mm, and 15 mm, all containing the same initial biomass) on the ability7of Bacillus NRRL B-l 4911 -containing bio-stickers to degrade PHB, bio-stickers of varying thicknesses were prepared and cultured them on Petri dishes containing PHB powder and 0.3 M CaCb. The degradation of PHB was quantitatively assessed using the clear zone method. While maintaining all parameters of the bio-sticker constant, the size of the clear zone in the Petri dish was measured for 22 consecutive days to determine the rate and extent of PHB degradation.

[0263] Panel A (FIG. 20A) shows the degradation of the bio-sticker over 22 days, and Panel B (FIG. 20B) reveals the clear zone radius of the bio-sticker with different sample thicknesses on day 22 of culture, indicating that changes in sample thickness (1 mm, 5 mm, 10 mm, and 15 mm) had no significant effect on the rate of PHB degradation.

[0264] Printing Radii Does Not Impact Biodegradation Efficiency of bio-stickers. To investigate the effect of different radii (10 mm, 20 mm, 25 mm) of 3D-printed bio-stickers on the degradation of PHB by Bacillus NRRL B-14911, bio-stickers were prepared with different radii and cultured them on Petri dishes containing PHB powder and 0.3 M CaCb. The degradation degree of PHB was quantitatively evaluated using the clear zone method. While keeping all parameters of the bio-stickers constant, the size of the clear zone in the Petri dishes was measured for 24 consecutive days to determine the rate and extent of PHB degradation.

[0265] Panel A (FIG. 21 A) shows the degradation changes of the bio-stickers cultured for 24 days, and Panel B (FIG. 2 IB) reveals the clear zone radius of bio-stickers withdifferent radii (10 mm, 20 mm, 25 mm) on day 24, indicating that the variation in bio-sticker radius has no significant effect on the rate of PHB degradation.

[0266] Bio-stickers with different radii, which were prepared by 3D printing, were cultured for 7, 14, 21, and 28 days on Petri dishes containing PHB, and then subjected to CFU detection (FIG. 21 C). The results of CFU detection showed that bio-stickers with different radii had the same decreasing trend, with a decrease of 1.68 log to 1.73 log CFU from week one to week four.

[0267] Modeling of PHB degradation by 3D-printed bio-stickers. To investigate the relationship between the survival capability curve of marine bacteria and PHB degradation, a model to predict the degradation efficiency of bio-stickers was created. The goal was to identify environmental factors influencing the degradation efficiency of bio-stickers in marine environments. This could potentially contribute to the production and deployment of bio-stickers in marine environments and reduce plastic pollution in marine ecosystems. f(x) = (N *k). / (N i ((k - N) *exp(-r *x)))Coefficients (with 95% confidence bounds)'.N = 0.0002769(0.0001325, 0.0004213) k = 0.05186(0.04125, 0.06248) r = 0.2659(0.2283. 0.3034)Goodnessoffit : SSE : 2.504c - 06R square : 0.9981R square Adjusted : 0.9977RMSE : 0.0005595The model utilizes degradation data of different concentrations of PHB degradation (ranging from 0.1% to 2%) (FIG. 16) on agar plates. The specific model used in this study was chosen to quantify the PHB degradation rate and to understand the kinetics of degradation. The logical growth curve model (FIG. 22A) has been widely used to analyze biological systems and has been applied in various studies to understand the dynamics of microbial growth, enzyme kinetics, and substrate degradation. The model is based on the assumption that thegrowth of microorganisms follows a sigmoidal curve, which includes a lag phase, an exponential phase, and a stationary phase. This model provides a simple and effective way to describe the kinetics of biological processes and to estimate parameters such as maximum growth rate, lag time, and saturation point. The logical growth curve model was applied to fit the data of PHB degradation by Bacillus NRRL B- 14911. allowing the determination of degradation rate (r), maximum degradation rate (K), and initial degradation rate (N) over time. The degradation rate (r) represents the rate at which PHB is degraded by the bacteria, the maximum degradation rate (K) represents the maximum rate of PHB degradation, and the initial degradation rate (N) represents the rate of PHB degradation at the beginning of the degradation process.

[0268] Panel B illustrates the potential correlation between the survival capability curve of marine bacteria and the degradation of PHB (FIG. 22B). This relationship revealed a potential correlation between the survival capability7of marine bacteria and their ability to degrade PHB, suggesting that environmental factors may influence the degradation process. By investigating this correlation, novel strategies can be developed to enhance PHB degradation in marine ecosystems. Regarding the growth curve, it is not uncommon for different models to yield different results, and the previous model may have been limited in its ability to capture certain phases of growth. Therefore, it is important to use multiple models to gain a comprehensive understanding of the system being studied.

[0269] These findings showed that the clear zone assay confirmed the correlation between microbial degradation of PHB and PHB concentration, incubation temperature, initial biomass, alginate concentration printing layers and printing radii. This enabled the assessment of the potential use of bio-stickers in biodegradable materials. This disclosure provides information for the optimization of bioprinting protocols for PHB degradation and potential applications in biodegradable materials and environmental remediation.

[0270] Viability and Degradation Rate Post-Transfer. Demonstrative Illustration of Bacillus NRRL B-14911 Bio-Stickers and their Biodegradative Capabilities. The UR-shaped bio-stickers containing Bacillus NRRL B-14917, fabricated using a bioprinter, showed excellent degradation performance on agar plates containing PHB. The biodegradation of PHB was visible on the agar plates, with white PHB powder in certain areas decomposed and disappeared, exhibiting clear zones in the shapes of the UR symbol (FIG. 23B). As time progresses, the degradation will gradually increase until the PHB pow der in the agar plate is completely degraded (FIG. 24).

[0271] To investigate the repeatability of the degradation process, the biodegradable bio-stickers were printed and transferred from the original agar plate containing PHB onto a new agar plate containing fresh PHB using sterilized tweezers. The bio-stickers could adhere to the sterilized tweezers and did not fall off during transfer (FIG. 23 A).

[0272] Viability and Degradation Rate Post-Transfer. To assess the reusability of biodegradable stickers during transportation and storage, a transfer experiment was conducted to investigate their ability to maintain biodegradation viability under different environmental conditions. Prolonged storage and transportation may potentially impact the biodegradability of these stickers in practical applications. The findings of this study could contribute to waste reduction by enabling the repeated use of biodegradable stickers in various settings.

[0273] The bio-stickers were cultivated on nutrient agar plates containing PHB powder for 1 day, 7 days, and 14 days. The bio-stickers were then transferred from original agar plates containing PHB onto new agar plates containing PHB powder, and the degradation rate of the clear-zone was measured to determine their degradation rate (FIG. 25 A and FIG. 25B). CFU counting was conducted to assess the survival rate of the biostickers after being transferred onto new nutrient agar plates containing PHB powder and cultivated for 7, 14, 21, and 28 days (FIG. 25C).

[0274] The clear-zone assay demonstrated that these biodegradable stickers were able to maintain their degradation activity even after being transferred to a new plastic substrate. This phenomenon could be attributed to the provision of a fresh culture medium that provides additional nutrients to the microorganisms, promoting their grow th as indicated by the CFU results (FIG. 25C).

[0275] The clear-zone assay demonstrated that these biodegradable bio-stickers were able to maintain their degradation activity when transferred onto new plastic substrates. The bio-stickers transferred after 7 and 14 days of cultivation exhibited a similar trend in degrading PHB powder as the non-transferred (control) bio-stickers (FIG. 25 A). This phenomenon may be attributed to the provision of new nutrient sources, which provided additional nutrients for microbial growth, as supported by the CFU results (FIG. 25C). The CFU results showed that the CFU profile of the bio-stickers transferred onto fresh nutrient media was 0.23 log CFU / mL higher than that of the control group in the fourth week. Panel B showed that the degradation radius of the transferred bio-stickers on day 10 after transfer w as lower than that of the non-transferred (control) bio-stickers (FIG. 25 A).

[0276] Mechanical properties of Bio-sticker. Under the shear stress of the ocean, the mechanical capacity of bioprinted stickers could pose a challenge. Poor mechanical properties may hinder the ability of bioprinted stickers to remain on the PHB surface and perform their biodegradation function.

[0277] To assess the feasibility and applicability of bio-stickers in real-world applications under seawater conditions, their mechanical properties were evaluated through testing. The bio-sticker, a novel adhesive material, exhibits mechanical properties such as ultimate tensile strength (UTS) and modulus of elasticity.

[0278] Modulus of elasticity (E) measures material stiffness using stress (o) and strain (E), where stress is the force applied per unit area and strain is the change in length per unit length.E = o / E

[0279] Ultimate tensile strength (UTS) is the maximum stress a material can bear before breaking. This parameter is crucial for designing and applying biodegradable stickers, where it is calculated as the maximum load (F t) applied to the material before it breaks, divided by the original cross-sectional area of the material (Ao).UTS = Fmax / AoTesting biodegradable sticker’s mechanical properties is crucial for optimizing their suitability7, material properties, and performance, impacting their practical development and use. A series of mechanical tests w ere conducted to evaluate the initial mechanical properties of dog bone samples injected with Bacillus NRRL B-14911 PEIB-degrading strains. A "dog bone structure” is a shape used in materials science and engineering to create test specimens for tensile strength testing. The shape resembles a dog bone, with a central region that ends are thicker and wider than the central region. The mechanical tests were conducted on biosticker samples with a “dog bone structure” formed by 3D printing a non-polymeric bio-ink composed of 4% alginate and Bacillus NRRL B-14911 into an agarose slurry containing calcium salts. Bio-sticker samples with a “dog bone structure” that did not contain Bacillus NRRL B-14911 or that contained E. coli BL-21 were utilized as the control groups. The printed bio-sticker samples were then incubated at 30°C for 14 or 21 days before being stored in a cold room in preparation for mechanical testing (FIG. 26A-C).

[0280] The preliminary results show that the 14-day and 21 -day culture samples exhibit similar stiffness and ultimate tensile strength (FIG. 52). Several every day materials demonstrate comparable stiffness to the bio-sticker. Notably, silicone finds wide utilization invarious domains, including medical devices, kitchenware, and consumer electronics. Silicone’s stiffness range aligns closely with the elastic modulus of the bio-sticker. FIG. 27 and FIG. 28 show results for elastic modulus, ultimate tensile strength, maximum load, and energy dissipation.

[0281] Consequently, both silicone and the bio-sticker exhibit a similar degree of flexibility and stiffness. Silicone’s low modulus of elasticity grants it flexibility', stretchability, and resistance to deformation, making it suitable for applications necessitating materials capable of conforming to diverse shapes, while providing cushioning and shock absorption.

[0282] Triggers for PHA (e.g., PHB) Degradation. Triggered degradation refers to Bacillus NRRL B-14911 bio-sticker’s response to specific stimuli or environmental signals, resulting in the release of enzymes or other substances that promote bioplastic degradation, accelerating the process. One approach is to prepare spores or freeze-dried bacteria and activate them in seawater after bioplastics have reached their end-of-life, promoting degradation.

[0283] Viability and Degradation Rate of Lyophilizing Storage. To ascertain the degradation capability of freeze-dried bacteria in the context of bioprinting, tests on the efficiency of rehydrated freeze-dried Bacillus NRRL B-14911 were conducted on degrading PHB powder on agar plates. Given the application of bioprinted stickers containing freeze- dried bacteria in oceanographic instruments, these devices can submerge themselves in seawater, thereby rehydrating and reviving the embedded bacteria, in order to initiate degradation.

[0284] To determine whether bio-stickers after lyophilization and storage remain effective over time, the viability and degradation rates were tested. Storage can potentially affect the viability of bacteria embedded in a bio-sticker, which may impact their ability to degrade PHB. Freezing and lyophilizing can cause physical damage to the cells due to the formation of ice crystals and dehydration, respectively. These stresses can lead to a loss of cell viability and a decrease in the efficiency of the biodegradation process. In addition, the freeze-drying process may affect the metabolic activity and membrane permeability of the cells, which can also affect their viability. By testing the viability and degradation rates after storage, it can be ensured that bio-stickers remain viable and effective when used in real- world applications.

[0285] To maintain the viability and degradation rates of bio-stickers after storage, they were stored in a controlled environment that is conducive to bacterial survival.lyophilization is performed at temperatures below the material’s glass transition temperature (Tg), which is the point at which the material transitions from a rubbery’ to a glassy state. For most biological samples, the lyophilization process is typically carried out at a temperature between -40 °C and -50 °C for the freezing step and then at a lower pressure and slightly higher temperature for the drying step. Freeze-drying was performed at low temperatures and low pressure to remove moisture from the sample, and freeze-drying protectants were used to maintain the viability of microorganisms during the freeze-drying process and maintain them at a low metabolic level. This helps stabilize proteins and other biological materials during the freeze-drying process.

[0286] To evaluate the survival rate of bacteria after storage, samples containing Bacillus NRRL B- 14911 were lyophilized in protectant media, protectant media with 4%- alginate (bio-ink, non-polymeric), protectant media with 4%-alginate and 0. IM CaC12 (biosticker, polymeric), and PBS as a control group, and performed CFU measurement to determine the survival rates of Bacillus NRRL B- 14911 (FIG. 29). The CFU profiles showed that the samples containing protectant media, including the protectant media, the protectant media bio-ink (non-polymeric) and protectant media bio-sticker (polymeric), had higher CFU counts than the control group, with increases of 2.37 log CFU / rnL, 1.51 log CFU / mL, and 0.26 log CFU / mL, respectively. These results indicate that the addition of protectant media during lyophilization can enhance the survival rate oil Bacillus NRRL B-14911. Furthermore, the CFU profile indicated that the CFU of the protectant media bio-sticker (polymeric) group increased by 0.26 log CFU / mL compared to the control group but decreased by 1 .46 log CFU / mL compared to the protectant media bio-ink (non-polymerized) group. It is speculated that the preparation process of the bio-sticker, which contained 0.1 M calcium chloride, may have had a potential adverse effect on the survival rate of Bacillus NRRL B-14911. Based on the CFU results, it was found that freeze-drying Bacillus NRRL B-14911 with protectant media had a higher survival rate, with an increase of 2.37 log CFU / mL compared to the control group (Figure 29). Therefore, freeze-dried Bacillus NRRL B-14911 with protectant media, which was rehydrated and mixed with 4%-alginate to form a bio-ink (nonpolymerized), was used to produce a bio-sticker (polymerized) on an agar plate containing PHB powder and 0.3 M CaCL. The formation of a transparent area was observed, and the degradation efficiency was measured by culturing the plate at a constant temperature of 30 °C (FIG. 30).

[0287] To evaluate the degradation rate of the bio-sticker (polymerized) in degrading PHB, the clear zone method was used to measure the change in the radius of the transparentarea over time (FIG. 30A). The results showed that freeze-dry ing Bacillus NRRL B-14911 with protectant media exhibited a faster degradation rate from day 4 to day 12, while freeze- drying Bacillus NRRL B-14911 with PBS (control) did not show any degradation. Panel B showed that the average radius of the transparent area of the bio-sticker produced by freeze- drying Bacillus NRRL B-14911 with protectant media had reached 10.705 mm by day 16 (FIG. 30B).

[0288] Viability and Degradation Rate of Sporulation. To determine the degradation capabilities of spores within a bioprinting framework, we evaluated the efficacy of bioprinted Bacillus NRRL B-14911 spores in degrading PHB powder on agar plates. The inherent stability-, resistance, and potential degradability of spores satisfy the experimental requirements for initiating degradation. During the injection molding or 3D printing processes, PHB can be mixed with Bacillus, leading to the direct incorporation of the spores into the bioplastic material. These resilient embedded spores remain dormant for extended periods of time until the PHB begins to exhibit physical cracks or fissures at the end of its useful life, allowing seawater to penetrate and rehydrate the spores. This triggers the degradation of the PHB. effectively preventing the release of microplastics into the marine environment.

[0289] To obtain a substantial quantify of Bacillus NRRL B-14911 spores, a CFU assay was employed to approximate the spore yield and to confirm that the non-sporulating live cells were remove via heat shock treatment (FIG. 31). Bacillus NRRL B-14911 was initially cultivated in LB medium or DSM sporulation medium was employed a heat shock treatment, a method where the bacterial culture w as rapidly heated to 95 °C and maintained for a predetermined period (e.g., 60 minutes). This temperature and time setting was devised to annihilate the live cells while preserving the spores. Following the heat shock treatment, the sample was swiftly cooled down to room temperature, and an adequate dilution was conducted for the CFU assay. Observation revealed that under heat shock at 95 °C, live cells reduced to 0 CFU / mL, and the spore CFU decreased by 2 log, remaining at 102CFU / mL (FIG. 32A). For testing the degradation efficacy of the spores, spore bio-ink was prepared by combining the spores with 4% alginate. The clear-zone method was then utilized to measure the size of the degradation clear zone of the spore-containing bio-sticker (polymerized) on plates containing 1% PHB and 0.3M CaCh medium (FIG. 32B). A degradation trend line w as plotted to quantify the amount of PHB degraded per unit time. The trend line indicated that the spore recovery and degradation exhibited a lag phase compared to live cells, followed by a similar rate of PHB degradation.

[0290] Construction of Plasmids for the Purification of PHA-Degrading Enzymes. It was hypothesized that the purified polyhydroxybutyrate (PHB)-degrading enzyme, once integrated into bioplastic or into a hydrogel, would promptly initiate the degradation of a PHA (e.g., PHB). It was expected that the utilization of inorganic protective carriers or dispersion with heterogeneous polymers would augment the enzyme’s stability and activity' within the PHB.

[0291] To verify this hypothesis, a plasmid encoding the hydrolytic enzyme phaZ, known for its PHB degradation properties, was crafted. The phaZ gene was bio-sourced from Bacillus megaterium N-18-25-9. This plasmid was named pSBlC3-T7-phaZ::chlr. Subsequently, this engineered plasmid was introduced into E. coli BL21(DE3) competent cells to establish the strain named BL21 / pSBlC3-T7-phaZ::chlr( Figure 33B). The BL21 (DE3) / pSB]C3-T7-phaZ strain is a genetically engineered Escherichia coli strain, tailor-made for specific functions (FIG. 34). Specifically, BL21 (DE3) is a widely adopted E. coli strain for gene cloning and protein expression. The DE3 designation denotes a lambda phage derivative carrying the T7 RNA polymerase gene, under the control of the lacUV5 promoter. The pSBlC3 is a standard BioBrick plasmid utilized as a vector in genetic engineering processes. The T7 promoter, a robust bacteriophage promoter, is often employed for high- level protein expression in E. coli. Moreover, the phaZ gene encodes the enzy me PHB depolymerase, tasked yvith breaking doyyn PHB.

[0292] Upon the addition of Isopropyl [3-D-l -thiogalactopyranoside (IPTG), a structural analogue of allolactose, the natural inducer of the lac operon in E. coli, it binds to the lac repressor protein, leading to a conformational change that dislodges the repressor from the lac operator sequence (FIG. 34). This unshackling enables the T7 RNA polymerase, produced from the DE3 prophage in BL21(DE3) cells, to transcribe the phaZ gene from the T7 promoter on the pSBlC3 plasmid (FIG. 34).

[0293] Consequently, with the addition of IPTG, the phaZ gene, controlled by the T7 promoter on the pSB!C3 plasmid in the BL21(DE3) E. coli strain, is expressed (FIG. 34). The ensuing phaZ mRNA is then translated into PHB depolymerase. This expressed PHB depolymerase contains a signal peptide and a 6x His tag (FIG. 33B), which enhance its functionality and practicality in subsequent research and applications.

[0294] The signal peptide (FIG. 33B) is a short peptide sequence, typically around 20-30 amino acids long. It attaches to the N-terminus of the protein during translation, directing the protein before it enters the cell membrane. For PHB depolymerase, the presence of the signal peptide could result in the protein being directed outside the cell to locationscontaining its substrate, i.e., PHB, thereby augmenting its catalytic efficiency. In various embodiments, use of the signal peptide is not necessary.

[0295] Furthermore, the 6x His tag (FIG. 33B) is a small protein tag composed of six consecutive histidine residues. This tag can form coordination bonds with certain metal ions, such as nickel or cobalt, enabling proteins with His tags to be purified through immobilized metal ion affinity chromatography (IMAC). This feature allows for purification of the expressed PHB depolymerase (PhaZ) via Fast Protein Liquid Chromatography (FPLC). This is beneficial for enzyme activity measurement, structural analysis, or other experiments requiring purified proteins. Confirmation of successful construction of the BL21 / pSBlC3-T7- phaZ::chlrstrain was achieved through plasmid (pSBlC3-T7-phaZ::chlr) PCR and full plasmid sequencing, as shown in FIG. 33A.

[0296] FIG. 33 depicts the clear zones on LB agar plates containing PHB and chloramphenicol after the overnight culture of the engineered strain BL21 pSB! C3-T7- phaZ::chlrwas treated with 0.5 mM IPTG and a bio-ink composed of 4% alginate. Following the addition of IPTG and the bio-ink, the culture was fermented for 3 hours. After 5 days of incubation at 37 °C, the presence of clear zones indicates the successful degradation of PHB by the constructed strain, thereby confirming its functionality in PHB degradation.

[0297] 3D Printing for Future Biodegrading Bioplastics. Described herein, 3D printing technology is used as a tool to create a specialized support structure for the marine bacteria, Bacillus NRRL B-14911, that is known for its ability to break down bioplastics.

[0298] 3D printing technology enables the creation of a custom support structure that is specifically designed to optimize the grow th and activity of the bacteria. The 3D-printed support structure can provide a high surface area-to-volume ratio, allowing for a larger number of bacteria to be immobilized and increasing the efficiency of the biodegradation process. Moreover, 3D printing technology allows for the precise placement of the bacteria within the support structure, which can further enhance the biodegradation process. The controlled distribution of the bacteria within the 3D-printed structure can ensure that the biodegradation of bioplastics occurs uniformly throughout the structure, which can be critical for its effectiveness in marine environments. Furthermore, 3D printing technology offers a cost-effective and scalable approach to creating the support structure for the bacteria. This is particularly important for the development of a practical and commercially viable solution for the biodegradation of bioplastics in marine environments.

[0299] The application of 3D-printed Bacillus NRRL B-14911 biofilms in marine environments can be achieved through two approaches. One is integrating biofilms intobiodegradable structures (e.g., nets, ropes, containers) and deploying them in plastic-polluted marine environments, where the biofilms actively degrade the bio-plastics, reducing their persistence and impact. Another approach is developing floating or submerged biofilm-based systems placed strategically in high-concentration bio-plastic areas, facilitating direct contact between biofilms and bio-plastics to accelerate the degradation process.

[0300] Printing Parameters on Bio-Sticker Degradation. PHB concentration. PHB Concentration and Biodegradation Efficiency. A principal finding from this research is that lower concentrations of PHB enhance the biodegradation efficiency of the Bacillus NRRL B- 14911 bio-sticker (FIG. 16). This suggests that the hydrolytic enzyme PhaZ, which is pivotal in catalyzing PHB degradation, degrades more rapidly at lower PHB concentrations. The underlying mechanisms for this amplified efficiency at reduced concentrations merit further investigation, as understanding these processes could inform the design and deployment of bio-stickers in varying environmental contexts.

[0301] Correlation between PHB Concentration and Clear Zone Radius. A clear correlation was established between PHB concentration and the clear zone radius (FIG. 22). This relationship could serve as a predictive tool for bio-sticker efficiency under disparate conditions, potentially optimizing their use. However, an exploration of the factors that could influence this correlation, including environmental factors and bio-sticker composition, would provide a more nuanced understanding of this relationship.

[0302] Impact of PHB Concentration on Bio-sticker Growth. The higher PHB concentrations adversely affected the growth and viability’ of bio-stickers, as evidenced by the CFU result (FIG. 16C). It appears that the toxicity associated with elevated PHB concentrations may disrupt nutrient availability or obstruct essential cellular processes, thereby inhibiting bio-sticker growth. Understanding the mechanisms driving this result could inform strategies to mitigate its effects, particularly in environments with high PHB concentrations.

[0303] Time Required for PHB Degradation. The data elucidating the timeframes required for complete PHB degradation at different concentrations provide valuable information for the strategic deployment of bio-stickers (FIG. 22). In-depth discussion around how these timeframes could be optimized, possibly by adjusting PHB concentration or biosticker amounts, would be beneficial for maximizing the effectiveness of bio-stickers in various settings.

[0304] Initial Biomass. Influence of Initial Biomass on Biodegradation Efficiency. The experimental data demonstrated a significant correlation between the initial biomass ofbio-stickers and the efficacy of PHB degradation (FIG. 18). The heightened degradation efficiency associated with a higher initial biomass could be attributed to either an increase in nutrient consumption or an accumulation of PHB-degrading enzymes. Exploring the causal mechanisms behind this correlation could offer valuable insights for optimizing bio-sticker design and deployment strategies.

[0305] Accumulation of PHB-degrading Enzymes with Increased Biomass. Higher cell density seemingly resulted in an accumulation of PHB-degrading enzymes, thereby enhancing the efficiency of PHB degradation (FIG. 18). This phenomenon could potentially be explained by the increased production of enzymes as the bacterial cell count rises. Further exploration of the mechanisms triggering this enzy me accumulation could provide valuable information for the design and application of bio-stickers.

[0306] Nutrient Availability and Biomass. These results indicated a direct relationship between the nutrient availability in the culture medium and bio-ink, and the final biomass of the bio-stickers. This relationship could have crucial implications for the formulation of bioink and the growth conditions necessary for bio-stickers.

[0307] Trend of CFU Decrease Across Varied Initial Biomass. The CFU results indicated a consistent decreasing trend from the first to the fourth week, regardless of the initial cell densities of the bio-stickers. This trend suggests that factors other than initial cell density may be influencing the final biomass content of the bio-stickers, warranting further investigation.

[0308] Culture Temperature. Temperature Effects on Bio-Sticker Viability and Degradation rate. Temperature is a crucial parameter that can greatly influence the activity of enzymes responsible for PHB degradation. Higher temperatures generally increase the metabolic activity of bacteria and the rate of enzyme-catalyzed reactions, which can result in faster PHB degradation (FIG. 17). However, if the temperature exceeds the optimal range for the bacterial strain used in the biosticker production, it may result in reduced biomass or denaturation of enzymes, leading to a decrease in the degradation rate.

[0309] For Bacillus NRRL B-14911, the optimal growth temperature may vary depending on the specific strain and the growth medium used, but it is typically within the range, around 20-40°C (FIG. 17). If the temperature deviates significantly from this optimal range, the viability of Bacillus NRRL B-14911 may be reduced, leading to decreased grow th and potential loss of viability over time. Higher temperatures can generally accelerate the degradation process by increasing the metabolic activity of the bacteria and the rate of enzyme-catalyzed reactions. However, if the temperature exceeds the optimal range forBacillus NRRL B-14911, it may result in reduced activity or even denaturation of the enzymes, leading to a decrease in the degradation rate.

[0310] Temperature-dependent regulation of genes. Bacillus NRRL B-14911. like other bacteria, may have temperature-dependent regulation of genes that are involved in various cellular processes, including degradation pathways. The expression of specific genes involved in the production of enzymes responsible for PHB degradation, as well as other cellular activities, may be influenced by temperature. Therefore, changes in temperature can impact the activity of these enzymes, which in turn can affect the degradation rate of Bacillus NRRL B-14911 biostickers.

[0311] Thermal stability of bio-sticker materials. The thermal stability of biostickers can also be affected by temperature. High temperatures may cause the components of the biosticker to undergo thermal degradation, leading to changes in their physical properties, which can impact the overall degradation rate of the biosticker.

[0312] Impact of Ocean Temperatures on Biosticker Efficacy. Ocean temperatures vary significantly by location and depth. Tropical waters, for instance, are typically warmer, while polar waters are colder. Furthermore, deeper water layers are generally colder than surface waters. This variability could indeed influence the rate of PHB degradation if biostickers are designed to degrade based on specific temperature ranges.

[0313] For instance, if bio-stickers are primarily designed for optimal degradation at tropical temperatures, they might degrade slower in colder, polar regions. Conversely, biostickers designed for colder temperatures might degrade faster than intended in warmer waters, which could be problematic if the biostickers are intended to last for a specific period.

[0314] Tailoring Bio-stickers to Specific Environments. The bespoke design of biostickers allows for tailoring them to specific environmental contexts through the incorporation of bacterial strains or enzymes that exhibit optimal functionality within distinct temperature ranges. This would enable strategic deployment of bio-stickers in diverse climatic zones including tropical, temperate, and polar waters.

[0315] Developing Temperature-Resilient Bio-stickers. The engineering of temperature-resilient bio-stickers, would ensure sustained performance across a wide thermal spectrum, enhancing their versatility and resilience in response to dynamic oceanic conditions.

[0316] Monitoring and Predicting Ocean Temperatures. The integrating of continuous oceanic temperature monitoring and predictive modelling into bio-sticker deploymentstrategies may be highly beneficial. This integration could facilitate anticipatory responses to environmental fluctuations, fostering strategic and proactive deployment planning.

[0317] Environmental Impact Assessment. The comprehensive environmental impact assessments of biosticker deployment are indispensable. These assessments should encompass a thorough evaluation of potential ecological impacts arising from the biosticker degradation products, ensuring that biosticker use respects the integrity of the varied, and potentially vulnerable, marine ecosystems they inhabit.

[0318] Alginate concentration. Alginate Concentration and Biodegradation Efficiency. FIG. 19 shows the concentration of alginate in the bio-stickers, within the range of 2% to 6%, does not significantly impact the rate of PHB degradation. This finding is significant as it suggests the bio-sticker formulation can be flexible in terms of alginate concentration without affecting their degradation efficiency.

[0319] Alginate is often used as a matrix material in biosticker production to encapsulate bacteria and PHB. The alginate concentration can affect the structural integrity and permeability of the biosticker, which can impact the access of enzymes to PHB for degradation. Higher alginate concentrations may result in reduced permeability and slower degradation rates, while lower alginate concentrations may result in faster degradation rates due to increased enzyme access to PHB.

[0320] Layer structure. Impact of 3D Printing Layer Thickness on Biodegradation Efficiency. In the realm of 3D printing layer thickness and its influence on the biodegradation efficiency of bio-stickers, the experimental results have demonstrated a noteworthy consistency. Despite the variation in layer thickness, ranging from 1 mm to 15 mm, the degradation rate of PHB remains unaffected, suggesting a remarkable flexibility in the manufacturing parameters of the bio-stickers. This finding essentially widens the scope for bio-sticker production, permitting a range of thicknesses without compromising the degradation efficiency of PHB.

[0321] While the study has tested thicknesses up to 15 mm, it leaves open the question of the upper limit of layer thickness that can be employed without affecting the degradation rate. Determining if such a threshold exists, and pinpointing its exact value, could inform the design parameters and manufacturing guidelines for bio-stickers, ensuring their optimal performance in diverse applications.

[0322] Consistency of Bio-sticker Efficacy. The constancy of bio-sticker efficacy, irrespective of the variations in layer thickness, underscores the robustness of the bio-stickerdesign. Such resilience to thickness modifications points to a potential resistance against other manufacturing or environmental variations.

[0323] The mechanism underpinning this unwavering efficacy across different layer thicknesses remains elusive. A deeper exploration of the causal mechanisms underlying this correlation may provide insights beneficial for the optimization of bio-sticker design and deployment strategies. In particular, understanding the interactions between the bio-sticker’s physical structure and the biological activity of the embedded bacteria could shed light on the principles guiding this consistent performance.

[0324] Printing Radii. Bio-sticker Radius and Biodegradation Efficiency. The results indicate that varying the radius of the bio-stickers (within the tested range of 10 mm, 20 mm, and 25 mm) does not significantly impact the rate of PHB degradation. This could suggest flexibility in the design parameters of the bio-stickers, potentially allowing for customization based on specific application needs.

[0325] Consistent Decrease in CFU Across Different Radii. The CFU results showed a similar decreasing trend over the course of one to four weeks across bio-stickers with different radii. This consistency implies that the overall bactenal activity and survival within the bio-stickers are not significantly influenced by the radius of the bio-sticker. This could offer more flexibility' in the production and use of bio-stickers.

[0326] Modeling of PHB degradation by bio-stickers. Survival Capability and Degradation Efficiency. These models (FIG. 16) have the potential to provide valuable insights into the mechanisms of PHB degradation, which may contribute to optimizing conditions for PHB degradation in various applications. By predicting the degradation efficiency of bio-stickers through a logical growth curve, optimal conditions were simulated for PHB degradation, the effectiveness of bio-stickers w as evaluated in degrading PHB, and the degradation rate of bio-stickers were predicted under different conditions. In addition, the potential correlation between the survival capability7of marine bacteria and the rate of PHB degradation is a significant finding. It emphasizes the importance of bacterial survival in the context of PHB degradation and suggests that environmental factors influencing bacterial survival may also impact the degradation process.

[0327] Implications for Marine Ecosystems. The findings of this study could be crucial for developing strategies to mitigate plastic pollution in marine ecosystems. Understanding the relationship between bacterial survival and PHB degradation could guide the development of bio-stickers that are both effective and resilient in diverse marine environments.

[0328] Parameter Interpretation. This model provides interpretations for the degradation rate, maximum degradation rate, and initial degradation rate.

[0329] Importance of Calcium in 3D Printed Bio-Sticker Degradation. The addition of calcium salts, such as CaCh, to the bio-ink solution is critical for successful alginate-based bio-3D printing as it facilitates ion gelation, which is essential for forming 3D printed biostickers. However, excessive calcium ions can lead to rigid gels that may hinder cell proliferation and differentiation. Therefore, precise control of calcium ion concentration is necessary for optimal gelation and maintaining cell viability.

[0330] Calcium ions (more than 0.3 M) (FIG. 12) have also been shown to affect the viability of Bacillus NRRL B-14911. Studies have demonstrated that the presence of CaCh can delay Bacillus growth, suggesting that calcium ions may impact the activity of PHB- degrading enzy mes in these bacteria.

[0331] The enzyme PhaZ, responsible for catalyzing PHB degradation, uses calcium ions as a cofactor to enhance the degradation rate of PHB. Calcium ions bind to the enzy me’s active site, stabilizing the enzyme-substrate complex and promoting effective hydrolysis of ester bonds in PHB, resulting in smaller oligomers and monomers.

[0332] The use of calcium ions as a cofactor in PhaZ-mediated PHB degradation has potential applications in developing sustainable biodegradable materials and bioremediation of plastic w aste. However, further research is needed to better understand the role of calcium ions in PHB degradation and optimize their concentration in bio-sticker materials for efficient PHB degradation. Careful control of calcium ion concentration in bio-ink solutions may be necessary' to balance the gelation process and bacterial viability7, and to achieve optimal PHB degradation rates in 3D printed bio-stickers.

[0333] Given the ocean’s average calcium concentration of around 10.5 mM. it may indeed be insufficient to maintain the structural integrity of the alginate hydrogel system used in the 3D bio-stickers. Alginate hydrogels ty pically require higher concentrations of calcium ions for cross-linking and gelation processes, which provide the structure for the 3D printed objects.

[0334] Therefore, future research should investigate strategies for overcoming this challenge. One potential approach could be to incorporate a slow-release calcium source within the bio-sticker itself to maintain the structural integrity7of the alginate hydrogel in calcium-deficient environments. Another approach could involve exploring alternative materials or cross-linking mechanisms that could be more compatible with the calcium levels present in the ocean.

[0335] Potential Factors Affecting PHB Degradation by 3D Printed Bio-Stickers in Marine Environment. The marine environment plays a crucial role in the degradation of PHB in 3D printed bio-stickers produced using Bacillus NRRL B-14911. Simulating the submersion environment in seawater, including temperature, rotational speed, and salinity, is essential to accurately assess the performance of these bio-stickers. Factors such as oxygen supply, nutrient availability, and bio-ink shape can also affect PHB degradation. Adjustments to environmental conditions and bio-ink patterns may be necessary to ensure optimal adhesion and colony growth. Monitoring marine organisms and biodegradation rates is important in obtaining reliable results.

[0336] Temperature. The temperature of the marine environment, particularly the seawater, can have a significant impact on the degradation rate of PHB. The temperature in the simulated marine environment should be carefully controlled to replicate the actual conditions where the bio-stickers are intended to be used. The Pacific Ocean Current Report 2018 suggests that a temperature of 15 °C should be considered when replicating the ocean’s surface conditions. Higher temperatures can generally accelerate the degradation process, but it is important to find the optimal temperature range for Bacillus NRRL B-14911 to ensure optimal PHB degradation.

[0337] The specific implication of ocean temperature of 15 °C is the need to carefully consider the deployment environments of these bio-stickers. In environments with an average temperature of around 15 °C, such as the surface conditions of the Pacific Ocean, the biostickers may exhibit a slower degradation rate than in warmer environments. This could be a limitation in their efficacy for bioremediation of PHB plastic waste in such environments.

[0338] However, it should be noted that while the temperature of the seawater surface is around 15 °C, temperatures can vary significantly at different depths and in different geographical locations. Therefore, the observed degradation rates might differ in other marine environments.

[0339] Salinity . Salinity, or the salt content of the seawater, can also influence PHB degradation. Different marine environments may have varying salinity levels, and it is important to replicate the salinity conditions of the intended application site. Salinity affects the availability of nutrients, enzymes, and other factors that can impact the degradation of PHB. A salinity level of 35 psu (practical salinity units) is mentioned in the Pacific Ocean Current Report 2018 as a reference for replicating ocean surface conditions.

[0340] Nutrient availability. The availability of nutrients, such as carbon, nitrogen, and phosphorus, in the marine environment can also influence the degradation of PHB byBacillus NRRL B-14911. Nutrients are essential for the grow th and metabolism of bacteria, including those involved in PHB degradation. The composition and availability’ of nutrients in the marine environment should be considered to ensure optimal bacterial grow th and PHB degradation.

[0341] Bioplastic Delivery Prototype Design. Described herein are three high-fidelity' prototypes for producing living PHB-based materials, including bioprinted stickers, enzyme / spore-embedded materials, and bio-inserts. These prototy pes wall exhibit different degradation rates, shelf stability, and physical durability, with instantaneous or inducible degradation activity’ activated by seawater purging (FIG. 37).

[0342] Bio-Stickers. Bio-stickers containing living marine bacteria offer a promising approach for real-time degradation coverings on plastic surfaces. These stickers can be peeled and replaced onto fresh surfaces to assess their degradability and ability’ to maintain their PHB depolymerase activity even after transfer to a new’ PHB surface. Additionally, the living bacteria’s ability to survive within stickers outside of the lab environment and the potential for these stickers to degrade at tunable rates can extend the application of these living materials as environmental remediation agents.

[0343] Enzyme / Spore-Embedded Material. Lyophilized cells are freeze-dried bacterial cells, while bacterial spores are dormant and resilient cells capable of withstanding harsh environmental conditions until triggered for active growth. These can be incorporated into a plastic matrix and also into bio-stickers to create long-lasting, dormant components. Upon reaching the end of their useful lives, marine instruments composed of PHB may develop physical cracks or fissures, which allow’ seawater to penetrate the matrix and trigger the spores to germinate, enter active metabolism, and accelerate PHB degradation. This process can effectively accelerate degradation in a triggerable manner and prevent the release of microplastics into the ocean environment.

[0344] Moreover, incorporating purified PHB-degrading enzy mes can further enhance the degradation of PHB into bioplastics while maintaining a durable structure. Inorganic protective carriers or dispersing enzymes with heterogeneous polymers can increase the enzymes’ stability and activity within PHB, offering an easier method for monitoring and controlling PHB degradation.

[0345] Bio-Insertion. Bio-stickers are used for assessing the degradability' of plastic surfaces while bio-insertion is used for creating marine bacteria-containing components with a specific level of durability and structural support strength through casting. A seawatercompatible mold is first created, and then filled with a bio-ink containing live organisms. Thebio-ink is then molded and cured into a final shape. The molded-bio-ink components are tested for their durability against seawater exposure and evaluated for their ability to maintain their metabolic activity under seawater submersion. When the bio-insert is added to plastic products, the bacteria begin to feed on the plastic and decompose it. The compatibility and durability of various materials blended with Bacillus NRRL B- 14911 are examined to create a living bioprinted material that is robust under different environmental conditions. This living material is composed of marine bacteria and biopolymers, such as alginate and / or gelatin. The compatibility of the biopolymers with the bacteria is tested in terms of their capability to form a stable material and their ability to maintain the metabolic activity of the bacteria in different environmental conditions. Additionally, the physical properties, such as adhesion and wear-resistance, of the materials are tested to ensure their long-term survival in seawater.

[0346] Co-Printing PHB and Bacteria. Using the innovative technique of 3D bioprinting, PHB will be merged with bacterial cultures, specifically marine bacteria such as Bacillus NRRL B-14911. This process will use a specialized bio-ink, composed of the PHB and bacterial cultures, which is then printed layer by layer into a desired structure.

[0347] The inherent features of PHA and bacteria will be utilized to create materials with tunable degradation rates. As PHA has a natural susceptibility to biodegradation, it provides a suitable base for our bio-ink. The bacteria, on the other hand, will be selected based on their ability to metabolize PHA. The resulting co-printed material will then maintain its structure and integrity until the bacteria are activated by an environmental trigger, such as seawater exposure, to start the biodegradation process.

[0348] This co-printing process aims to create materials that exhibit varying degradation rates, shelf stability, and physical durability. The unique properties of these coprinted materials make them highly suitable for applications in environmental remediation, such as the production of biodegradable marine instruments and bio-stickers for real-time degradation coverings on plastic surfaces.

[0349] Optimization of printing conditions. Optimizing printing conditions is critical for 3D printed Bacillus NRRL B-14911 in degrading bioplastics in marine environments, as they significantly impact bio-sticker performance. This is particularly relevant in marine environments with varying temperature, humidity, and salinity conditions.

[0350] The optimization of printing conditions for Bacillus NRRL B-14911 has been completed by formulating an optimal bio-ink through the selection of the appropriate bacterial strain, adjustment of bacterial concentration, and addition of nutrients and additivesto promote bacterial growth and activity. The bio-ink formulation has been optimized to ensure sufficient bacterial activity and degradability for bioplastic degradation.

[0351] Parameters, including speed, temperature, and layer thickness, will be optimized to improve the adhesion, distribution, colonization, and activity of Bacillus NRRL B-14911 on bioplastics to enhance degradation performance. The design of biostickers, including shape, pattern, and structure, can be designed facilitate bacterial colonization and degradation, avoiding complex or intricate designs that may hinder growth. Maintaining stability and integrity of 3D printed bio-stickers during printing, handling, and underwater deployment will also be a focus.

[0352] Additionally, the selection of appropriate bioplastics for 3D printing and Bacillus NRRL B-14911 degradation will be considered in optimizing printing conditions. Different bioplastics have varying chemical compositions and properties that can impact colonization and degradation behavior. For example, before 3D printing can occur, the properties of PHB must be considered, such as its melting point, thermal stability, and viscosity when molten. These properties will inform the printing conditions, such as the temperature of the print head and the speed of extrusion. To optimize the process, different concentrations of PHB might be trialed, with the goal of finding the concentration that offers the best balance of printability and post-printing performance in terms of colonization by Bacillus NRRL B-14911 and subsequent degradation.

[0353] Moreover, it is reasonable to expect that different bioplastics would be degraded by these bio-stickers, but the efficiency and speed of degradation may vary. This is because bioplastics have diverse chemical structures, and the degradation process depends on the specific microorganisms and enzymes involved. For instance, Polyhydroxybutyrate (PHB) and Polyhydroxy valerate (PHV) are both biodegradable polyesters, but they have different side groups on their polymer chains, which might impact how quickly they can be broken down by Bacillus NRRL B-14911. Hence, selecting compatible bioplastics and optimizing printing conditions accordingly will enhance the overall performance of 3D printed bio-stickers for bioplastics degradation in marine environments.

[0354] Optimization of storage and transportation conditions. Optimizing storage and transportation conditions is critical to ensure bio-stickers reach marine environments in optimal condition, maximizing biodegradation performance. Deviations may reduce bacterial viability, activity, and performance, impacting bioplastics degradation efficiency in marine environments.

[0355] Storage condition optimization primarily involves controlling certain environmental factors like temperature, humidity, and light exposure to maintain Bacillus NRRL B-14911 viability and activity. For instance, freeze-drying has proven to be a successful method of preventing bacterial growth and preserving the bacteria’s effectiveness. Transportation conditions optimization focuses on safe and efficient transportation of 3D printed bio-stickers to marine environments, involving packaging, handling, and transportation methods that protect against contamination, damage, or degradation. For instance, using airtight and sterile packaging can maintain Bacillus NRRL B-14911 viability and activity, potentially utilizing advanced materials or coatings.

[0356] Further experimental testing and analysis will be performed to identify the specific temperature, humidity, and medium or buffer solution optimal for freeze-drying Bacillus NRRL B-14911. The CFU profile (FIG. 29) showed that the protectant media biosticker (polymerized) had a 0.26 log CFU / ml increase compared to the control group, but a 1.46 log CFU / ml decrease compared to the protectant media bio-ink (non-polymerized) group. It was hypothesized that the presence of 0. 1 M calcium chloride in the bio-sticker production process might adversely affect the survival rate of Bacillus NRRL B-14911. Therefore, strategies for optimizing freeze-drying storage are essential and need to be further investigated.

[0357] Compatibility with marine environment. The marine environment presents unique challenges to the viability and activity of Bacillus NRRL B-14911, including factors such as saltw ater, temperature fluctuations, varying pH levels, and UV radiation that can affect its survival and performance. While Bacillus NRRL B-14911 exhibits considerable resilience, marine stressors such as high salinity, temperature variability', and UV radiation present significant challenges. Concurrently, enhancing Bacillus NRRL B-1491TS bioplastic colonization capacity' is essential, yet any modifications to either the bacterium or bioplastic surface must not compromise biodegradation efficacy or bioplastic structural stability.Therefore, ensuring compatibility of Bacillus NRRL B-14911 with the marine environment is crucial to maximize its potential for bioplastics degradation.

[0358] Diverse biomaterials compatibility. The mechanical performance of Bacillus NRRL B-14911 bio-stickers is pivotal for their adhesion to bioplastics, surface colonization, durability, strength, flexibility, mechanical stability and subsequent biodegradation.Enhancing these aspects and the overall efficiency in degrading bioplastics could potentially be achieved by incorporating various biomaterials during the 3D printing process.

[0359] One potential approach to optimize the mechanical performance of Bacillus NRRL B- 14911 bio-stickers without introducing genetically modified organisms is through manipulation of the 3D printing process itself. This may involve adjusting printing parameters such as layer thickness, printing speed, and material composition to optimize the mechanical performance of the resulting bio-stickers. For example, incorporating higher viscosity bio-inks or reinforcing materials, such as nanocellulose or chitosan, into the bio-ink can enhance the mechanical properties of the bio-stickers, including strength, flexibility, and durability. By adjusting the composition and concentration of reinforcing materials in the bioink, bio-stickers with improved mechanical strength, flexibility, and durability can be obtained, allowing them to withstand harsh conditions in marine environments and effectively degrade bioplastics.

[0360] Another approach is to use bio-inks of varied compositions and properties, such as those containing purified enzy mes or more efficient PHB-degrading marine strains, could enhance the bio-stickers’ versatility and efficacy. These adjustments could potentially broaden the range of degradable bioplastics or accelerate the degradation rate. The increased efficiency might be attributable to improved enzymatic activity, accelerated growth rates, or superior adhesion to the bioplastic surface. This approach could optimize the bio-stickers’ performance in marine environments with diverse bioplastic compositions and structures.

[0361] In addition, advanced techniques such as multi-material 3D printing or biomanufacturing may provide opportunities to optimize the mechanical performance of Bacillus NRRL B-14911 bio-stickers. These techniques allow precise control over the material properties and structures of printed bio-stickers, enabling the creation of structures that can withstand harsh conditions in marine environments, including mechanical stresses, water flow, and wave action. This allows for the creation of complex structures with different material properties and structures that can be designed to provide optimal performance in different marine environments for bioplastic degradation. For example, using softer materials on the surface of the bio-stickers targeting bioplastics can enhance adhesion, while using harder materials in the core can provide mechanical stability.

[0362] Triggered degradation with monitoring and feedback mechanisms. The shelf life of the spores will be quantified. This will be achieved by preparing spore medium, spore bio-ink, and 3D-printed spore stickers, and using CFU detection and grow th curves to determine the shelf life of the spores in different nutrient media.

[0363] Monitoring and feedback mechanisms can be integrated into 3D-printed Bacillus NRRL B-14911 bio-stickers for real-time monitoring of the marine environment andfeedback on degradation progress. This could involve integrating sensors or monitoring devices capable of detecting specific environmental cues, such as temperature, pH. or marine chemicals, into Bacillus NRRL B- 14911 bio-stickers. These sensors can trigger responses when environmental conditions favor bioplastic degradation, such as releasing enzymes or signaling molecules. This feedback mechanism provides information on degradation progress and efficiency, allowing for adjustments based on real-time monitoring data. Remote sensing technologies, such as satellite-based monitoring or underwater drones, can also be used for collecting performance data of Bacillus NRRL B-14911 bio-stickers in degrading bioplastics in the marine environment, which can be analyzed to optimize deployment strategy', dosage, and effectiveness of Bacillus NRRL B-14911.

[0364] Scaling up and application of bio-stickers. To scale up the application of Bacillus NRRL B-14911 biostickers, key considerations include large-scale production of biostickers for widespread use. This can be achieved through optimization of the fermentation process, such as improving the yield and viability of spores or freeze-dried bacteria and optimizing the conditions for biofilm formation. Furthermore, advancements in 3D printing technology can enable efficient and cost-effective production of Bacillus NRRL B-14911 biostickers with optimized thickness, porosity', and composition tailored for specific applications.EXAMPLE 2

[0365] This example provides a description of the bioplastics of the present disclosure.

[0366] Bioplastics encompass a family of materials with properties similar to conventional plastics. They can be bio-based, biodegradable, or both: bio-based products are derived from materials of biological origin, while biodegradable refers to materials that microorganisms can break down into natural substances, such as water and carbon dioxide. Some bioplastics are bio-based, but non-biodegradable, such as bio-based polyethylene; others derived from fossil resources are not bio-based but still biodegradable, such as polybutylene adipate terephthalate (PBAT). Bio-based and biodegradable plastics include polylactic acid (PLA), polybutylene succinate (PBS), starch blends, and polyhydroxyalkanoates (PHAs).

[0367] A leading class of biopolymer, PHAs have gained widespread attention due to similar physical and mechanical properties to traditional plastics like polypropylene. PHAs are polyesters naturally synthesized by bacteria and stored as intracellular carbon and energyreserves. Unlike traditional plastics and some other bioplastics, PHAs will biodegrade in a marine environment, making them the most promising candidates for ocean applications requiring a rigid material. When formulated and processed appropriately, PHAs have the potential to replace a wide variety of persistent, petroleum-based plastics.

[0368] The typical end-of-life for PHA is biodegradation; PHA can break down in both aerobic and anaerobic environments, including marine environments where it is respired by microorganisms into carbon dioxide and water. PHB and its copolymers have been shown to biodegrade in saltwater, showing a maximum of 88-99% biodegradation after 49 days. While PHB is known to biodegrade in certain marine environments, marine testing has been limited and unstandardized. Degradation rate constants for PHB have been determined, and it as found found that a 2 kg PHB object would degrade in temperate ocean conditions in about 8 years - 35-100 times faster than the '‘biodegradable” plastic PL A

[0369] Engineered living materials will facilitate the rapid degradation of bioplastics in marine environments. Engineered living materials (ELMs) are a class of functional materials composed of engineered biological systems that create, modify, or maintain their own material structures or properties. ELMs are a promising new discipline at the interface of synthetic biology and material science.

[0370] 3D printing (additive manufacturing) of bacteria has been used as a new' class of ELM. This method can be used to deposit bacteria in specific three-dimensional patterns via straightforward alginate chemistry. The precise actuation of commercial 3D printers allow s for accurate and reproducible positioning of the printhead during the fabrication process.

[0371] This printing strategy allow s for the deposition of bacteria-containing bio-ink onto a printing substrate. After printing, the bacteria are fixed in place within a solidified hydrogel matrix. Individual lines of bio-ink printed can be as narrow as 0.1 mm, and multilayer structures can consist of 50 or more layers (FIG. 38B). Printed microbes can survive for tw o w eeks or more w ithout sustaining major losses of viability. Freeze-drying and sporulation of the printed bacteria may also expand this ‘shelf life’ further. Printed microbes are physically robust, retaining physical integrity upon twisting or crumpling, and are able to be repeatedly adhered to new surfaces. These ELMs feature embedded microbes that create or supplement their ow n material structure, allow ing manufacture of three-dimensional living materials that can degrade their surrounding structures. It has been demonstrated that natural PHB-degrading bacteria can be printed into living materials that can degrade PHB bioplastics, even when transferred to new surfaces. Future work will engineer the PHB-degrading ability of the engineered living materials to have tunable, triggerable degradation rates, producing a range of materials with personalized lifetimes (FIG. 39).

[0372] Microorganisms require sources of carbon, nitrogen, and phosphorus to grow and reproduce. How these needs are met can vary considerably among microbes depending on both biological and physical aspects of their environment. A lack of any one nutrient or growth factor, such as trace metals, vitamins, or amino acids can limit an organism’s ability to acquire other nutrients. In this way. biodegradation of high carbon-containing materials such as plastics may be limited by the availability of specific forms of nitrogen and phosphorus, or by the absence of specific grow th factors. The need for a growth factor that an organism is unable to synthesize itself is called auxotrophy; research has identified numerous metabolic auxotrophies in different marine microbial lineages that may lead to growth limitation in the environment.

[0373] PHB is a polymer that is synthesized by naturally-occurring microbes in many ecosy stems, including the ocean. As a result, PHB exists as a potential energy source in the environment, and diverse microbes have evolved to degrade it, including several readily culturable taxa that are found in the marine environment. Yet, the vast ma|ont of microbial diversity in the ocean is uncultivated and cannot be easily grown in the laboratory. Understanding of microbial metabolism in the ocean has grown exponentially in the last decade, aided by large-scale oceanographic programs that used metagenomic sequencing to uncover the metabolic potential of uncultivated organisms. A recent metagenomic analysis specifically compared the occurrence of PHB depolymerases in uncultivated microorganisms across different environments, including marine environments. This information has not previously been used to inform the smart design of materials to enable their degradation in the ocean. It has been previously established that there are few previously-cultured marine PHB-degrading bacteria, but demonstrated they can be rapidly isolated from coastal marine environments (Fig. 39B). These isolates will be used together with metagenomic data to develop living materials that will accelerate the degradation of PHB-based products on timescales of weeks to months.

[0374] Starting with the bioplastic PHB. these materials will be tested under real ocean conditions in Narragansett Bay, the New England shelf, and coastal CA. Embedding ocean-derived live cells or spores known to degrade PHB or applying them as an adhesive sticker will further speed the degradation of these materials.

[0375] Closed-system tuning and degradation testing. PHB formulations will evaluate the impact of additives such as nitrogen-containing compounds (e.g., amino acids) ondegradation rates informed by ongoing metagenomic analyses. Manufacturers need materials that conform to international degradability standards, but also need to know what degradation rates are under real environmental conditions. Because ASTM standards for plastic biodegradability in seawater are based on degradation of a fixed fraction of the original plastic mass (e.g., 80% degradation), testing can take months. An automated oxygen-based respiration system (an ‘autoBOD’) will be used to measure oxygen consumption during microbial respiration of our formulations (FIG. 40). It is anticipated that each cycle of formulation, respirometry, and analysis will take three months, allowing for up to four cycles in a year.13C-labeled PHB will be synthesized using13C-methanol in a bench-scale bioreactor, separated from the non-polymer cell mass, and compounded it various formulations. The labeled material will be used in closed system respirometry experiments with timepoints taken and analyzed for <5'I3C-DIC and c>13C of the microbial cell biomass using isotope ratio mass spectrometry.

[0376] Open-system degradation testing. The closed-system design poorly mimics the ocean, where materials are subject to constant exchange of seawater and. depending on the deployment location, photochemical transformations. To address this, experiments will be conducted in simulated environmental conditions prior to moving to the in-ocean experiments. First, flow-through microcosms using a seawater system will be used. Flow rates will be varied to determine the effect of flow conditions on degradation. Temperature can be controlled within a range of 4-25 °C. Respiration rates in the microcosms will be monitored with high-precision measurements of Ch:Ar using membrane-inlet mass spectrometry (MIMS). The MIMS can be set up in a flow-through mode to provide high- precision continuous monitoring throughout an experiment. Second, an Accelerated Weathering Tester will be used to simulate different UV exposures and seawater wetting prior to degradation testing. Previous work has shown that photochemical processes can strongly affect both the mechanism and rate of plastic degradation in seawater. Following each of these experiments, the degraded materials compared to non-degraded materials will be visualized using scanning electron microscopy to demonstrate that degradation of the polymer structure is occurring versus the polymer simply breaking into smaller microplastic pieces. Electron microscopy will also provide insight into the physical mechanisms of degradation, including how microbes are physically interacting with the degraded material.

[0377] Marine bacteria isolations. Seven marine bacterial strains capable of depolymerizing PHB in liquid and solid media were isolated (FIG. 39B).

[0378] Whole-cell living materials. Previous experiments indicated that marine PHB- degrading bacteria species Bacillus NRRL B-14911 and Marinobacter NK1 can be printed into living materials that degrade PHB (FIG. 39C). Newly isolated strains will also be evaluated for their ability to be printed into PHB-degrading living materials. Microbial viability7within 3D-printed bio-ink over time, physical stability of 3D-printed hydrogels, and shelf life of the materials during storage will be quantitatively tested in various mimicked marine habitats via determining colony forming units, autofluorescence, clear-zone analysis, or flow cytometry. If needed, improvements in viability or hydrogel integrity will be engineered through tuning the bio-ink matrix density or increasing nutrient availability, composition, or delivery post-printing. PHB degradation will be measured through changes in mass of rinsed and dried PHB materials, the clearance of PHB within agar plates, or molecular weight reduction measured by gel permeation chromatography. Efficiency of PHB degradation will be tuned via titrating and spatially patterning nutrient levels and bacteria concentrations, and also adjusting the concentration and chemistries of printing matrix polymers. 3D-printed living materials that degrade PHB in seawater with a range of degradation rates, ranging from days to months to years will be developed.

[0379] Delivery method prototyping. The above methods for living bioplastic production will be employed to develop prototypes, each with different degradation rates, shelf stability, and physical robustness. Prototypes will be engineered to have either immediate or triggerable degradation activity, with triggering achieved by purging seawater into an internal reservoir of the float, thus activating the living material (FIG. 41).

[0380] Stickers. Thin, flexible 3D-printed adhesive materials containing PHB- degrading bacteria that can be applied to target PHB materials before deployment. Stickers containing living bacteria can be used to immediately kick off degradation. Stickers containing freeze-dried bacteria or bacterial spores can be applied for triggered degradation by engineering oceanographic instruments with the ability to flood themselves with seawater to rehydrate and revive the embedded bacteria. Revival of freeze-dried bacteria will be enhanced where needed by the addition of cryoprotectants.

[0381] Inserts. PHB will be 3D-printed with hollow plugs or screws, which can be filled with PHB-degrading bacteria just prior to usage. The plugs or screws can be inserted into a target PHB item before deployment to accelerate degradation in applications where stickers would not be durable or adherent enough. If spores or freeze-dried bacteria are used in the plugs, they can be re-activated via seawater that enters via a small hole in the end of the Plug.

[0382] Co-printed PHB & bio-ink. A dual-head 3D printer that can extrude PHB and bio-ink in separate channels will be used. PHB-degrading bacteria will be deposited within the PHB structure as separate layers or alternating segments, to degrade the PHB from within. If spores or freeze-dried bacteria are co-printed, they will be re-activated upon contact with seawater during deployment.

[0383] Directly -embedded spores or enzymes. PHB will be mixed with bacterial spores during the injection-molding or 3D-printing process so that the spores are directly embedded within the bioplastic material. Durable, embedded spores will remain inert over extended timescales (months / years), until the PHB begins to physically develop cracks or fissures at end-of-life, which will allow seawater to access and revive the spores, further accelerating the PHB degradation and preventing the creation of microplastics. An alternative, complementary approach is to embed purified PHB-degrading enz mes within the bioplastic, which will begin to degrade the PHB immediately. The stability and activity of the enzy mes within the PHB will be enhanced via the use of inorganic protective carriers or via dispersion with heteropolymers.

[0384] To quantify the degradability of the prototypes in real ocean conditions, a series of experiments will be conducted in an estuary (at the sediment-water interface), on the coastal shelf, and in deep ocean water. In-ocean degradation will be quantified with both closed system and open flow. This work package will provide a mechanism to assess the materials developed, while also establishing a standardized method that could be used across diverse fields for measuring degradation of plastics in dark ocean environments.

[0385] Coastal degradation rates. For degradation experiments in water up to 300 m depth, an Automated Chamber System (Unisense) will be utilized, which enables completely automated in situ chamber incubations for deployments of up to 2 months. The in situ sampler will enable experiments that parallel those completed, but in real ocean conditions. The Unisense system consists of a main chamber (either sealed or open to ambient seawater) with multiple microsensors (O2, N2O, pH, H2S) mounted in the chamber lid. An automated array of syringe samplers takes in water from the main chamber at pre-programmed intervals. A series of ~one-week deployments will be conducted in estuary and coastal settings mimicking earlier experiments. The syringes will be pre-loaded with13C-labeled PHB developed. After seawater is drawn into the syringes from the open-flow main chamber, degradation rates can be inferred from O2 sensors attached to a subset of the syringes. The sampler will later be deployed with larger, rigid objects based on other prototypes. These will be placed inside segmented regions of the main chamber to observe degradability of solid,molded objects versus the standard powder. It is anticipated that over 2 months, some objects may only experience a small percentage of total mass loss. By creating objects with different shapes and textures (e.g., intentional pitting), strategies that alter degradability further will be developed. Before deployment, and following each recovery, degraded materials using scanning electron microscopy to assess degradation progress will be performed.

[0386] Deep-water degradation rates. Pressure-rated oxygen-based respirometers will be used to quantify degradability at the New England shelf break, a Santa Barbara Channel mooring, and two sites within CINMS. In the closed-system incubators, a polymerspecific respiration is obtained by subtracting the control from the rate of change of oxygen inside a PHB-containing reservoir. Following each of these ocean-based degradation experiments, the degraded materials will be visualized and compared to non-degraded materials using scanning electron microscopy, providing insight into the physical mechanisms of degradation under real ocean conditions.

[0387] Formulated PHB materials will be characterized prior to molding objects for marine deployment. Planned characterization methods to assess structural properties include tensile (ASTM D638) and flexural properties (ASTM D790) to assess strength, toughness, and flexibility of molded objects and impact resistance (ASTM D256) to assess resistance of molded objects to cracking on impact. The properties of melt flow rate (ASTM DI 238) and mold shrinkage (ASTM D955) will be assessed to understand material characteristics for injection molding prototype objects.

[0388] Additionally, material properties after marine deployment will be characterized in collaboration with NOAA-PMEL. Standardized dogbone test specimens will be deployed in various ocean environments and then will be recovered and tested for tensile properties (ASTM D638). Water absorption (ASTM D570) and thermal expansion (ASTM E831) will be assessed to understand density and / or dimensional changes to molded objects after marine deployment. PHB-based molded objects will also be characterized for molecular weight by gel permeation chromatography and imaged by scanning electron microscopy before and after marine deployment. Additionally, an environmental assessment of the breakdown of PHB-containing products will also be completed by performing toxicity testing; this will be accomplished by external laboratories.EXAMPLE 3

[0389] FIG. 37 shows surface erosion of solid PHB discs incubated with Bacillus NRRL B- 14911 biostickers for 28 days, or negative control incubated without biostickers,assayed by scanning electron microscopy. Solid PHB discs were incubated and covered by a Bacillus NRRL 14911 biosticker for 28 days. Then, the bio sticker was removed and the surface of the PHB disc was analyzed by SEM. The discs that were covered by the bio sticker showed surface erosion indicated by the rough surface, compared to the smooth surfaces of the negative control discs.

[0390] Initially, Bacillus NRRL 14911 is inoculated into a marine broth medium until it attains the stationary phase. Following this, 1mm PHB thin films are immersed in the bacterial bioink for degradation over 28 days.

[0391] Upon completion of incubation, PHB films were harvested and thoroughly rinsed to dislodge residual bacterial cells. The films were then freeze-dried, careful to eliminate moisture while maintaining film integrity. Post-degradation, PHB films were collected by centrifugation and stored at 4 °C overnight for SEM preparation.

[0392] For SEM analysis, freeze-dried films were securely positioned onto an SEM stub. As needed, films were sputter-coated with gold powder (5 mA for 300 seconds) to enhance conductivity. SEM parameters were configured to an accelerating voltage of 5 kV, and backscattered electron images of the most evidently degraded regions are captured at varying magnifications.

[0393] Subsequent SEM analysis involved meticulous interpretation of images to ascertain PHB thin film surface changes due to bacterial degradation. Significant surface features indicative of the degradation process were emphasized, and image processing software is utilized for quantitative evaluation.

[0394] PHB flakes were sterilized with a 75% ethanol solution for 15 minutes and rinsed thrice with PBS to remove excess ethanol. The samples underwent dehydration through a graded ethanol series, with optional sodium hypochlorite solution treatment followed by PBS washing, based on sample needs. Flakes are freeze-dried or air-dried at room temperature, as appropriate.

[0395] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A method for degrading a polyhydroxyalkanoate polymer comprising contacting the polyhydroxyalkanoate polymer with an article, wherein after contacting the degradation of the polyhydroxyalkanoate polymer is initiated.

2. The method according to claim 1, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3- hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate-co-3- hy dr oxy hexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or combinations thereof.

3. The method according to claim 1, wherein the polyhydroxyalkanoate polymer comprises or is a copolymer of 3 -hydroxy butyrate.

4. A method according to claim 1, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).

5. The method according to claim 1, wherein the article is 3D printed or injection molded.

6. The method according claim 1. wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, or insert.

7. The method according to claim 1, wherein the article comprises bacteria.

8. The method according to claim 7, wherein at least a portion of the bacteria are of the genus Bacillus, Nocardioides, Comamonas , Marinobacter, Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia, Microbulbifer , Alteromonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalter omonas, Arenicella, or Marisediminitalea, or a combination thereof.

9. The method according to claim 8, wherein the bacteria are Bacillus NRRL-14911 , Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1.Shewanella sp. JKCM-AJ-6, la. Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp., Ralstonia sp. , Marinobacter maritimus, Microbulbifer sp. , Alteromonas sp., Bowmanella sp., Alcanivorax sp., Vibrio diabolicus, Pseudoalteromonas sp..Arenicella sp. , Marisediminitalea aggregate. Pseudoalteromonas Hpolytica A. , or a combination thereof.

10. The method according to claim 8, wherein the bacteria are of the genus Bacillus.

11. The method according to claim 9. wherein the bacteria are Bacillus NRRL-14911.

12. The method according to claim 7, wherein the bacteria are sporulated prior to incorporation into the article.

13. The method according to claim 1. wherein the article comprises one or more enzymes.

14. The method according to claim 13, wherein at least a portion of the one or more enzymes are a polyhydroxy alkanoate hydrolase, PHB depolymerase, or PHA depolymerase, or combinations thereof.

15. The method according to claim 14, wherein the polyhydroxy alkanoate hydrolase is a PHB monomer hydrolase.

16. The method according to claim 15, wherein the PHB monomer hydrolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.

17. The method according to claim 16, wherein the PHB monomer hydrolase is PhaZ.

18. The method according to claim 1, wherein polyhydroxyalkanoate polymer is contacted with water prior to, during, and / or after contacting the article.

19. The method according to claim 18, wherein the water is salt water.

20. The method according to claim 18, wherein the water is a body of water.

21. The method according to claim 20, wherein the body of water is a lake, sea, bay, ocean, pond, river, stream, tributary', lagoon, creek, delta, estuary, drainage basin, reserv oir, gulf, puddle, canal, wetland, fjord, strait, wastewater, soil, sludge, mud, or the like.

22. An article suitable for degradation of a polyhydroxyalkanoate polymer, wherein the article comprises one or more bacteria and / or one or more enzymes.

23. The article according to claim 22, wherein the article is disposed on a surface of or in contact with the polyhydroxyalkanoate polymer.

24. The article according to claim 23, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybuty rate (P4HB), poly-3- hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate-co-3- hy dr oxy hexanoate) (PHBHHx). poly-3-hydroxybutyrate-co-valerate (PHBV), polyhydroxybutyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD). or combinations thereof.

25. The article according to claim 23, wherein the polyhydroxyalkanoate polymer comprises or is a copolymer of 3-hydroxybutyrate.

26. The article according to claim 24, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).

27. The article according to claim 22, wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, or insert.

28. The article according to claim 22, wherein the article is a sticker.

29. The article according to claim 28, wherein the sticker is a hydrogel.

30. The article according to claim 29. wherein the sticker further comprises gelatin methacryloyl, pluronic F127, collagen, cellulose, gelatin, fibrin ogen / fibrin, gellan gum, silk, hyaluronic acid, dextran, agarose, alginate, chitosan, polyvinyl alcohol, polyethylene glycol, polycaprolactone, poly(lactic-co-glycolic acid), or combinations thereof.

31. The article according to claim 28. wherein the sticker further comprises alginate and / or calcium chloride.

32. The article according to claim 31, wherein calcium chloride has a concentration of 0.05 to 0.5 M.

33. The article according to claim 29, wherein the sticker has an elastic modulus of 1 kPa to 10 MPa.

34. The article according to claim 22. wherein the article is a screw.

35. The article according to claim 22, wherein at least a portion of the bacteria of the one or more bacteria are of the genus Bacillus, Nocardioides, Comamonas, Marinobacter, Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia. Microbulbifer.Alter omonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas, Arenicella, or Marisediminitalea, or any combination thereof.

36. The article according to claim 22, wherein at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6,la, Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp., Ralstonia sp. , Marinobacter maritimus, Microbulbifer sp.,Alteromonas sp., Bowmanella sp., Alcanivorax sp. , Vibrio diabolicus, Pseudoalteromonas sp.. Arenicella sp.. Marisediminitalea aggregata, Pseudoalteromonas Hpolytica A, or any combination thereof.

37. The article according to claim 35, wherein the bacteria are of the genus Bacillus.

38. The article according to claim 36, wherein the bacteria are Bacillus NRRL-14911.

39. The article according to claim 22. wherein the one or more bacteria are sporulated prior to incorporation into the article.

40. The article according claim 22, wherein the article is 3D printed.

41. The article according to claim 22. wherein the one or more enzymes are a polyhydroxyalkanoate hydrolase, PHB depolymerase, or PHA depolymerase, or any combination thereof.

42. The article according to claim 41. wherein the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase.

43. The article according to claim 42, wherein the PHB monomer hy drolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.

44. The article according to claim 43, wherein the PHB monomer hydrolase is PhaZ.

45. The article according to claim 44, wherein the article further comprises a polyhydroxyalkanoate polymer.

46. The article according to claim 44, wherein the article further comprises an exterior layer comprising a polyhydroxyalkanoate polymer.

47. The article according to claim 45, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).

48. An article comprising a polyhydroxyalkanoate polymer and one or more bacteria and / or one or more enzymes embedded therein.

49. The article according to claim 48, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3- hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), poly-3-hydroxybutyrate-co-valerate (PHBV),polyhydroxy butyrate-co-hexanoate (PHBH), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx), Poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxydecanoate) (PHD), poly(3-hydroxydodecanoate) (PHDD), poly(3-hydroxyoctanoate-co-3- hydroxydecanaote) (PHO-co-PHD), or any combination thereof.

50. The article according to claim 48, wherein the polyhydroxyalkanoate polymer comprises or is a copolymer of 3-hydroxybutyrate.

51. The article according to claim 48, wherein the polyhydroxyalkanoate polymer comprises or is poly-3-hydroxybutyrate (PHB).

52. The article according to claim 48, wherein the article is a sticker, screw, plug, dowel, patterned structure, patch, fastener, rivet, nail, fixture, attachment, film, sheet, pellet, bead, fiber, thread, foam, coating, paint, granule, or insert.

53. The article according to claim 52. wherein the article is a screw.

54. The article according to claim 48, wherein at least a portion of the bacteria of the one or more bacteria are of the genus Bacillus, Nocardioides, Comamonas, Marinobacter, Shewanella, Pseudomonas, Paucimonas, Cupriavidus, Ralstonia. Microbulbifer.Alter omonas, Bowmanella, Alcanivorax, Vibrio, Pseudoalteromonas, Arenicella, or Marisediminitalea, or any combination thereof.

55. The article according to claim 48, wherein at least a portion of the bacteria are Bacillus NRRL-14911, Nocardioides sp. BAA-499, Comamonas testosterone, Marinobacter sp. NK-1, Shewanella sp. JKCM-AJ-6, la, Pseudomonas stutzeri YM1006, Pseudomonas sp. DSDY0501, Paucimonas lemoignei, Marinobacter algicola DG893, Cupriavidus sp., Ralstonia sp. , Marinobacter maritimus, Microbulbifer sp., Alteromonas sp., Bowmanella sp., Alcanivorax sp. , Vibrio diabolicus, Pseudoalteromonas sp.. Arenicella sp.. Marisediminitalea aggregata, and Pseudoalteromonas lipolytica A, or any combination thereof.

56. The article according to claim 54, wherein the bacteria are of the genus Bacillus.

57. The article according to claim 55, wherein the bacteria are Bacillus NRRL-14911.

58. The article according to claim 48. wherein the one or more bacteria are sporulated prior to incorporation into the article.

59. The article according to claim 48, wherein the article is 3D printed.

60. The article according to claim 48. wherein the one or more enzymes are a polyhydroxyalkanoate hydrolase, PHB depolymerase, or PHA depolymerase, or combinations thereof.

61. The article according to claim 60. wherein the polyhydroxyalkanoate hydrolase is a PHB monomer hydrolase.

62. The article according to claim 61, wherein the PHB monomer hy drolase is PhaZ, PhaZ2, PhaZ5, PhaZ7, or a combination thereof.

63. The article according to claim 62, wherein the PHB monomer hydrolase is PhaZ.

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