Method for treatment of a post-consumer product, and system for carrying out the method

Description

68 METHOD FOR TREATING A POST-CONSUMER PRODUCT, AND SYSTEM FOR IMPLEMENTING THE METHOD - FUNDAMENTALS

[001] It is estimated that more than 300 million metric tons of petroleum-based polymers are produced each year, with global production continuing to increase. A significant portion of these polymers is used to produce single-use products such as plastic beverage bottles, straws, packaging, and personal hygiene products. Most of these plastic products are discarded and do not enter the recycling stream. As the global epidemic of disposable plastics worsens, it becomes crucial to identify fully renewable plastics and develop methods and materials that enable the industrial processing of renewable plastics.

[002] Biodegradable polymers produced from renewable resources (also called “biopolymers”) hold great promise for reducing the global accumulation of petroleum-based plastics in the environment. One such class of biopolymers is polyhydroxyalkanoates (PHAs). Much work has been done on the PHA family, most notably polyhydroxybutyrate (PHB) polymers, including poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), and their copolymers. Of particular advantage, PHA exhibits thermoplastic properties that are very similar to some petroleum-based polymers and therefore represent viable substitutes for petroleum-based polymers such as polypropylene and polyethylene.

[003] PHAs are naturally produced in many bacterial, fungal, and archaeal strains, including Azotobacter, Ralstonia, Burkholderia, Protomonas, Bacillus, and Schlegelella for use as an energy dissipator. The production of PHA polymers involves a mechanism Petition 870240024923, dated 03 / 22 / 2024, page 7 / 96 / 68 three-step enzymatic process that begins with acetyl coenzyme A (acetyl-CoA). As shown in FIG. 1, the first step is the catalysis of acetyl-CoA by PhaA (a β-ketothiolase) to form acetoacetyl-CoA. This, in turn, is converted in an NADP-dependent reaction to R-3-hydroxybutyryl-CoA by the enzyme PhaB (a β-ketoacyl-CoA reductase). The final step is catalyzed by PhaC (a PHB synthase) and is the polymerization of the monomer to form PHA, for example, PHB as shown in FIG. 1. Biosynthesized polyhydroxyalkanoates accumulate in the bacterial cell as large molecular weight granules and can represent about 60% to about 90% of the cell dry mass.

[004] In nature, to recover the energy stored in the polymer, biodegradation is carried out by a PHA depolymerase (PHADase) that is expressed by the organism and that degrades the polymer back to the hydroxyalkanoate (HA) monomer and small PHA oligomers, as shown in FIG. 2 for the degradation of PHB to form HB monomer. In nature, the monomer is then degraded to provide a source of carbon and energy for the organism. Unfortunately, although the monomer can be fully degraded environmentally, it is a dead end from an industrial point of view, since it cannot be directly repolymerized by any known chemical process.

[005] Although biopolymers are capable of biodegrading significantly faster than petroleum-based polymers, biopolymers still remain in landfills or soil for significant periods of time once discarded. Thus, there is a need for systems and processes that can fully recycle biopolymers. A truly circular use of a bioplastic that is capable of breaking down the polymer into monomeric units and then using that monomer as a carbon source to create a new polymer would bring significant advances in waste disposal processes. It would be an additional benefit if the Petition 870240024923, dated 03 / 22 / 2024, page 8 / 96 / 68 recycled and reformed biopolymer would be suitable for use in consumer products and industrial processes. Specifically, it would be economically and environmentally advantageous to use post-consumer HA monomer obtained according to a post-consumer recycling process as a carbon source for microorganisms capable of producing PHA suitable for reuse in the formation of new products. A process and system that incorporates both depolymerization and metabolic polymer formation aspects would be particularly beneficial and would provide a truly cyclical biopolymer utilization approach. SUMMARY

[006] In general, this disclosure is directed to methods and systems for the degradation of PHA polymers and the production of new PHA polymers through the metabolic use of the degradation products. The PHA polymers fed into a process can be components of post-consumer materials, such as post-consumer personal care products, food industry products, packaging, post-consumer medical products, post-consumer industrial products, and other articles containing recyclable PHA. This disclosure is directed to a truly cyclical process that can be used for single-system biodegradation combined with the formation of new biopolymers in small or large environments.

[007] A process may involve contacting a post-consumer product containing a PHA with a PHA-Ase. Upon contact, the HA monomer may be released from the PHA. A process may also involve providing the post-consumer HA monomer thus obtained as a carbon source to a microorganism capable of metabolizing HA. The microorganism is one that can produce PHA, and the HA monomer may be provided to the microorganism under growth conditions configured to stimulate the metabolic production of PHA by the microorganism. For example, the monomer Petition 870240024923, dated 03 / 22 / 2024, page 9 / 96 / 68 of HA can be supplied to the microorganism under stressful metabolic conditions, for example, with HA as the only available carbon source or with any other carbon source in low concentration and / or with low or no presence of other nutrients, such as nitrogen and / or phosphorus.

[008] In one aspect, PHA and HA can be PHB and HB. In another aspect, one or both of the depolymerization component and the microorganism monomer metabolism component can be carried out under extreme conditions, such as extreme temperature, pressure, salt content, etc., or combinations thereof. In such an embodiment, a procedure can decontaminate the raw material as well as provide new biopolymer. Furthermore, the two components of a process can be carried out under the same conditions as each other or under conditions that differ in some aspect from each other (e.g., temperature, pressure, salt content, etc.). The depolymerization of a post-consumer polymer and the utilization of the produced monomer as a carbon source for microorganism metabolism can be carried out simultaneously with each other (e.g., in parallel bioreactors), immediately sequentially with each other (e.g., direct feeding between bioreactors in series), or at different times and / or locations.In some applications, these two components of a process can be carried out together in a single bioreactor.

[009] The depolymerization of a post-consumer product to obtain a post-consumer HA monomer can be carried out according to an enzymatic process that uses one or more natural PHADases, one or more modified PHADases, or combinations thereof. For example, a post-consumer product can be contacted with a purified enzyme to provide the HA monomer, and the purified enzyme can be a naturally expressed enzyme or a modified purified enzyme that includes one or more modifications compared to an expressed enzyme. Petition 870240024923, dated 03 / 22 / 2024, page 10 / 96 / 68 naturally. In some respects, a post-consumer product may come into contact with a microorganism that produces an enzyme, and the microorganism may be one that naturally produces the enzyme, a microorganism that has been transformed to produce the enzyme, or a combination thereof. A microorganism transformed to produce a modified enzyme used in a process may be a microorganism that naturally produces a PHADase (the unmodified PHADase or a different PHADase) or a microorganism that does not naturally produce a PHADase.

[0010] In some aspects, a process may incorporate an enzymatic depolymerization component that may utilize a PHADase produced by a microorganism (or a modified version thereof), and the same microorganism may utilize the HA monomer thus produced as a carbon source in the production of metabolic polymer. The PHADase of such a process may include only enzyme produced by (or derived from) a microorganism that metabolizes HA. For example, a process may involve feeding a post-consumption feed to a single microorganism capable of expressing a PHADase to depolymerize the PHA from a feed and subsequently metabolize the released HA to produce new PHA under the process conditions of a single bioreactor system. In some embodiments, the PHADase of such a process may include enzyme produced by (or derived from) the microorganism that metabolizes HA, in addition to additional PHADase produced by (or derived from) one or more additional microorganisms.As used herein, the term “derived” in relation to an enzyme derived from a microorganism is intended to refer to a natural enzyme that has been purified after expression from a microorganism that naturally produces the enzyme, to a natural enzyme (purified or not) that has been expressed from a microorganism that has been transformed to produce the enzyme, as well as a modified enzyme (purified or not) that includes one or more amino acid modifications compared to an enzyme. Petition 870240024923, dated 03 / 22 / 2024, p. 11 / 96 / 68 natural.

[0011] In some respects, co-cultures of microorganisms may be used which may include one or more microorganisms that can produce a PHADase for depolymerization of a post-consumer product together with one or more microorganisms that can utilize the monomer thus produced as a metabolic carbon source and that can produce a PHA under the culture conditions. Co-cultures may utilize natural microorganisms, transformed microorganisms or combinations thereof.

[0012] Other features and aspects of this publication are discussed in more detail below. BRIEF DESCRIPTION OF THE FIGURES

[0013] A full and informative disclosure of this disclosure is set out, more particularly, in the remainder of the descriptive report, including reference to the attached figures, in which: FIG. 1 illustrates a natural metabolic reaction process to form polyhydroxybutyrate (PHB) as it is known in the art.

[0014] FIG. 2 illustrates a natural metabolic reaction process for PHB degradation as known in the art.

[0015] FIG. 3 provides the sequence (SEQ ID NO: 1) of a purified and cleaved recombinant Lysobacter enzymogenes PHBDase as it can be used in the disclosed methods.

[0016] FIG. 4 schematically illustrates one embodiment of a bioreactor system that can be used to carry out a process as described here.

[0017] FIG. 5 schematically illustrates another embodiment of a bioreactor system that can be used in carrying out a process as disclosed here.

[0018] FIG. 6 graphically illustrates the formation of the HB monomer. Petition 870240024923, dated 03 / 22 / 2024, p. 12 / 96 / 68 following the depolymerization of a PHB polymer using a purified enzyme as described in more detail in this document.

[0019] FIG. 7 graphically illustrates the growth of Lysobacter enzymogenes bacteria as a function of time (closed circles) and the simultaneous reduction of HB in the flask (open circles) using HB produced by the depolymerization of a PHB polymer using a purified enzyme.

[0020] FIG. 8 graphically illustrates the growth of L. enzymogenes bacteria as a function of time (closed circles) and the simultaneous concentration of HB in the flask (open circles) under simultaneous depolymerization and polymerization conditions using a PHBDase added together with the presence of L. enzymogenes bacteria.

[0021] FIG. 9 graphically illustrates the growth of L. enzymogenes bacteria as a function of time (closed circles) and the simultaneous concentration of HB in the flask (open circles) during depolymerization and polymerization after the use of the bacteria to produce the depolymerase.

[0022] FIG. 10 graphically illustrates the growth of a mixed culture of P. fluorescent and L. enzymogenes bacteria as a function of time (closed circles) and the simultaneous concentration of HB in the flask (open circles) during depolymerization and polymerization after the use of the bacteria to produce the depolymerase.

[0023] FIG. 11 graphically illustrates the growth of a mixed culture including E. coli expressing P. geniculate PHBDase and L. enzymogenes bacteria as a function of time (closed circles) and the simultaneous concentration of HB in the flask (open circles).

[0024] FIG. 12 graphically illustrates the mass of PHB extracted from L. enzymogenes in each of the five different methods performed and described in the examples section of this document.

[0025] FIG. 13 graphically overlays the formation of HB through the Petition 870240024923, dated 03 / 22 / 2024, page 13 / 96 / 68 depolymerization of PHB formed in each of the five different methods performed and described in the example section of this document.

[0026] The repeated use of reference characters in this descriptive report and in the drawings is intended to represent features or elements that are the same as, or analogous to, the present invention. DETAILED DESCRIPTION

[0027] Reference will now be made in detail to various embodiments of the disclosed subject matter, one or more examples of which are presented below. Each embodiment is provided by way of explanation of the subject matter, not as a limitation thereof. Indeed, it will be evident to those skilled in the art that various modifications and variations can be made to the present description without departing from the scope or spirit of the subject matter. For example, features illustrated or described as part of one embodiment can be used in another embodiment to produce yet another embodiment.

[0028] This disclosure is directed to a truly circular use of a polyhydroxyalkanoate (PHA) that includes the degradation / depolymerization of a PHA polymer to release the hydroxyalkanoate (HA) monomer and the use of the released monomer as a metabolic carbon source for a microorganism capable of producing new PHA polymer. While by no means limited to this, in one embodiment, the method can be directed to the treatment of post-consumer products incorporating polyhydroxybutyrate (PHB).

[0029] The methods provide a route for the recycling of post-consumer PHA in conjunction with the production of new industrially / commercially viable PHA. The methods incorporate an enzyme-based approach to depolymerization in conjunction with the exploitation of metabolic processes to utilize the depolymerization product to produce new polymer. Thus, a fully recycled use of a biopolymer can be provided. The Petition 870240024923, dated 03 / 22 / 2024, page 14 / 96 / 68. Newly produced polymers are suitable for producing all different types of products, including single-use products such as, without limitation, packaging, straws, cups, bottles, shopping bags, cutlery, trays, and personal hygiene products such as personal hygiene clothing (e.g., diapers, children's training pants, disposable swim trunks, feminine hygiene products, adult incontinence products), tampon dispensers, medical supplies, etc. Post-consumer products for use as a feedstock in disclosed processes may be from any source (e.g., commercial, industrial, medical, etc.) and may include PHA in conjunction with other components, including other polymers and / or other non-polymeric materials, waste, labeling materials, etc.The use and reuse of biopolymers through a fully circular use of polymer components to replace petroleum-based polymers will provide significant advances in creating a sustainable economy.

[0030] Any product including a polymer incorporating a recyclable linear polyester PHA as produced in nature by bacterial fermentation of sugar or lipids can be processed according to this disclosure. Similarly, PHA produced by the disclosed methods can be used in the formation of any PHA-based polymer as known in the art. As is known, more than 100 different monomers can be combined within this family to produce materials. Examples of monomeric units that can be incorporated into a PHA, as well as those that can be degraded and / or formed according to the disclosed methods, may include, without limitation, 2-hydroxybutyrate, glycolic acid, 3-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate, 3-hydroxydecanoate, 3-hydroxydodecanoate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, and Petition 870240024923, dated 03 / 22 / 2024, page 15 / 96 / 68 6-hydroxyhexanoate.

[0031] A polymer can be a homopolymer or a copolymer. Examples of PHA homopolymers that can be processed and / or produced according to the disclosed methods may include, without limitation, poly 3-hydroxyalkanoates (e.g., poly 3-hydroxypropionate (PHP), poly 3-hydroxybutyrate (PHB), poly 3-hydroxyvalerate (PHV), poly 3-hydroxyhexonoate (PHH), poly 3-hydroxyoctanoate (PHO), poly 3-hydroxydecanoate (PHD) and poly 3-hydroxy-5-phenylvalerate (PHPV)), poly 4-hydroxyalkanoates (e.g., poly 4-hydroxybutyrate (hereinafter referred to as PHB) and poly 4-hydroxyvalerate (hereinafter referred to as PHV)), or poly 5-hydroxyalkanoates (e.g., poly 5-hydroxyvalerate (hereinafter referred to as PHV)). A common type of PHA covered in this disclosure is Poly(3-hydroxybutyrate) (PHB).

[0032] In certain respects, a post-consumer product degraded according to disclosed methods and / or formed from PHA produced according to disclosed methods may include a PHA copolymer that may include only PHA components or PHA components in combination with other types of polymeric components. Examples of PHA copolymers include, without limitation, poly 3-hydroxybutyrate-co-3-hydroxypropionate (hereinafter referred to as PHB3HP), poly 3-hydroxybutyrate-co-4-hydroxybutyrate (hereinafter referred to as P3HB4HB), poly 3-hydroxybutyrate-co-4-hydroxyvalerate (hereinafter referred to as PHB4HV), poly 3-hydroxybutyrate-co-3-hydroxyvalerate (hereinafter referred to as PHB3HV), poly 3-hydroxybutyrate-co-3-hydroxyhexanoate (hereinafter referred to as PHB3HH) and poly 3-hydroxybutyrate-co-5-hydroxyvalerate (hereinafter referred to as PHB5HV). Depolymerization

[0033] Regardless of the specific PHA(s) included in a post-consumer product, a process may include depolymerization. Petition 870240024923, dated 03 / 22 / 2024, page 16 / 96 / 68 enzymatic breakdown of PHA from post-consumer product. The present methods may utilize one or more depolymerase enzymes alone (e.g., purified PHADase), one or more microorganisms that can express one or more depolymerase enzymes, or a combination of one or more microorganisms expressing PHADase with one or more added (purified) PHADases to break down the PHA from a post-consumer product and provide the HA monomer, which can then be used as a carbon source for a microorganism in the production of newly formed polymer.

[0034] Microorganisms that express PHADase may include those that naturally express the enzyme, as well as those that have been modified to express the enzyme, which, similarly, may be a naturally occurring enzyme or a modified enzyme. When using one or more microorganisms that naturally express the enzymes discussed, or that have been modified to express the desired enzymes, the depolymerization component of a process may be referred to as a bacteria-based enzymatic component of a process, rather than an enzyme-based component.

[0035] The microorganism expressing PHADase or PAHDase used to degrade PHA from a post-consumer product is not particularly limited. In one embodiment, the enzyme that degrades a PHA may be a naturally occurring enzyme. For example, in one aspect, the microorganism, or enzyme expressed from it, incorporated into a depolymerization component of a process may include a microorganism from a bacterial genus including, but not limited to, Halomonas, Lihuaxuella, Lysobacter, Alteromonas, Arthrobacter, Azospirillum, Empedobacter, Desulfovibrio, Halobacillus, Halobacteriovorax, Haloechinothrix, Halomarin, Halorussus, Haloterrigena, Isoptericola, Marinobacter, Methyloligella, Micromonospora, Natronococcus, Nocardiopsis, Paracoccus, Roseivivax, Saccharomonospora, Petition 870240024923, dated 03 / 22 / 2024, p. 17 / 96 / 68 Shewanella, Alicyclobacillus, Natranaerobius, Halobacteriaceae, Hyphomonas, Amycolatopsis, Georgenia, Acidothermus and Thermobifida, as well as any combination thereof.

[0036] As an example, one or more of the following organisms may be used in accordance with this disclosure to provide enzymes of the present disclosure (either as the microorganism itself or as the purified enzyme): Lysobacter aestuarii, Lysobacter antibioticus, Lysobacter bugurensis, Lysobacter capsica, Lysobacter enzymogenes, Lysobacter lacus, Lysobacter lycopersici, Lysobacter maris, Lysobacter niastensis, Lysobacter profundi, Lysobacter sp., Lysobacter sp. A03, Lysobacter sp. cf310, Lysobacter sp. H21R20, Lysobacter sp. H21R4, Lysobacter sp. H23M41, Lysobacter sp. R19, Lysobacter sp. Root604, Lysobacter sp. Root690, Lysobacter sp. Root916, Lysobacter sp. Root983, Lysobacter sp.TY2-98, Lysobacter spongiae, Lysobacter spongiicola, Lysobacter, Lysobacter alkalisoli, Lysobacter arseniciresistens, Lysobacter daejeonensis, Lysobacter dokdonensis, Lysobacter enzymogenes, Lysobacter enzymogenes, Lysobacter gilvus, Lysobacter gummosus, Lysobacter maris, Lysobacter oculi, Lysobacter panacisoli, Lysobacter penaei, Lysobacter prati, Lysobacter psychrotolerans, Lysobacter pythonis, Lysobacter ruishenii, Lysobacter segetis, Lysobacter silvestris, Lysobacter silvisoli, Lysobacter soli, Lysobacter sp., Lysobacter sp. 17J7-1, Lysobacter sp. Alg18-2.2, Lysobacter sp. Cm-3-T8, Lysobacter sp. H23M47, Lysobacter sp. HDW10, Lysobacter sp. II4, Lysobacter sp. N42, Lysobacter sp. OAE881, Lysobacter sp. Raíz494, Lysobacter sp. URHA0019, Lysobacter sp. WF-2, Lysobacter sp.yr284, Lysobacter tabacisoli, Lysobacter telluris, Lysobacter tolerans, Lysobacter tolerans, Lysobacter xinjiangensis, unclassified Lysobacter, Aliivibrio finisterrensis, Aliivibrio fischeri, Aliivibrio sifiae, Aliivibrio sp., Aliivibrio sp. 1S128, Aliivibrio sp. EL58, Aliivibrio sp. SR45-2, Caballeronia arvi, Caballeronia calidae, Caballeronia hypogeia, Caballeronia insecticola,. Petition 870240024923, dated 22 / 03 / 2024, p. 18 / 96 / 68 Caballeronia pedi, Caballeronia terrestris, Dokdonella koreensis, Dyella caseinilytica, Dyella choica, Dyella dinghuensis, Dyella flava, Dyella jiangningensis, Dyella kyungheensis, Dyella mobilis, Dyella monticola, Dyella nitratireducens, Dyella psychrodurans, Dyella soli, Dyella solisilvae, Dyella sp. 7MK23, Dyella sp. ASV21, Dyella sp. ASV24, Dyella sp. C11, Dyella sp. C9, Dyella sp. DHC06, Dyella sp. EPa41, Dyella sp. G9, Dyella sp. M7H15-1, Dyella sp. M7H15-1, Dyella sp. OK004, Dyella sp. S184, Dyella sp. SG562, Dyella sp. SG609, Dyella sp. YR388, Dyella tabacisoli, Fluoribacter bozemanae, Fluoribacter dumoffii NY 23, Fluoribacter gormanii, Microscilla marina, Pseudomonas aeruginosa, Pseudomonas thermotolerans, Pseudomonas mediterranea, Psychrobacter sp., Psychromonas sp. MB-3u-54, Psychromonas sp. psych-6C06, Psychromonas sp. RZ22, Psychromonas sp.Urea-02u-13, Rhodanobacter denitrificans, Rhodanobacter fulvus, Rhodanobacter glycinis, Rhodanobacter lindaniclasticus, Rhodanobacter panaciterrae, Rhodanobacter sp. 7MK24, Rhodanobacter sp. A1T4, Rhodanobacter sp. B04, Rhodanobacter sp. B05, Rhodanobacter sp. C01, Rhodanobacter sp. C03, Rhodanobacter sp. C05, Rhodanobacter sp. C06, Rhodanobacter sp. DHB23, Rhodanobacter sp. DHG33, Rhodanobacter sp. L36, Rhodanobacter sp. MP1X3, Rhodanobacter sp. OK091, Rhodanobacter sp. OR444, Rhodanobacter sp. PCA2, Rhodanobacter sp. Raíz480, Rhodanobacter sp. Raíz627, Rhodanobacter sp. Raíz627, Rhodanobacter sp. SCN 67-45, Rhodanobacter sp. SCN 68-63, Rhodanobacter sp. Soil772, Rhodanobacter sp. T12-5, Rhodanobacter sp. TND4EH1, Rhodanobacter sp. TND4FH1, Rhodanobacter spathiphylli, Rhodanobacter thiooxydans, Stenotrophomonas chelatiphaga, Stenotrophomonas maltophilia, Stenotrophomonas panacihumi, Stenotrophomonas pavanii, Stenotrophomonas rhizophila, Stenotrophomonas sp. DDT-1, Stenotrophomonas sp.RIT309, Stenotrophomonas sp. SKA14, Vibrio aestuarianus, Vibrio antiquaries, Vibrio aquaticus, Vibrio. Petition 870240024923, dated 22 / 03 / 2024, p. 19 / 96 / 68 tasmaniensis, Xanthomonadales bacterium, Xanthomonas albilineans, Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas bromi, Xanthomonas campestris, Xanthomonas cannabis, Xanthomonas citri, Xanthomonas euvesicatoria, Xanthomonas fragariae, Xanthomonas hortorum,, Xanthomonas hyacinthi, Xanthomonas rice, Xanthomonas phaseoli, Xanthomonas pisi, Xanthomonas sugar, Xanthomonas sp. Leaf131, Xanthomonas sp. NCPPB 1128, Xanthomonas translucens, Xanthomonas vasicola, Xanthomonas vesicatoria, or any combination of these. It should be understood that the following list is only exemplary.

[0037] In some embodiments, a method may utilize extremophilic depolymerase enzymes (also called extremozymes) that can function in extreme environmental conditions. As used herein, and as will be discussed in greater detail below, extremophilic refers to a microorganism, or an enzyme obtained from an extremophilic microorganism, that can tolerate one or more hostile environments, such as high salt content, high temperature, high pressure, acid, basic or similar.

[0038] Enzymes derived from extremophilic microorganisms or the microorganisms themselves can be used under selected environmental conditions during a depolymerization component of a process to improve any aspect of the process, for example, production kinetics, production efficiency, production purity, etc. In some embodiments, the use of extremophilic enzymes can reduce / eliminate the presence of contaminants in processed materials, such as, for example, post-consumer diapers, since processing at this stage can be carried out under extreme conditions (high temperature, high salt concentration, high pressures, etc.) in which the contaminant can be degraded. Eliminating or reducing contaminants in materials can reduce the risk to process operators and reduce the risk of contamination of Petition 870240024923, dated 22 / 03 / 2024, p. 20 / 96 / 68 process equipment, as well as allowing subsequent processing to be carried out without additional sterilization procedures prior to further downstream product treatment (e.g., polymer culture, recovery and purification, etc.). Thus, this disclosure provides an efficient way to simultaneously sterilize post-consumer products, as well as depolymerize a PHA from the materials and form a new PHA biopolymer.

[0039] Contaminants that may be present in post-consumer products that can be rendered non-pathogenic by the use of an extremozyme (or microorganism expressing the extremozyme) under selected environmental conditions may include mesophilic pathogens such as, without limitation, viruses, bacteria, fungi and protozoa, methods disclosed. As used herein, the terms “mesophile” and “mesophilic” refer to organisms that exist naturally in environmental conditions in which humans generally coexist with the organism, including temperatures close to human body temperature (e.g., from about 20°C to about 45°C), a saline content in water of about 5 to about 18 parts per thousand (also referred to as mesohaline), pressure of about one atmosphere (e.g., from about 20 kPa to about 110 kPa), and pH close to neutral (e.g., from about pH 5 to about pH 8.5, also referred to as neutrophils or neutrophilic).Typical bacterial pathogens may include those commonly found in human feces, such as, but not limited to, those of the genus Streptococcus, Bifidobacterum, Lactobacillus, Staphylococcus, Clostridium, Enterobacteriaceae, or Bacteroides.

[0040] The microorganism or collection of microorganisms for use may be selected not only to secrete a specific PHA depolymerase, but may also be used based on the environmental conditions in which the post-consumer product exists (e.g., in a waste disposal site) or that a bioreactor contains. For example, the post-consumer material may be Petition 870240024923, dated 03 / 22 / 2024, page 21 / 96 / 68 contained in an environment or a bioreactor can be designed to operate in an environment that can be defined by a specific temperature range, salinity levels, oxygen levels, etc., or combinations thereof. Such specific environmental variables can be combined with one or more microorganisms, such as bacteria and / or archaea, best suited to the specific environment. For example, the microorganism can be selected based on the salt or temperature tolerance of the selected microorganism, or by modifying a microorganism having the desired salt or temperature tolerance to express an appropriate depolymerase enzyme. For example, when a microorganism has a specific salt tolerance, the expression of an appropriate depolymerase enzyme and therefore the degradation of PHA can be further increased and / or delayed based on the desired degradation rate.The microorganism, for example, could be one that naturally produces the desired enzyme, or it could be a microorganism that has been genetically modified or cloned to express the desired depolymerase gene.

[0041] In one aspect, a microorganism and / or enzyme produced by the microorganism may be tolerant to a salt concentration of about 0.5 M or higher, such as about 1 M or higher, such as about 2 M or higher, such as about 2.5 M or higher, such as about 3 M or higher, such as about 3.5 M or higher, such as about 4 M or higher, such as about 4.5 M or higher, such as about 5 M or higher, such as about 5.5 M or higher, such as about 6 M or higher, such as about 6.5 M or higher, such as about 7 M or higher, or any intervals or values ​​between them.

[0042] In another aspect, a microorganism and / or enzyme produced by the microorganism may be tolerant to a temperature of about 40°C or higher, such as about 50°C or higher, such as about 60°C or Petition 870240024923, dated 22 / 03 / 2024, p. 22 / 96 / 68 higher, such as approximately 70°C. °C or higher, such as approximately 80°C or higher, such as approximately 90°C or higher, such as approximately 100°C or higher, such as approximately 110°C or higher, such as approximately 120°C or higher, such as approximately 130°C or higher, such as approximately 150°C or higher, or any intervals or values ​​between them.

[0043] In one aspect, a microorganism and / or enzyme produced by the microorganism may be tolerant to elevated pressure of about 0.5 MPa or higher, such as about 1 MPa or higher, such as about 5 MPa or higher, such as about 10 MPa or higher, such as about 15 MPa or higher, such as about 20 MPa or higher, such as about 30 MPa or higher, such as about 40 MPa or higher, such as about 50 MPa or higher, such as about 60 MPa or higher, such as about 70 MPa or higher, such as about 80 MPa or higher, such as about 90 MPa or higher, such as about 100 MPa or higher, such as about 150 MPa or higher, such as about 200 MPa or higher, such as about 250 MPa or higher, such as about 300 MPa or higher, such as approximately 350 MPa or higher, such as approximately 400 MPa or higher, such as approximately 450 MPa or higher, such as approximately 500 MPa or higher, such as approximately 550 MPa or higher,such as approximately 600 MPa or less, or any range or values ​​in between.

[0044] Naturally, combinations of extreme conditions, as well as modifications of conditions during a depolymerization component of a process, are covered here. As an example, a post-consumer product may begin to degrade upon contact with a low-salinity environment. After a period of time in a low-salinity environment, the post-consumer product may be placed in a high-salinity environment that includes a highly salt-tolerant microorganism (or enzyme expressed from it), and the degradation process may be completed. Similarly, a post-consumer product may begin to degrade in a Petition 870240024923, dated 03 / 22 / 2024, p. 23 / 96 / 68 relatively low temperature environment. After a period of time, the product can then be placed in a high temperature environment that includes a high temperature tolerant microorganism (or enzyme expressed from it) and the degradation process can be completed.

[0045] In one aspect, an extremophilic enzyme for use in disclosed methods and processes may be a thermophilic enzyme that exhibits a Topt (that at which a maximum reaction rate can be achieved given a suitable substrate) of about 40°C or higher, about 50°C or higher, about 60°C or higher, about 70°C or higher, about 80°C or higher, or about 90°C or higher in some aspects. Exemplary thermophiles (and thermophilic enzymes produced by them, accession numbers for them given in parentheses in the listings provided) covered herein may include, without limitation, Alicyclobacillus pomorum (WP084453829), Amycolatopsis thermoflava (WP-123687648), Amycolatopsis thermalba (WP-094002797), Amycolatopsis rumanii (WP-116109633), Azospirillum thermophilum (WP-109324320), Deinococcus actinosclerus (WP-082689076), Fervidobacterium gondwanense (SHN54810), Gandjariella thermophila (WP-137812779), Georgenia satyanarayanai (WP146237554), Hyphomanas sp.(HAO37884), Lihuaxuella thermophila (WP089972404), Microbulbifer thermotolerans (P-197462976), Minwuia thermotolerans (WP-206420073), Rhodopseudomonas thermotolerans (WP114356866), Rhodopseudomonas pentothenatexigens, (WP-114356866), Streptomyces thermovulgaris (WP-067396676), Thermanaeromonas toyohensis (WP-084666479), Thermoactinomyces sp. CICC 10523 (WP198056464), Thermoactinomyces daqus (WP-033100012), Thermoactinospora sp. (NUT44302), Thermoactinospora rubra (WP084965756), Thermobifida halotolerans (WP-068692693), Thermobifida fusca (WP-011290529), Thermobispora bispora (WP-206206594), Thermocatellispora tengchongensis, (WP-185055796), Thermochromatium. Petição 870240024923, de 22 / 03 / 2024, pág. 24 / 96 / 68 tepidum (WP-153975900), Thermocrispum municipal (WP-028851041), Thermoflavimicrobium dichotomicum (WP-093229000), Thermogemmatispora carboxidivorans (WP-081839208), Thermogemmatispora aurantia (WP-151728970), Thermogemmatispora tikiterensis (WP-11243376), Thermogemmatispora onikobensis (WP084659191), Thermoleophilaceae bacterium (MBA2429278), Thermomonospora echinospora (WP-160147065), Thermomonospora cellulosilytica (WP-182704610), Thermomonospora amylolytica (WP198679325), Thermostaphylospora chromogena (WP-093263254), Thermus thermophilus (WP-197735236), Thermus aquaticus (WP-053768217), Thermus islandicus (HEO42284).

[0046] The temperature-based enzymes covered here are not limited to high-temperature thermophilic enzymes (and their expressing microorganisms) and, in some respects, low-temperature cryophilic enzymes (also referred to as psychrophilic enzymes), or a microorganism expressing them, may be used. For example, many bacterial strains will not be able to multiply but will still survive after exposure to a temperature of about 10°C for a period of about 6 hours. Thus, in some respects, a microorganism (or a cryophilic enzyme expressed from it) capable of activity at a temperature of about 10°C or less, for example 7°C or less, or from about -15°C to about 10°C in some respects, may be used. The exemplary psychrophiles covered here may include, without limitation, Alteromonas oceani (WP-123325050), Alteromonas alba (WP-105936495), Alteromonas sp.38 (WP-201299304), Alteromonas macleodii (WP156078157), Alteromonas ponticola (WP-169211550), Alteromonas lipolytica (WP-070178363), Arthrobacter crystallopoietes (WP-005270754), Bosea psychrotolerans (WP-181011807), Glaciecola amylolytica (WP-164472126), Hyphomonas sp. (HAO37884), Janthinobacterium psychrotolerans (WPPetition 870240024923, dated 22 / 03 / 2024, p. 25 / 96 / 68. 065307954), Massilia psychrophile (WP-099914383), Paraglaciecola psychrophile (WP-007642709), Polaromonas sp. SP1 (WP-164483751), Polaromonas sp. AER18D-145 (WP-096697750), Polaromonas sp. CF318 (WP-007872516), Polaromonas vacuolate (WP-168920719), Polaromonas naphthalenivorans (WP-157040436), Polaromonas sp. JS666 (WP011482994), Polaromonas glacialis (WP-084181426), Polaromonas sp. EUR3 1,2.1 (WP-197028649), Polaromonas sp.CG_9.2 (WP-196864241), Polaromonas sp. CG_9.11 (WP-196869863), Polaromonas eurypsychrophila (WP-188708524), Polaromonas sp. (MBC7445758), Polaromonas jejuensis (WP-068832216), Polaromonas sp. AET17H-212 (WP-096671180), Polaromonas sp. YR568 (WP-092127764), Polaromonas sp. C04 (WP077562980), Pseudorhodobacter psychrotolerans (WP-08235149), Psychrobacillus lasiicapitis (WP-142537823), Psychrobacillus sp. OK032 (WP-093265425), Psychrobacillus sp. OK028 (WP-093060398), Psychrobacillus sp. FJAT-21963 (WP-056833301), Psychrobacter jeotgali (WP-201583776), Psychrobacter sp. H8-1 (WP-201574875), Psychrobacter sp. Cmf 22.2 (WP-075103245), Psychrobacter sp. ENNN9_III (WP058368887), Psychrobacter sp. P2G3 (WP-068327306), Psychrobacter sp. P11G5 (WP-068035467), Psychrosphaera haliotis (WP-155693683), Shewanella psychrophile (WP-077755816), Simplicispire psychrophile (WP051603004), Sphingobium psychrophilum (WP-169570392), Sphingomonas psychrolutea (WP-188445826), Clostridium homopropionicum (WP074782965), Clostridium sp. DL-VIII (WP-009169886), Clostridium clostridioforme CAG:132 (CDB63357), Zunongwangia atlantica 22II14-10F7 (ORL47196).

[0047] Extremophilic enzymes produced by halophiles can be used in some aspects. Examples of halophiles (and halophilic enzymes produced by them) covered here may include, without limitation, Alteromonas halophila (WP-189403400), Arthrobacter crystallopoietes (WP Petition 870240024923, dated 03 / 22 / 2024, p. 26 / 96 / 68 005270754), Arthrobacter sp. NEB 688 (WP-173027059), Azospirillum halopraeferens (WP-029007775), Empedobacter haloabium (TXE30443), Desulfovibrio sulfodismutans (NDY59052), Halobacillus hunanensis (WP139377117), Halobacillus ihumii (WP-16352794), Halobacteriovorax marinus (WP-157868258), Haloechinothrix halophila (WP-051400222), Halomarina oriensis (WP-158204529), Halomonas cerina (WP-183325502), Halomonas korlensis (WP-089794761), Halomonas sp. PR-M31 (WP048308188), Halomonas aquamarine (WP-089674669), Halomonas zhanjiangensis (WP-040460201), Halomonas aestuarii (WP-071946866), Halomonas endophytica (WP-102654199), Halomonas heilongjiangensis (WP-102629242), Halomonas campaniensis (WP-088701082), Halomonas alkaliphile (WP-038486873), Halomonas sp. ALS9 (WP-064233856), Halomonas sp. GFAJ-1 (WP-009098816), Halomonas sp. KHS3 (WP041159480), Halomonas alkaliphile (WP-162218603), Halomonas sp.ZH2S (WP-160419650), Halomonas alkaliantarctica (WP-133732469), Halomonas zincidurans (WP-031384106), Halomonas chromatireducens (WP083517585), Halomonas sp. KO116 (WP-035563078), Halmonas sp. A40-4 (WP-199285424), Halomonas ventosae (WP-035579360), Halomonas sp. HAL1 )WP-008958555), Halomonas sp. MES3-P3E (WP-101146070), Halomonas sp. 1513 (WP-083700770), Halomonas sp. GT (WP-083007892), Halomonas sp. PA5 (QJQ97022), Halomonas songnenensis (WP106373458), Halomonas subglaciescola (WP-079553041), Halomonas sp. HL-92 (WP-074398447), Halomonas xinjiangensis (WP-197053288), Halomonas saliphila (WP-104202516), Halomonas sp. HL-48 (WP027336292), Halomonas qijiaojingensis (WP-189471950), Halomonas urumqiensis (WP-102588859), Halomonas lutea (WP-019020614), Halomonas lutescens (WP-188638020), Halomonas salicampi (WP179930793), Halomonas sp. FME66 (WP-193092800), Halomonas sp. 156 (CAD5269671), Halomonas sp. L5 (WP-149329933), Halomonas. Petição 870240024923, de 03 / 22 / 2024, pág. 27 / 96 / 68 nanhaiensis (WP-127060197), Halomonas titanicae (WP-144810212), Halomonas sp. SH5A2 (WP-186255949), Halomonas sp. TD01 (WP009722522), Halomonas sp. PC (WP-127040515), Halomonas sp. RC (WP126951333), Halomonas sp. DQ26W (WP-114573011), Halomonas sp. TQ8S (WP-114486842), Halomonas sp. PYC7W (WP-114478819), Halomonas sp. LBP4 (WP-181421925), Halomonas sp. QX-1 (WP-176303735), Halomonas sp. QX-2 (WP-180092182), Halomonas glaciei (WP-179915254), Halomonas zhaodongensis (WP-179927495), Halomonas xianhensis (WP-092845804), Halomonas gudaonensis (WP-089686750), Halomonas humidisoli (WP095603093), Halomonas boliviensis (WP-083825729), Halomonas sp. QHL1 (WP-083571058), Halomonas ilicicola (WP-072822829), Halomonas saccharevitans (WP-089847692), Halomonas muralis (WP-089729617), Halomonas arcis (WP-089706930), Halomonas boliviensis (WP-040480056), Halomonas andesensis (WP-126944084), Halomonas sp. G5-11 (WP168017113), Halomonas sp.THAF5a (QFU03326), Halomonas taeanensis (SDG32001), Halorussus sp. RC-68 (WP-128475846), Halorussus ruber (WP-135825713), Halorussus sp. ZS-3 (WP-158056449), Halorussus sp. HD8-83 (WP-135830119), Halorussus salinus (WP-135854680), Halorussus amylolyticus (WP-132060623), Halorussus sp. MSC15.2 (WP-163523881), Haloterrigena limicola (WP-008010666), Haloterrigena hispanica (WP149782231), Haloterrigena sp. H1 (WP-138782397), Isoptericola halotolerans (WP-171781920), Marinobacter sp. X15-166B (WP198929205) PJ-16 (WP-137435339), Marinobacter nanhaiticus (WP-004579452), Marinobacter bohaiensis (WP111497193), Marinobacter sp. ANT_B65 (WP-202971753), Marinobacter sediminum (WP-203299860), Marinobacter fonticola (WP-148861082), Marinobacter sp. JB02H27 (WP-150989051), Marinobacter maritimus (WP144775354), Marinobacter nitratireducens (WP-036130189), Marinobacter. Petition 870240024923, dated 03 / 22 / 2024, p. 28 / 96 / 68 aromaticivorans (WP-100686899), Marinobacter sp. MCTG268 (WP081899301), Marinobacter profundi (WP-099614009), Marinobacter sp. R17 (WP-123633665), Marinobacter sp. F3R11 (WP-113816648), Marinobacter lipolyticus (WP-012136507), Marinobacter sp. LV10MA510-1 (WP098421792), Marinobacter sp. LV10R520-4 (WP-143751449), Marinobacter antarcticus (WP-072795398), Marinobacter zhejiangensis (WP-092022278), Marinobacter sp. LZ-8 (WP-138439039), Marinobacter sp. LZ-6 (WP138437074), Marinobacter sp. DS40M8 (WP-169052525), Marinobacter shengliensis (WP-106694886), Marinobacter algicola (WP-007152654), Marinobacter salicampi (WP-166253549), Marinobacter sp. JSM 1782161 (WP-165857264), Methyloligella halotolerans (WP-069095898), Micromonospora halophytica (WP-091291516), Natronococcus sp.LS1_42 (WP-148858780), Nocardiopsis halotolerans (WP-017570132), Paracoccus halophilus (WP-036743786), Roseivivax halodurans (WP-037257008), Saccharomonospora halophila (WP-157601674), Shewanella vesiculosa (NCO72699), Shewanella psychrophila (WP-077755816), Shewanella frigidimarina (WP-123883413), Shewanella scurvy (WP-126168307), Shewanella halifaxensis (WP-108946642), Shewanella waxmani (WP028774143), Shewanella salivary gland (WP-188922486), Shewanella ulleungensis (WP-188954542), and Shewanella litoralis (WP-160052797).

[0048] Extremophilic enzymes produced by acidophiles can be used in some aspects. For example, acidophilic enzymes that exhibit activity at a pH of about 1 to about 5.5 can be used. Examples of acidophiles (and acidophilic enzymes produced by them) covered here may include, without limitation, Acidibrevibacterium fodinaquatile (WP-162800754), Acidicaldus sp (HGC43174), Acidiphilium cryptum (WP050751056), Acidisphaera rubrifaciens (WP-084623200), Acidisphaera sp. S103 (WP-158926549), Acidobacteria bacterium (MBI4850940), Acidobacteriales bacterium (MBA3914351), Acidimicrobiaceae bacterium Petition 870240024923, dated 22 / 03 / 2024, p. 29 / 96 / 68 (TPW09344), Acidothermus cellulolyticus (WP-011719018), Acidovorax sp. (RZJ59385), Acidovorax sp. Leaf160 (WP-156382378), Acidovorax citrulli (WP-116212334), Acidovorax sp. ST3 (WP-110960035), Acidovorax sp. SD340 (WP-055393692), Acidovorax sp. JHL-9 (WP-026434583), Acidovorax sp. JHL-3 (WP-024815995), Acidovorax sp. 59 (WP-099731663), Acidovorax sp. T1 (WP-087747071), Acidovorax radices (WP-145694120), Acidovorax citrulli (MVT28077), Acidovorax konjaci (WP-184273732), Acidovorax sp. YL-MeA13-2016 (WP-179683865), Acidovorax sp. JMULE5 (WP-176888736), Acidovorax carolinensis (WP-086926820), Acidovorax sp. Raíz219 (WP-057264729), Acidovorax sp. Raíz217 (WP-057200451), Acidovorax sp. Raíz70 (WP-056639581), Acidovorax sp. Raíz267 (WP057271450), Acidovorax sp. Raiz275 (WP-057228519), Acidovorax sp. Raiz568 (WP-056742554), Acidovorax sp. Raiz402 (WP-056056880), Acidovorax sp. Leaf78 (WP-056167938), Acidovorax sp.CF316 (WP007848954), Acidovorax sp. NO-1 (WP-008904688), Acidovorax sp. KKS102 (WP-015015374), Acidovorax sp. BoFeN1 (WP-114656624), Acidovorax sp. MR-S7 (WP-020227330), Acidovorax sp. GW101-3H11 (WP-063462297), Acidovorax sp. 100 (WP-121942233), Acidovorax sp. 94 (WP-121421729), Acidovorax sp. 93 (WP-121508058), Acidovorax sp. IB03 (WP-198847087), Acidovorax facilis (WP-182119389), Acidovorax cattleya (WP-196290774), Acidovorax soli (WP-184855240), Acidovorax sp. TP4 (BAA35137), Acidovorax sp. HMWF018 (WP-199227795), Acidovorax sp. 107 (WP108624875), Acidovorax sp. 69 (WP-100412617), Acidovorax sp. RAC01 (WP-069104250), Acidovorax avenae (WP-107129247), Acidovorax sp. ACV01 (WP-192426852), Acidovorax sp. ACV02 (WP-192419383), Acidovorax sp. SRB_14 (WP-173025722), Acidovorax sp. 99 (WP116748450), Acidovorax delafieldii (WP-060985808), Acidovorax sp. 16-355 (WP-175506463), Acidovorax valerianellae (WP-092740663), Acidovorax temperans (WP-142084895), Acidovorax oryzae (WP-026433360),. Petition 870240024923, dated 03 / 22 / 2024, p. 30 / 96 / 68 Acidovorax sp. SRB_24 (WP-169168665), Acidovorax cavernicola (WP119555154), Acidovorax temperans (WP-044398345), Acidisoma sp. S159 (WP-159014448), Acidisoma sp. L85 (WP-158802619), Acidisphaera sp. L21 (WP-158747166), Acidiphilium cryptum JF-5 (ABQ28771), Actinospica acidiphila (WP-193455356), Alicyclobacillus pomorum (WP-084453829), Amycolatopsis acidiphila (WP-144638401), Azospirillum baldaniorum (WP014240680), Bacillus megaterium (WP-013057692), Catenulispora acidiphila (WP-015793547), Delftia sp.UME58 (WP-183018265), Delftia acidovorans (WP-202760212), Delftia lacustris (WP-016453321), Methylocapsa acidiphila (WP-026607232), Paraburkholderia acidophila (WP-084908171), Paraburkholderia acidisoli (WP-158957882), Paraburkholderia acidipaludis (WP-027796272), Priestia megaterium (WP016764703), Rhizobium acidisoli (WP-054183259), Rhodoblastus acidophilus (WP-088519736), Stenotrophomonas acidaminiphila (WP054666853), Streptomyces acidiscabies (WP-078480871), Streptomyces acidicola (WP-152864677).

[0049] Extremophilic enzymes produced by alkalophiles can be used in some aspects. For example, alkalophilic enzymes that exhibit activity at a pH of about 7.5 to about 11.5 can be used. Examples of alkalophiles (and alkalophilic enzymes produced by them) covered here may include, without limitation, Alkalilacustris brevis (WP114966465), Alkalihalobacillus macyae (WP-152670966), Alkalihalobacillus pseudofirmus (WP-012960136), Alkalihalobacillus shacheensis (WP082676287), Alkalihalobacillus xiaoxiensis (WP-204463621), Alkalilimnicola sp. S0819 (WP-152144452) (WP-062478888), Lysobacter alkalisoli (QDH70273), Massilia alkalitolerans (WP-036214799), Methylobacter sp. B2 WP-174627553), Petition 870240024923, dated 03 / 22 / 2024, page 31 / 96 26 / 68 Neorhizobium alkalisoli (WP-105385441), Nocardiopsis alkaliphile (WP051045978), Ramlibacter alkalitolerans (WP-201687394), Spinactinospora alkalitolerans (WP-179641803).

[0050] Extremophilic enzymes produced by piezophiles can be used in some respects. For example, piezophilic enzymes that exhibit activity at a pressure of about 110 kPa or higher, or about 50 MPa or higher in some respects, can be used. Examples of piezophiles (and piezophilic enzymes produced by them) covered here may include, without limitation, Oceanobacillus piezotolerans (WP-121525044), Oceanobacillus profunda (WP-169713018), Colwellia marinimaniae (WP082606415), Salinimonas sediminis (WP-108566897).

[0051] Radiation-resistant extremophiles are also included here. For example, radiation-resistant organisms such as Deinococcus radiotolerans which produces a radiation-resistant enzyme (WP_189068351) can be used. A radiation-resistant organism and a radiation-resistant enzyme covered here can generally be active at an acute ionizing radiation level (gamma rays, high-energy UV rays, X-rays, etc.) of about 1,000 Gy or more, or about 2,000 Gy or more in some respects.

[0052] In one aspect, extremophilic depolymerase enzyme or extremophilic microorganism for use as disclosed herein may include polyextremophiles that exist in a combination of extreme environmental conditions. For example, a halophilic alkaline thermophile, which ideally exists in high saline and alkaline conditions, or a psychrotrophic halophile, which ideally exists in high saline and low temperature conditions. Most piezophilic (pressure-loving) extremophiles are found on the ocean floor and are therefore also halophilic (salt-loving) and psychrophilic (cold-loving), all conditions that can be generated and maintained simultaneously within a reaction chamber to provide Petition 870240024923, dated 03 / 22 / 2024, page 32 / 96 27 / 68 decontamination of mesophilic pathogens in conjunction with depolymerization and HA monomer production. In this respect, mesophilic contamination can be treated through multiple mechanisms in conjunction with a depolymerization reaction catalyzed by a single polyextremophilic enzyme.

[0053] Examples of polyextremophiles (and polyextremophilic enzymes thus produced) covered herein may include, without limitation (some of which are also included in those referred to above), Acidothermus cellulolyiicus (WP_Q\ \1 \9Q\S), Arthrobacter crystallopoietes (WP_005270754), Arthrobacter sp. NEB 688 (WP_173027059), Amycolatopsis decaplanina (WP_007028471), Amycolatopsis azurea (WP_039919726), Amycolatopsis orientalis (WP_044853678), Amycolatopsis regifaucium (WP_061985795), Amycolatopsis alba (WP_020632115), Amycolatopsis sp. CB00013 (WP_073845662), Amycolatopsis sp. WAC 04182 (WP_125683401), Amycolatopsis sp. WAC 04197 (WP_125733174), Amycolatopsis sp. WAC 1.416 (WP_125797595), Amycolatopsis lurida (WP_034314791), Amycolatopsis australiensis (WP_072479564), Amycolatopsis sp. WAC 01375 (WP-125786221), Amycolatopsis sp. YIM 10 (WP_194239921), Amycolatopsis australiensis (WP_072480012), Amycolatopsis sp. WAC 01376 (WP_125797552), Amycolatopsis sp. WAC 01376 (WP_125791151), Amycolatopsis sp. BJA-103 (WP_168214428), Amycolatopsis sp. WAC 04169 (WP-125694889), Amycolatopsis sp. YIM 10 (WP_153034611), Amycolatopsis xylanica (WP_091289432), Amycolatopsis thailandensis (WP_093938547), Amycolatopsis tolypomycina (WP_091314877), Amycolatopsis (WP_094002797), Amycolatopsis mediterranean (WP_013227677), Amycolatopsis tolypomycina (WP_091316988), Amycolatopsis mediterranei (WP_013225900), Amycolatopsis sp. MJM2582 (WP_037335097), Amycolatopsis pretoriensis (WP_086680613), Petition 870240024923, dated 22 / 03 / 2024, p. 33 / 96 / 68 Amycolatopsis mediterranei (WP_014467631), Amycolatopsis mediterranei (WP_013227743), Amycolatopsis lexingtonensis (WP_086861387), Amycolatopsis balhimycina (WP_026468360), Amycolatopsis tolypomycina (WP_091309318), Amycolatopsis mediterranei (WP_013225589), Amycolatopsis lexingtonensis (WP_086864508), Amycolatopsis balhimycina (WP_020640708), Amycolatopsis balhimycina (WP_020639925), Amycolatopsis japonica (WP_038521005), Amycolatopsis vancoresmycina (WP_051767789), Amycolatopsis vancoresmycina (WP_162146255), Amycolatopsis vancoresmycina (WP_003055279), Amycolatopsis vancoresmycina (WP_003059137), Amycolatopsis arida (WP_177216885), Amycolatopsis orientalis (WP_037305638), Amycolatopsis mediterranei U32 (ADJ49174), Amycolatopsis balhimycina (WP_020640186), Amycolatopsis balhimycina (WP_020646797), Amycolatopsis regifaucium (WP_158070237), Amycolatopsis umgeniensis (WP_184896802), Amycolatopsis mediterranei (WP_176742238), Amycolatopsis orientalis (WP_037318494), Amycolatopsis taiwanensis (WP_027941815), Amycolatopsis thermoflava (WP_037323546), Amycolatopsis nigrescens (WP_157357235), Amycolatopsis benzoatilytica (WP_020658806), Amycolatopsis thermoflava (WP_123687648), Amycolatopsis sp. MtRt6 (WP_206788940), Amycolatopsis nigrescens (WP_020673950), Amycolatopsis sp. MtRt-6 (WP_206796628), Amycolatopsis sp. MtRt-6 (WP_206785025), Amycolatopsis sp. 195334CR (WP 206808196), Amycolatopsis sp. SID8362 (WP_166641473), Amycolatopsis wastetatis (WP_167441766), Amycolatopsis sp. MtRt-6 (WP_206794433), Amycolatopsis sp. 195334CR (WP_206804625), Amycolatopsis sp. SID8362 (WP_160695402), Amycolatopsis sp. 195334CR (WP_206805671), Amycolatopsis mediterranei S699 (AEK42609), Amycolatopsis sp. SID8362 (WP_160697844), Amycolatopsis ruanii (WP_116109633), Amycolatopsis Petition 870240024923, dated 22 / 03 / 2024, p. 34 / 96 / 68 waste (WP_093953441), Amycolatopsis antarctica (WP_094864937), Amycolatopsis sp. SID8362 (WP_160697847) Amycolatopsis wastetatis (WP_093953762), Amycolatopsis keratiniphila (WP_043848437), Amycolatopsis rifamycinica (WP_043776526), ​​Amycolatopsis sp. ATCC 39116 (WP_039791697), Amycolatopsis sp. CA-126428 (WP_199191631), Amycolatopsis sp. CA-128772 (WP_199199004), Amycolatopsis rifamycinica (WP_043775110), Amycolatopsis sp. CA-128772 (WP_103347542), Amycolatopsis sp. CA-126428 (WP_103341161), Amycolatopsis sp. CA-126428 (WP_103338297), Amycolatopsis sp. CA128772 (WP_103347494), Amycolatopsis sp. CA-128772 (WP_103351389), Amycolatopsis sp. CA-126428 (WP_10334050), Amycolatopsis sp. CA126428 (WP_103337215), Amycolatopsis sp. BJA-103 (WP_101611121), Amycolatopsis rifamycinica (WP_043775220), Amycolatopsis bullii (WP_191309718), Amycolatopsis alkalitolerans (WP_139096058), Amycolatopsis sp. CA-126428 (WP_103340450), Amycolatopsis sp. A23 (WP_155542679), Amycolatopsis sp. A23 (WP_155546301), Amycolatopsis bullii (WP_191313482), Amycolatopsis oliviviridis (WP_191256639), Amycolatopsis bullii (WP_191317041), Amycolatopsis sp. A23 WP_155546374), Amycolatopsis bullii (WP_191309628), Amycolatopsis sp. H6(2020) (MBE8525409), Amycolatopsis sp. H6(2020) (MBE8516875), Amycolatopsis acidiphila (WP_144638401), Amycolatopsis deserti (WP_191242759), Amycolatopsis sp. H6(2020) (MBE8523464), Amycolatopsis roodepoortensis (WP_192744003), Amycolatopsis lexingtonensis (WP_086861614), Amycolatopsis sp. H6(2020) (MBE8523449), Amycolatopsis lexingtonensis (WP_086861672), Amycolatopsis sp. H6(2020) (MBE8519699), Amycolatopsis eburnean Petition 870240024923, dated 03 / 22 / 2024, page 35 / 96 / 68 (WP_125314097), Amycolatopsis sp. PIP199 (WP_181777181), Amycolatopsis eburnean

[0054] (WP_125313793), Amycolatopsis sp. YIM 10 (WP_153034239), Amycolatopsis rhizosphaerae (WP_144585784), Amycolatopsis eburnean (WP_191984376), Amycolatopsis australiensis (WP_072479963), Amycolatopsis eburnean (WP_125313723), Amycolatopsis sp. Hca4 (WP_176178332), Amycolatopsis pretoriensis (WP_086674376), Amycolatopsis sp. YIM 10 (WP_153033440), Amycolatopsis sp. Hca4 (WP_176171164), Amycolatopsis thermalba (WP_115944128), Amycolatopsis tolypomycina (WP_091313624), Amycolatopsis sacchari (WP_09150482), Amycolatopsis kentuckyensis (WP_086849953), Amycolatopsis pretoriensis (WP_086676731), Amycolatopsis kentuckyensis (WP_086838850), Amycolatopsis vancoresmycina (WP_033262149), Amycolatopsis sacchari (WP_091509483), Amycolatopsis eburnean (RSD12104), Amycolatopsis vancoresmycina (WP_033262457), Amycolatopsis tolypomycina (WP_091314771), Amycolatopsis kentuckyensis (WP_086842561), Amycolatopsis tolypomycina (SED02538), Amycolatopsis kentuckyensis (WP_086850817), Amycolatopsis keratiniphila (SDU59319), Amycolatopsis sp.SID8362 (NBH10816), Amycolatopsis sacchari (SFI91313), Amycolatopsis keratiniphila (AGM10176), Amycolatopsis vancoresmycina DSM 44592 (EOD69417), Amycolatopsis vancoresmycina DSM 44592 (EOD63279), Colwellia psychrerythraea (WP_033095470), Colwellia psychrerythraea (WP_033082346), Colwellia chukchiensis (WP_085285385), unclassified Colwellia (WP_182245161), unclassified Colwellia (WP_108456828), Colwellia (WP_082606415), unclassified Colwellia (WP_182136131), no classified Colwellia (WP_182222214), Colwellia psychrerythraea (WP_138140233), unclassified Colwellia (WP_182213899), unclassified Colwellia (WP_182191078), Colwellia psychrerythraea (WP_033082290), Colwellia. Petição 870240024923, de 22 / 03 / 2024, pág. 36 / 96 / 68 sp. Arc7-635 (WP_126668020), Colwellia aestuarii (WP_143323591), Colwellia sp. BRX8-4 (WP_182258889), Colwellia sp. (MBL4900302), Colwellia sp. (MBL0710453), Colwellia sp. PAMC 21821 (WP_081180401), Colwellia sp. (MBL4764635), Colwellia sp. 12G3 (WP_101233926), Colwellia Polaris (WP_085306422), Colwellia sp. Bg11-28 (WP_157825823), Colwellia sp. BRX10-3 (WP_182133028), Colwellia sp. MB02u-6 (WP_182233718), Colwellia sp. BRX8-2 (WP_182231462), Colwellia sp. MB3u-4 (WP_182185277), Colwellia sp. BRX9-1 (WP_182230151), Colwellia sp. BRX8-7 (WP_182242732), Colwellia sp. (NQZ90610), Colwellia sp. MB02u-10 (WP_182238471), Colwellia sp. (NQZ28611), Colwellia sp. (QY47923), Colwellia sp. Bg11-12 (WP_182229555), Colwellia sp. (NQY89088), Colwellia beringensis (WP_081152231), Colwellia sp. (NQZ82584), Colwellia demingiae (WP_146789187), Candidatus Colwellia aromaticivorans (WP_114327742), Colwellia sp. MB02u-9 (WP_182197537), Colwellia mytili (WP_085299583), Colwellia sp. (NQY47915), Colwellia sp. (NQZ28619), Haladaptatus paucihalophilus (WP_007977720), Haladaptatus littoreus (WP_076429835), Haladaptatus paucihalophilus (WP_007977722), Haladaptatus sp. R4 (WP_066143160), Haladaptatus cibarius (WP_049970104), Haladaptatus sp. (W1 WP_069450211), Haladaptatus cibarius (WP_049971911), Haladaptatus paucihalophilus DX253 Halobacillus ihumii (WP_163527944), Halobacillus (SHK49397), hunanensis (WP_139377117), Halomarina oriensis oriensis (WP_158204529), Halomonas (WP_124957125), Halomarina (ventosae) (WP_035579360), Halomonas sp. (WP_008956714), chromatireducens 156 (CAD5269671), unclassified Halomonas (WP_083602247), (WP_074398447), Halomonas (WP_035577590), (WP_083517585), unclassified Halomonas sp. GFAJ-1 Halomonas Halomonas Halomonas meridiana (sp. HL-92) (WP_009101808), Halomonas Petition 870240024923, of 22 / 03 / 2024, p. 37 / 96 / 68 chromatireducens (WP_066448186), Halomonas sp. KO116 (WP_035563078), Halomonas sp. KO116 (WP_035565981), Halomonas arcis (WP_089708323), Halomonas sp. TD01 (WP_009724586), Halomonas arcis (WP_089706930), Halomonas korlensis (WP_089792833), Halomonas alkaliantarctica (WP_133732469), Halomonas ilicicola (WP_0728), Halomonas bolensis 222 (WP_007114283), Halomonas sp. HL -48 (WP_027336292), Halomonas alkaliphila (WP_038486873), unclassified Halomonas (WP_074394764), Halomonas sp. HAL1 (WP_008958555), Halomonas subglaciescola (WP_079553041), Halomonas korlensis (WP_089797758), Halomonas cerina (WP_183325502), unclassified Halomonas (sp. RC) (WP_133), Halomonas sp.TD01 (WP_009722522), Halomonas titanicae (WP_089691351), Halomonas aquamarine (WP_089674669), Halomonas gudaonensis (WP_089686750), Halomonas alkaliantarctica (WP_133731111), Halomonas saccharevitans (WP_089847692), Halomonas xianhensis (WP_092845804), Halomonas songnenensis (WP_106373458), Halomonas zincidurans (WP_031384106), Halomonas lutea (WP_019020614), Halomonas boliviensis (WP_083825729), Halomonas sp. GFAJ-1 (WP_009098816), Halomonas muralis (WP_089729617), Halomonas boliviensis (WP_040480056), Halomonas sp. (HAA45741), Halomonas zhanjiangensis (WP_040460201), Halomonas campaniensis (WP_088701082), Halomonas alkaliphile (WP_162218603), Halomonas sp. ZH2S (WP_160419650), Halomonas endophytica (WP_102654199), Halomonas sp. ALS9 (WP_064233856), Halomonas sp. KHS3 (WP_041159480), Halomonas salicampi (WP_179930793), Halomonas salicampi (WP_179928774), Halomonas heilongjiangensis (WP_102629242), Halomonas campaniensis (WP_088701419), Halomonas sp.MES3-P3E (WP_101146070), Halomonas alkaliantarctica (WP_030070137), Halomonas xinjiangensis (WP_197053288), Halomonas alkaliantarctica (WP_030072571),. Petição 870240024923, de 03 / 22 / 2024, pág. 38 / 96 / 68 Halomonas sp. GT (WP_083002052), Halomonas sp. A40-4 (WP_199285424), Halomonas sp. GT (WP_083007892), Halomonas sp. 1513 (WP_076746720), Halomonas sp. HL - 48 (WP_027335517), Halomonas sp. 1513 (WP_083700770), Halomonas sp. (MBL1266350), Halomonas urumqiensis (WP_102588859), Halomonas lutescens (WP_188638020), Halomonas lutescens (WP_188638515), Halomonas sp. FME66 (WP_193092800), Halomonas saliphila (WP_104202516), Halomonas sp. (MBE0488383), Halomonas qijiaojingensis (WP_189471950), Halomonas sp. 3(2) (WP_151442249), Halomonas sp. FME20 (WP_192536925), Halomonas sp. SH5A2

[0055] (WP_186255949), Halomonas sp. TQ8S (WP_114486842), Halomonas titanicae (WP_144812651), Halomonas sp. PYC7W (WP_114478819), Halomonas sp. PYC7W (WP_114478692), Halomonas sp. LBP4 (WP_181421925), Halomonas sp. TQ8S (WP_114487405), Halomonas glaciei (WP_179915254), Halomonas sp. QX-2 9 (WP_180092182), Halomonas sp. SH5A2 (WP_186253301), Halomonas zhaodongensis (WP_179927495), Halomonas titanicae (WP_144810212), Halomonas nanhaiensis (WP_127060197), Halomonas pantelleriensis (WP_089659512), Halomonas zhaodongensis (WP_179926908), Halomonas humidisoli (WP_095603093), Halomonas sp. QHL1 (WP_083571058), Halomonas sp. PC (WP_127040515), Halomonas sp. DQ26W (WP_114573011), Halomonas shengliensis (WP_089679049), Halomonas sp. QX-1 (WP_176303735), Halomonas sp. QHL1 (WP_071693265), Halomonas korlensis (WP_089794761), Halomonas aestuarii (WP_071946866), Halomonas sp. PR - M31 (WP_048308188), Halomonas sp. PA5 (QJQ97022), Halomonas andesensis (WP_126944084), Halomonas sp. PA5 (QJQ94877), Halomonas sp.L5 (WP_149329933), Halomonas corlensis (SFU56513), Halomonas sp. G5 -11 (WP_168017113), Halomonas subterranean (WP_092824778), Halomonas sp. (HDZ47214), Halomonas sp. THAF5a (QFU03326),. Petition 870240024923, of 22 / 03 / 2024, p. 39 / 96 / 68 Halomonas sp (HDZ46744), Halomonas chromatireducens (AMD02558), Halomonas andesensis (WP_126948398), Halomonas korlensis (SFU93166), Halomonas taeanensis (SDG32001), Halorussus salinus (WP_13385), Halorussus sp. MSC15.2 (WP_163523881), Halorussus salinus (WP_135854680), Halorussus amylolyticus (WP_132060623), Halorussus sp. ZS-3 (WP_158056449), Halorussus amylolyticus (WP_132060625), Halorussus sp. ZS-3 (WP_158056448), Halorussus sp. RC-68 (WP_128475846), Halorussus ruber (WP_135825713), Halorussus ruber (WP_135825712), Halorussus sp. HD8-83 (WP_135830119), MariInobacter sp. LV10R520-4 (WP_143751449), Marinobacter zhejiangensis (WP_092022278), unclassified Marinobacter (WP_150989051), Marinobacter nitratireducens (WP_036130189), Marinobacter salaries (WP_091640839), Marinobacter unclassified (WP_098419392), Marinobacter algicola (WP_007152654), Marinobacter antarcticus (WP_072795398), unclassified Marinobacter (WP_152438805), Marinobacter (WP_075197007), Marinobacter profundi (WP_099614009), Marinobacter sp. LPB0319 (WP_206643988), Marinobacter sp. DS40M8 (WP_169052525), Marinobacter sp. X15-166B (WP_198929205), unclassified Marinobacter (WP_081899301), Marinobacter sp. PJ-16 (WP_137435339), Marinobacter bohaiensis (WP_111497193), Marinobacter sediminum (WP_203299860), Marinobacter lipolyticus (WP_012136507), Marinobacter sp. ANT_B65 (WP_202971753), Marinobacter nanhaiticus (WP_004579452), Marinobacter salários (WP_126811858), Marinobacter maritimus (WP_144775354), Marinobacter sp. F3R11 (WP_113816648), Marinobacter sp. LZ-8 (WP_138439039), Marinobacter sp. LZ-6 (WP_138437074), Marinobacter shengliensis (WP_106694886), Marinobacter fondicola (WP_148861082), Marinobacter

[0056] sp. JSM 1782161 (WP_165857264), Marinobacter sp. R17 (WP_123633665), Marinobacter salicampi (WP_166253549), Marinobacter Petition 870240024923, dated 03 / 22 / 2024, p. 40 / 96 35 / 68 sp. LV10MA510-1 (WP.098421792), Thermobifida fusca (WP_016187994), Zunongwangia atlantica 22II14-10F7 (ORL471960).

[0057] Naturally, any combination of microorganisms or enzymes thereof can be used in disclosed methods and systems, and any combination of environmental conditions corresponding to the active conditions for the enzymes can also be used to provide a multidimensional approach for simultaneous decontamination of a post-consumer product and degradation of PHA in the post-consumer product.

[0058] In addition to or as an alternative to microorganisms that naturally express one or more of the enzymes that may be useful for providing HA monomer from a post-consumer product, one or more genetically modified microorganisms may be used that can express exogenous enzyme(s) capable of degrading a post-consumer product and producing HA monomer from PHA included in the product. For example, genetically modified PHADase variants can be recombined into a bacterial chromosome under the control of the native promoter and ribosomal binding site. Such bacteria can then produce a desired variant of PHADase, for example, in response to environmental conditions that may instigate PHA depolymerization, for example, deprivation of the energy / carbon source.

[0059] In general, any suitable organism can be modified to express a PHADase, for example, from a constitutive vector coupled to the correct signal sequence. For example, any suitable Gram-positive or Gram-negative bacterium can be used to produce and secrete a PHADase, which can be a Gram-positive PHADase. In this way, an expression system can be customized based on, for example, the environmental variables of the depolymerization component, the type and quantity of post-consumer materials to be processed, as well as their combinations. Furthermore, the enzyme sequence can be combined Petition 870240024923, dated 03 / 22 / 2024, p. 41 / 96 / 68 with the environment and / or the PHA being processed by selecting one or more of approximately 6,400 depolymerase sequences that are known (e.g., NCBI database) or with a wholly or partially engineered variant. In one aspect, an organism can be transformed with a plasmid vector harboring a constitutively expressed gene encoding a PHA that contains an appropriate N-ter signal sequence. Alternatively, an organism can have a depolymerase gene inserted into the chromosome by transduction, linear recombination, or any other suitable method.

[0060] An enzyme can be expressed by transformation of a suitable host organism through the use of prokaryotic or eukaryotic host cells. Examples of host cell types include, but are not limited to, bacterial cells (e.g., E. coli), yeast cells (e.g., pichia, S. cerevisiae), cultured insect cell lines (e.g., Drosophila), plant cell lines (e.g., maize, tobacco, rice, sugarcane, potato tuber), or mammalian cell lines (e.g., Chinese hamster ovary (CHO)). In one aspect, a recombinant host cell system can be used that processes and post-translationally modifies nascent polypeptides in a desired manner to produce the final catalytic enzyme.

[0061] However, any suitable organism may be used. For example, a suitable gram-positive or gram-negative bacterium may be used, such as a bacterium obtained from the genus Streptomyces. Particular examples of microorganisms of the above genus include Streptomyces thermovulgaris, Streptomyces thermoolivaceus, Streptomyces thermohygroscopicus, Streptomyces thermocarboxydovorans, or mixtures thereof.

[0062] Other genera may still be used in accordance with the present disclosure to express modified enzymes including, without Petition 870240024923, dated 03 / 22 / 2024, p. 42 / 96 / 68 limitation, Firmicutes (Bacillus, Lihuaxuella, and Clostridium),

[0063] Proteobacteria (Bradyrhizobium, Sphingomonas, Azotobacter, Azospirillum, Nitrobacter, Lysobacter, Stenotrophomonas, Rhizobium, Acinetobacter, Thiobacillus, Schlegelella, Janthinobacterium, Sinorhizobium, Pseudomonas, Agrobacterium and Escherichia (e.g. Escherichia coli)), Actinobacteria (Rhodococcus, Arthobacter, Streptomyces, Conexibacter, Rhodococcus, Solirubrobacter, Micrococcus, Rubrobacter and Actinomyces), Bacteroidetes (Flavobacterium and Pedobacter), Deinococcus-thermus (Deinococcus and Thermus), Gemmatimonadetes (Gemmatimonas and Gemmatirosa, (Spirochaetes (Tumeriella and Leptospira), Verrucomicrobia (Pedosphaera, Chthoniobacter and Verrucomicrobia), Chloroflexi (Thermogemmatispora and Dictyobacter) and Armatimonadetes (Fimbriimonas).

[0064] A nucleic acid sequence encoding an enzyme can be placed in an expression vector for expression in the host. Such expression vectors may generally comprise a transcription initiation region linked to the nucleic acid sequence encoding the enzyme. An expression vector may also include a plurality of restriction sites for nucleic acid insertion under the transcriptional regulation of various control elements. The expression vector may additionally contain selectable marker genes. Suitable control elements, such as enhancers / promoters, splice junctions, polyadenylation signals, etc., may be placed close to the coding region to allow proper transcription initiation and / or correct processing of the primary transcript, i.e., the coding region for the enzyme.Alternatively, the coding region used in an expression vector may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of endogenous and exogenous control elements.

[0065] An expression vector usually includes the 5'-3' direction of Petition 870240024923, dated 03 / 22 / 2024, page 43 / 96 / 68 transcription, a promoter, a transcription and translation initiation region, a DNA sequence encoding the enzyme, and a functional transcription and translation termination region in the host cell. In one aspect, a T7-based vector may be used, which may include at least the following components: an origin of replication, a selectable antibiotic resistance gene (e.g., -ampr, tetr, chlrr), a multiple cloning site, T7 initiator and terminator sequences, a ribosomal binding site, and a T7 promoter.

[0066] In general, any suitable promoter capable of operative binding to heterologous DNA can be used so that DNA transcription can be initiated from the promoter by an RNA polymerase that can specifically recognize, bind to, and transcribe the DNA into an open reading frame. Some useful promoters include constitutive promoters, inducible promoters, regulated promoters, cell-specific promoters, viral promoters, and synthetic promoters. Furthermore, while promoters may include sequences to which an RNA polymerase binds, this is not a requirement. A promoter can be obtained from a variety of different sources. For example, a promoter may be derived entirely from a native host cell gene, be composed of different elements derived from different promoters found in nature, or be composed of nucleic acid sequences that are entirely synthetic.A promoter can be derived from many different types of organisms and adapted for use in a particular cell. For example, a promoter may include regions to which other regulatory proteins can bind in addition to regions involved in controlling protein translation, including coding sequences.

[0067] A translation initiation sequence can be derived from any source, for example, any expressed E. coli gene. Usually, the gene is a highly expressed gene. A translation initiation sequence Petition 870240024923, dated 03 / 22 / 2024, pp. 44 / 96 / 68, can be obtained via standard recombinant methods, synthetic techniques, purification techniques, or combinations thereof, all of which are well known. Alternatively, translation initiation sequences can be obtained from various commercial vendors (Operon Technologies; Life Technologies Inc.).

[0068] The termination region may be native to the transcription initiation region, may be native to the coding region, or may be derived from another source. Transcription termination sequences recognized by the transformed cell are regulatory regions located 3' from the translation termination codon and thus, together with the promoter, flank the coding sequence. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli, as well as other biosynthetic genes.

[0069] Vectors that can be used include, but are not limited to, those capable of being replicated in prokaryotes and eukaryotes. For example, vectors could be used that are replicated in bacteria, yeast, insect cells, and mammalian cells. Examples of vectors include plasmids, phagemids, bacteriophages, viruses (e.g., baculovirus), cosmids, and F factors. Specific vectors can be used for specific cell types. In addition, shuttle vectors can be used for cloning and replication in more than one cell type. Such transport vectors are known in the art. The vector can, if desired, be a bifunctional expression vector that can function in multiple hosts.

[0070] An expression vector encoding a PHADase of interest may be introduced into a host cell by any method known to one skilled in the art, and the nucleic acid constructs may be transported extrachromosomally within a host cell or may be integrated into a host cell chromosome, as desired. A vector for use in a prokaryotic host, such Petition 870240024923, dated 03 / 22 / 2024, p. 45 / 96 / 68, as a bacterial cell, includes a replication system that allows its maintenance in the host for expression or for cloning and amplification. A vector may be present in the cell in a high or low copy number. Generally, about 5 to about 200, and normally about 10 to about 150 copies of a high copy number vector are present within a host cell. A host cell containing a high copy number vector will preferentially contain at least about 10, and more preferably at least about 20 plasmid vectors. Generally, about 1 to 10, and normally about 1 to 4 copies of a low copy number vector will be present in a host cell.

[0071] In many respects, bacteria are used as host cells. Examples of bacteria include, but are not limited to, Gram-negative and Gram-positive organisms. In one respect, an E. coli can be used as a suitable expression system for the expression of the T7 protein. Examples of T7-expressing strains may include, but are not limited to, BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, Tuner(DE3)pLysS, Tuner(DE3), Lemo21(DE3), NiCO2(DE3), Oragami2(DE3), Origami B(DE3), Shuffle T7 Expres, HMS174(DE3), HMS174(DE3)pLysS, DH5aplhaE, Rosetta2(DE3), Rosetta2(DE3)pLysS, NovaBlue(DE3), Rosetta-gami B, Rosetta-gami B(DE3), Rosetta-gami B(DE3)pLysS, Rosetta Blue (DE3), Novagen(DE3), Novagen(DE3)pLysS.

[0072] An expression vector can be introduced into bacterial cells by commonly used transformation / infection procedures. A nucleic acid construct containing an expression cassette can be integrated into the genome of a bacterial host cell through the use of an integration vector. Integration vectors generally contain at least one sequence that is homologous to the bacterial chromosome, allowing for vector integration. Integration vectors Petition 870240024923, dated 03 / 22 / 2024, pp. 46 / 96 / 68, may also contain bacteriophage or transposon sequences. Extrachromosomal and integration vectors may contain selectable markers to allow selection of bacterial strains that have been transformed.

[0073] Useful vectors for an E. coli expression system can contain constitutive or inducible promoters to target the expression of fusion or non-fusion proteins. With fusion vectors, several amino acids are typically added to the expressed target gene sequence. In addition, a proteolytic cleavage site can be introduced at a location between the target recombinant protein and the fusion sequence. Once the fusion protein is purified, the cleavage site allows the target recombinant protein to be separated from the fusion sequence. Suitable enzymes for use in cleaving the proteolytic cleavage site include TEV, Factor Xa, and thrombin.Fusion expression vectors that may be useful at present may include those expressing, for example and without limitation, Maltose Binding Protein (MBP), Thioredoxin (THX), Chitin Binding Domain (CBD), Hexahistadine marker (His marker), glutathione-protein S-transferase (GST), FLAG peptide, N-utilization substance (NusA) or modified small ubiquitin (SUMO) fused to the recombinant target enzyme.

[0074] Methods for introducing exogenous DNA into a host cell are available in the art and may include transformation of bacteria treated with CaCl2 or other agents such as divalent cations and DMSO. DNA can also be introduced into host cells by electroporation, use of a bacteriophage, ballistic transformation, coprecipitation with calcium phosphate, spheroplast fusion, electroporation, treatment of host cells with lithium acetate, or by electroporation. Transformation procedures generally vary according to the bacterial species to be transformed.

[0075] After transformation or transfection of a nucleic acid Petition 870240024923, dated 03 / 22 / 2024, p. 47 / 96 / 68 In a cell, the cell can be selected for the presence of nucleic acid through the use of a selectable marker. A selectable marker is usually encoded in the nucleic acid being introduced into the recipient cell. However, co-transfection of a selectable marker can also be used during the introduction of nucleic acid into a host cell.Selectable markers that can be expressed in the recipient host cell may include, but are not limited to, genes that make the recipient host cell resistant to drugs such as actinomycin C1, actinomycin D, amphotericin, ampicillin, bleomycin, carbenicillin, chloramphenicol, geneticin, gentamicin, hygromycin B, kanamycin monosulfate, methotrexate, mitomycin C, neomycin B sulfate, novobiocin sodium salt, penicillin G sodium salt, puromycin dihydrochloride, rifampicin, streptomycin sulfate, tetracycline hydrochloride, and erythromycin. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways. After transfection or transformation of a host cell, the cell is brought into contact with an appropriate selection agent.

[0076] In one aspect, a depolymerization component of a process may utilize a combination of different microorganisms and / or enzymes, including combinations of transformed and natural microorganisms and / or modified and natural enzymes. For example, in one aspect, a depolymerization component of a process may include one or more microorganisms that naturally secrete the depolymerase enzyme combined with one or more microorganisms that have been genetically modified to secrete the depolymerase enzyme. A genetically modified microorganism, for example, may be used to adjust a system based on environmental conditions and feed supply, among other characteristics. Petition 870240024923, dated 03 / 22 / 2024, page 48 / 96 43 / 68

[0077] By way of example, one or more natural and / or transformed microorganisms and / or their respective enzymes may be used based on one or more of the following factors: easy and rapid to grow at high density, does not require special media, aerobic, kinetically fast, stable, tolerant to an extreme environment (e.g., high salt environment, extreme temperature environment, etc.), capable of producing easily purifiable enzymes, does not have an unusual isoelectric point, does not require enhanced biosafety measures, does not contain excess cysteine ​​residues, is generally non-esoteric and available for commercial purchase. For example, the present disclosure has found that by limiting the amount of cysteine ​​residues, improved folding can be achieved. Selection based on one or more of the above factors may further improve the speed and efficiency of the depolymerization component, allowing for better industrial process yield. Metabolic Polymer Production

[0078] According to the disclosed methods, the HA monomer released after the depolymerization of PHA contained in a post-consumer product (hereinafter referred to as post-consumer HA monomer) can be brought into contact with a microorganism capable of producing PHA. The contact can occur under conditions that stimulate the metabolism of the HA monomer by the organism as a carbon source and the subsequent metabolic production of PHA by the microorganism. The PHA thus produced can be suitable for use in the formation of new consumer products. Thus, a circular and sustainable polymeric system can be created.

[0079] Culture conditions can be controlled to stimulate HA metabolism and PHA production by the organism. Such conditions may include deprivation of nutrient sources, optionally in conjunction with other environmental conditions that may stress the microorganism and Petition 870240024923, dated 03 / 22 / 2024, p. 49 / 96 44 / 68 encourage PHA production as an energy sink.

[0080] In one embodiment, the culture conditions may include limiting carbon sources to the microorganism. For example, post-consumption HA may be supplied to the body as the sole metabolic carbon source. In some embodiments, other carbon sources may be included in the culture, but may be present in limited amounts, for example, at micromolar concentrations. For example, any metabolic carbon source (e.g., sugars, organic acids, etc., or combinations thereof) present in a culture other than the post-consumption HA monomer may be present at a concentration of about 2 mM or less, such as about 1 mM or less, or about 500 mM or less.

[0081] Stressful culture conditions that can encourage HA metabolism and PHA production by a microorganism may include the deprivation of other nutrients, for example, nitrogen- and / or phosphate-containing nutrients, in order to encourage PHA formation as an energy sink for the organism. In one embodiment, culture conditions may include the deprivation of phosphate-containing nutrients in order to prevent the formation of polyphosphate by the microorganism, which is another common metabolic process used by microorganisms as an energy sink during times of stress. Deprivation of a nutrient source may include the complete absence of the nutrient source in the culture or the presence of the nutrient source in amounts that stress the microorganism's metabolic processes, for example, about 50% or less of the nutrient concentration present in an unstressed culture environment.

[0082] Stressful cultivation conditions may include environmental conditions, optionally in conjunction with stressful nutritional conditions. For example, a culture may be carried out near the limits of pressure, temperature, salinity, pH, etc., within which the microorganism normally functions. As an example, a mesophilic organism may be Petition 870240024923, dated 03 / 22 / 2024, p. 50 / 96 45 / 68 cultivated under temperature conditions of about 15°C to about 25°C or about 40°C to about 50°C (near the end of the normal growing range), under saline conditions of about 0.5 parts per thousand (ppt) to about 8 ppt or about 15 ppt to about 22 ppt, under pH conditions of about pH 4 to about pH 6 or about pH 9 to about pH 10, under pressure conditions of about 10 kPa to about 30 kPa or about 100 kPa to about 120 kPa, or any combination thereof.

[0083] Microorganisms for use in the PHA production component of a process can be selected and / or modified to overcome problems or deficiencies in order to form PHA by using a metabolic process using HA as a carbon source. In particular, microorganisms can be selected based on factors such as those mentioned above in relation to microorganisms for use in a depolymerization component of a process. Such factors may include, but are not limited to, easy and rapid growth at high density, not requiring special media, aerobic, kinetically fast, stable, tolerant to an extreme environment (e.g., high salt environment, extreme temperature environment, etc.).), capable of producing easily purifiable enzymes, do not have an unusual isoelectric point, do not require enhanced biosafety measures, do not contain excess cysteine ​​residues, are generally non-esoteric, are commercially available for purchase, or a combination of these.

[0084] In some respects, a microorganism for metabolic production of PHA using post-consumer PA monomer can also produce a PHADase that can be used (as produced or purified) or modified for use in the depolymerization component of a process. Species of the genus Lysobacter can be used in one embodiment, as they can produce PHADase capable of actively hydrolyzing PHB and also contain the biochemical machinery to metabolize HB and Petition 870240024923, dated 03 / 22 / 2024, page 51 / 96 / 68 to internally form PHB. Lysobacter species are naturally found in soil, and by using such species, a modality of a process can be conducted as part of a compostable process instead of requiring the formation and processing of a liquid culture in a bioreactor or series of bioreactors.

[0085] Exemplary Lysobacter species that produce both a PHA polymerase and a depolymerase that can be used in a process may include, without limitation, those given in Table 1 below: Table 1 Organism Accession number Polymerase Despolymerase Lysobacter aestuarii WP_141519092 QDH70273 Lysobacter antibioticus WP_057917797 WP_075575206 Lysobacter antibioticus WP_064749485 WP_057971776 Lysobacter antibioticus WP_031370714 WP_057970457 Lysobacter bugurensis WP_189454736 WP_189453172 Lysobacter capsici WP_036103061 WP_036102479 Lysobacter capsici WP_191821024 WP_082723829 Lysobacter enzymogenes WP_057947866 WP_074867011 Lysobacter enzymogenes WP_206409599 WP_206412663 Lysobacter enzymogenes WP_123648422 WP_096378935 Lysobacter enzymogenes WP_078996336 WP_096378891 Lysobacter lacus WP_149351326 WP_149353094 Lysobacter lycopersici WP_143878270 WP_111268077 Lysobacter maris WP_111268029 WP_141481346 Lysobacter niastensis WP_194931164 WP_194930566 Lysobacter profundi WP_159015985 WP_199268782 Lysobacter sp. MBA2238340 MBA3486130 Lysobacter sp. NOT90012 NOT88901 Lysobacter sp. TXI44079 TXI49260 Lysobacter sp. TBR06965 TBR07230 Lysobacter sp.A03 WP 043958955 WP 043958589 Lysobacter sp. cf310 WP_091637072 SFK67843 Lysobacter sp. H21R20 WP_193987019 WP_193986963 Lysobacter sp. H21R4 WP_194342245 WP_194342197 Lysobacter sp. H23M41 WP_194035564 WP_194035504 Lysobacter sp. R19 WP 200614426 MBK3415203 Lysobacter sp. Root604 WP 056175356 WP 056174125 Lysobacter sp. Root690 WP_056115057 WP_056115653 Lysobacter sp. Root916 WP_057163275 WP_082578417 Lysobacter sp. Root983 WP_057159495 WP_057162992 Lysobacter sp. TY2-98 WP_115646306 WP_057159102 Lysobacter spongiae WP 182687030 WP 182685163 Lysobacter spongiicola WP_078757079 WP_200809237.

[0086] Naturally, the species for use in the metabolic formation of new PHA from post-consumption HA monomer are not limited. Petition 870240024923, dated 03 / 22 / 2024, page 52 / 96 / 68 to those that also produce a PHADase, and a PHA-producing microorganism may be used in conjunction with one or more other microorganisms and / or PHADases that differ from each other, as discussed previously. Exemplary Lysobacter species that are documented only as producing a PHA polymerase as may be used in a process may include, without limitation, those provided in Table 2 below: Table 2 Organism Polymerase Accession Number Lysobacter WP_036193982 Lysobacter alkalisoli WP_141625093 Lysobacter arseniciresistens WP_036208009 Lysobacter daejeonensis WP_036135021 Lysobacter dokdonensis WP_036168095 Lysobacter enzymogenes WP_207524961 Lysobacter enzymogenes WP_096377760 Lysobacter enzymogenes WP_074869551 Lysobacter gilvus WP_156641946 Lysobacter gummosus WP_057943197 Lysobacter maris WP_141483002 Lysobacter oculi WP_112926105 Lysobacter panacisoli WP 200604936 Lysobacter penaei WP 182668477 Lysobacter prati WP_158731614 Lysobacter psychrotolerans WP_123087040 Lysobacter pythonis WP_122100479 Lysobacter ruishenii WP_144812683 Lysobacter segetis WP_133478701 Lysobacter silvestris WP_103075695 Lysobacter silvisoli WP_115858207 Lysobacter soli WP_157029884 Lysobacter sp. NUO78313 Lysobacter sp. 17J7-1 WP_133500014 Lysobacter sp. Alg18-2.2 WP 147890376 Lysobacter sp. Cm-3-T8 WP 206859118 Lysobacter sp. H23M47 WP_194037433 Lysobacter sp. HDW10 WP_166296513 Lysobacter sp.II4 WP_187713470 Lysobacter sp. N42 WP_132328958 Lysobacter sp. OAE881 WP 192630396 Lysobacter sp. Root494 WP 056131727 Lysobacter sp. URHA0019 WP_027083001 Lysobacter sp. WF-2 WP_117202823 Lysobacter sp. yr284 WP_091793341 Lysobacter tabacisoli WP_119719022 Lysobacter telluris WP_166211016 Lysobacter tolerans WP_076587639 Lysobacter tolerans SIP87483 Lysobacter xinjiangensis WP_189447436 Lysobacter unclassified WP_055899693. Petition 870240024923, dated 03 / 22 / 2024, p. 53 / 96 / 68

[0087] Other organisms may be used, in conjunction with or instead of those of the genus Lysobacter. As an example, and without limitation, Table 3 presents examples of other species and ascending numbers for their PHA polymerase that may be used and that may be encouraged to metabolize the HA monomer as a carbon source in the production of new PHA. Table 3 Organism No. of Ascent Organism No. of Ascent Aliivibrio finisterrensis WP_151654375 Rhodanobacter lindaniclasticus WP_ .136257156 Aliivibrio fischeri WP_065624776 Rhodanobacter panaciterrae WP_ .189440331 Aliivibrio sifiae WP 105055326 Rhodanobacter sp. 7MK24 WP 192155134 Aliivibrio sp MBL4831209 Rhodanobacter sp. A1T4 WP 184673302 Aliivibrio sp. 1S128 WP 065600195 Rhodanobacter sp. B04 WP 077555812 Aliivibrio sp. EL58 WP_122034402 Rhodanobacter sp. B05 WP_ .077513483 Aliivibrio sp. SR45-2 WP_182699437 Rhodanobacter sp. C01 WP_ .077442012 Caballeronia arvi WP_061150199 Rhodanobacter sp. C03 WP_ .077518181 Caballeronia calidae WP_062608567 Rhodanobacter sp. C05 WP_ 077443954 Caballeronia hypogeia WP_061169280 Rhodanobacter sp. C06 WP_ .077485236 Caballeronia insecticola BAN58336 Rhodanobacter sp. DHB23 WP_ .192106892 Caballeronia pedi WP_061178553 Rhodanobacter sp. DHG33 WP_ .192163461 Caballeronia terrestris WP_087660849 Rhodanobacter sp. L36 WP_ .158885070 Dokdonella koreensis WP_067647850 Rhodanobacter sp. MP1X3 WP_ .184604847 Dyella caseinilytica WP 188798656 Rhodanobacter sp. OK091 WP 072760944 Dyella choica WP 126682794 Rhodanobacter sp. OR444 WP 027492196 Dyella dinghuensis WP_126672795 Rhodanobacter sp. PCA2 WP_ .181302403 Dyella flava WP_204681682 Rhodanobacter sp. Raíz480 WP_ .056080179 Dyella jiangningensis AHX12796 Rhodanobacter sp. Raíz627 WP_ .082545971 Dyella kyungheensis WP_204634561 Rhodanobacter sp. Raíz627 KRA35976 Dyella mobilis WP_204632428 Rhodanobacter sp. SCN 67-45 ODT97084 Dyella monticola WP_115496150 Rhodanobacter sp. SCN 68-63 ODV10878 Dyella nitratireducens WP_188792429 Rhodanobacter sp. Soil772 WP_ .056386006 Dyella psychrodurans RDS86489 Rhodanobacter sp. T12-5 WP_ .149365305 Dyella soli WP_131407398 Rhodanobacter sp. TND4EH1 WP_ .099652471 Dyella solisilvae WP_114823339 Rhodanobacter sp. TND4FH1 WP_ .133950922 Dyella sp. 7MK23 WP 192556083 Rhodanobacter spathiphylli WP 007805234 Dyella sp.ASV21 WP 199100073 Rhodanobacter thiooxydans WP 008435591 Dyella sp. ASV24 WP_199038667 Stenotrophomonas chelatiphaga WP_ .057508611 Dyella sp. C11v WP_157956602 Stenotrophomonas maltophilia WP_ .019338202 Dyella sp. C9 WP_114241222 Stenotrophomonas panacihumi WP_ .057643119 Dyella sp. DHC06 WP_130620551 Stenotrophomonas pavanii WP_ .057494653 Dyella sp. EPa41 WP 201314821 Stenotrophomonas rhizophila WP 038687867 Dyella sp. G9 WP 187056353 Stenotrophomonas sp. DDT-1 WP 061479060 Dyella sp. M7H15-1 WP_164931796 Stenotrophomonas sp. RIT309 WP_ .032976188 Dyella sp. M7H15-1 QAU23859 Stenotrophomonas sp. SKA14 WP_ .008265690 Dyella sp. OK004 WP_090451505 Vibrio aestuarianus WP_ .168520800 Dyella sp. S184 WP_158755276 Vibrio antiquarius WP_ .074190087 Dyella sp. SG562 WP 167257616 Vibrio aquaticus WP 126574305 Dyella sp. SG609 WP_168647555 Vibrio tasmaniensis WP_ .102248967 Dyella sp.YR388 WP_147455377 Xanthomonadales Bacteria OZB58863 Dyella tabacisoli WP_114845894 Xanthomonas albilineans WP_ .012916138 Fluoribacter bozemanae WP_058459 Xanthomonas ar boricola WP_144 .039511932 Fluoribacter dumoffii NY 23 KTC90057 Xanthomonas axonopodis WP_ .042822558. Petition 870240024923, of 22 / 03 / 2024, p. 54 / 96 49 / 68 Fluoribacter gormanii KTD05403 Xanthomonas bromi PPV05022 Microscilla marina WP_002702565 Xanthomonas campestris WP_011037305 Pseudomonas aeruginosa AHJ25666 Xanthomonas cannabis WP_047694901 Pseudomonas thermotolerans WP_027896668 Xanthomonas citri WP_046832369 Pseudomonas mediterranea WP_047699726 Xanthomonas euvesicatoria WP_136732577 Psychrobacter sp. QCF41916 Xanthomonas fragariae WP 002802267 Psychromonas sp. MB-3u-54 WP 101038601 Xanthomonas hortorum WP 006450930 Psychromonas sp. psych6C06 WP_101107093 Xanthomonas hyacinthi WP_046978386 Psychromonas sp. RZ22 WP_134276148 Xanthomonas oryzae WP_014503544 Psychromonas sp. Urea-02u- 13 WP_101081048 Xanthomonas phaseoli WP_017157553 Rhodanobacter denitrificans NMW25143 Xanthomonas pisi WP_046964104 Rhodanobacter fulvus WP_040670830 Xanthomonas sacchari WP_043092075 Rhodanobacter glycinis WP_140650985 Xanthomonas sp. Leaf! 31 WP_055826366 Xanthomonas sp.NCPPB 1128 WP_048489717 Xanthomonas translucens WP_003466505 Xanthomonas vasicola WP_039434864 Xanthomonas vesicatoria WP_039424128.

[0088] In some embodiments, the selection of microorganisms for the PHA production component can be chosen to meet the needs of the depolymerization process. For example, if there is a need / desire to run both components of a process simultaneously or separately, but at the same elevated temperature, then the enzyme(s) and / or microorganisms can be thermophilic, functioning in the same temperature range. Similarly, if there is a need or desire to run both components of a process in the presence of high salt content, then the enzyme(s) and / or microorganisms to be used can be halophilic.Similarly, if less extreme conditions are desired for the decontamination and depolymerization process, for example, due to known contaminants, and it is desired to run the metabolic production component under the same conditions, then extremophilic enzyme(s) and / or microorganisms can be used for both components that exhibit high activity under these less extreme conditions.

[0089] Alternatively, in those modalities in which the two components of a process are carried out separately from each other, that is, at different times and / or locations, the selection of microorganisms for the metabolic component of PHA production can be selected based Petition 870240024923, dated 03 / 22 / 2024, page 55 / 96 50 / 68 in the desired production parameters, for example, kinetics, efficiency, etc. In such an embodiment, the two components of a process may be carried out under the same conditions or under different conditions and, when carried out under different conditions, the conditions may differ in one or more environmental conditions, including, without limitation, temperature, salt content, pressure, acid / alkaline content, radiation, etc. Bioreactor System

[0090] A bioreactor system can be used to carry out the disclosed methods. A bioreactor system may include a single reaction vessel or multiple reaction vessels, as it may be used to carry out a depolymerization component and a metabolic production component, as discussed in this document. By way of example, FIG. 4 illustrates a bioreactor system including a single vessel 10 within which the depolymerization of a feed 2 containing PHA and of a polymer can be carried out. FIG. 5 illustrates another bioreactor system including a first vessel 110 within which the depolymerization of a PHA feed can be carried out and a second vessel 120 within which a microorganism can be grown together with the HA monomer 4 obtained from the first vessel 110 to produce new PHA.Furthermore, a single container 10 may be composed of two or more internal compartments that are tightly connected, so as to separate components of an overall process and allow separation operations (e.g., filtration), modification of environmental factors, modification of processing aids, etc., between components of the overall process.

[0091] In single or multiple vessel modalities, a process can be controlled manually or automatically. For example, a process can be carried out according to a continuous, semi-discontinuous, discontinuous process or perfusion mode with any combination of Petition 870240024923, dated 03 / 22 / 2024, pp. 56 / 96 / 68 automatic and manual control mechanisms.

[0092] By way of example, ration 2 containing PHA may contain discarded incontinence products or other post-consumer polymer-based products that include one or more PHAs. Incontinence products include, for example, diapers, training diapers, swim trunks, adult incontinence products, feminine hygiene products and the like. These products typically include a water-permeable liner, an outer cover and an absorbent structure positioned between the water-permeable liner and the outer cover. Incontinence products may contain biopolymers in amounts exceeding about 5% by weight, such as in amounts exceeding about 10% by weight, such as in amounts exceeding about 20% by weight, such as in amounts exceeding about 30% by weight.weight, such as in quantities exceeding about 40% by weight, such as in quantities exceeding about 50% by weight, such as in quantities exceeding about 60% by weight, such as in quantities exceeding about 70% by weight.

[0093] Feed 2 containing PHA can be fed into a 10 / 110 container in any suitable form. For example, feed containing PHA may include the polymer in its original consumable form, or a post-consumer product may be pre-treated, for example, it may be chopped or crushed, before the feed is added to a 10 / 110 container. For example, as discussed above, in some embodiments, little or no pre-cleaning or sanitizing step is performed before the feed containing PHA 2 is added to a 10 / 110 container, for example, in those embodiments where one or both components of a process are carried out in an extreme environment in which contaminants can be degraded or otherwise rendered non-hazardous.

[0094] As shown in FIG. 4 and FIG. 5, feed 2 including one or more post-consumer PHA polymers can be fed into Petition 870240024923, dated 22 / 03 / 2024, p. 57 / 96 / 68 container 10 / 110 within which the PHA of the feed can be depolymerized by the action of one or more suitable PHADases. A PHADase 8 can be supplied to container 10 / 110 as a purified PHADase in an enzyme-based depolymerization.

[0095] Alternatively, a depolymerization component may be a bacteria-based process, in which case a PHADase may be expressed within a vessel 10 from a suitable microorganism culture, which may optionally be fed to a vessel 10 before, simultaneously with, or after the introduction of feed 2 into the vessel 10. As discussed previously, combinations of PHADase sources are also covered in this document, including combinations of purified natural and / or modified PHADase that may optionally be combined with one or more microorganisms expressing PHADase, and such microorganisms may equally express any combination of natural and modified PHADase.

[0096] Additional materials, as they would be known in the art, can be combined with the feed 2 and the PHADase of the depolymerization component. For example, suitable growth media may be required if a bacteria-based depolymerization component is used. Similarly, in those embodiments where a depolymerization component is carried out in an extreme environment, suitable materials, for example, salts, acids, bases, etc., can be fed to a 10 / 110 container.

[0097] The conditions within a 10 / 110 vessel can be controlled as needed to encourage the depolymerization of a PHA polymer. For example, a 10 / 110 vessel may include a stirrer that can run continuously through a depolymerizing component or can be started and stopped intermittently, as is known in the art. Petition 870240024923, dated 03 / 22 / 2024, pp. 58 / 96 / 68

[0098] In a single-vessel system, as illustrated in FIG. 4, a depolymerization component and a PHA-forming component can be carried out simultaneously or separately. For example, in one embodiment, a PHA depolymerase reaction can proceed until all or a portion of the PHA has been depolymerized, after which the HA monomer can be used as a carbon source in the metabolic production of PHA polymer by microorganisms. For example, and as will be discussed in greater detail in relation to the examples below, the reaction can proceed until a decrease in PHA depolymerase is observed, for example, according to visual observation of a system, fluorescence emission from a system, or according to the passage of a predetermined reaction time period. In one aspect, the completion of the depolymerization component of a process can be determined by determining a plateau in enzymatic activity.Alternatively, one or more optical or visual measurements can be performed. For example, PHB is insoluble in most solvents, creating a high optical density at 600 nm, where HB is generally soluble due to its lower molecular weight. Therefore, a decreased optical density, such as identifying an optical density of approximately 0.9 or less, approximately 0.8 or less, approximately 0.7 or less, approximately 0.6 or less, approximately 0.5 or less, approximately 0.4 or less, approximately 0.3 or less, approximately 0.2 or less, or approximately 0.1 or less, as measured at 600 nm, can be used to determine that the PHB from a feed 2 has been depolymerized to provide HB monomer.

[0099] After determining the completion of a depolymerization component of a process, a microorganism 9 can be fed into the container along with any additional components needed for the culture and in conjunction with any variation in the container environment. Petition 870240024923, dated 03 / 22 / 2024, p. 59 / 96 / 68, is necessary for the culture. The previously produced HA monomer can then be available as a carbon source for the microorganism culture.

[00100] In one aspect, microorganisms for use in any component of a process can be encapsulated in a carrier, such as a polymer carrier. A polymer carrier can be a highly water-absorbent material without being water-soluble. In one aspect, for example, a polymer carrier is in the form of a gel when combined with water, can be dehydrated and converted into a solid form, and then capable of being rehydrated when it comes into contact with moisture. In this way, one or more microorganisms can be combined with the polymer carrier in the form of a gel. Once mixed, the water can be removed to form a solid. The solid can be formed into any suitable shape and come into contact with post-consumer product waste materials 104. To degrade the polymers contained in the waste material, the solid material comes into contact with moisture which causes the carrier polymer to be rehydrated.Once rehydrated, the microorganisms can be released from the polymeric gel or they can secrete enzymes that are released from the polymeric gel.

[00101] A single-vessel system as illustrated in FIG. 4 can, in one embodiment, be used to perform both components of a process simultaneously. For example, a single microorganism that expresses a PHADase and is also capable of metabolizing the HA monomer thus released as a carbon source to produce new PHA can be cultivated within the single reaction vessel 10 in conjunction with a feed 2. As discussed previously, such a microorganism can be used alone or in conjunction with additional enzyme and / or co-cultivated with additional microorganism to fit a system.

[00102] A process can continue until the carbon source of Petition 870240024923, dated 03 / 22 / 2024, pages 60 / 96 / 68 HA is depleted and microorganism growth and PHA production cease, after which the microorganism can be removed from the container and post-processing carried out, including, for example, polymer extraction, polymer purification, residue removal, etc., to produce a PHA product suitable for use in the formation of a new product. The determination of the completion of a production batch can be determined according to standard procedures, such as those discussed above and in the example section below, to determine a plateau in growth and / or a depletion of the carbon source in the culture medium. In some embodiments, a continuous system can be used in which microorganisms are removed from a container continuously or in a semi-batch approach and subjected to post-processing for new polymer extraction.

[00103] FIG. 5 illustrates a two-vessel system in which a first depolymerization component of a process is carried out in a first vessel 110 and after depolymerization of PHA from a feed 2 by the use of one or more purified PHADase 8 fed to vessel 10, PHADase expressed by one or more microorganisms grown within vessel 10 together with the feed, or any combination thereof, the HA monomer thus formed can be removed from the first vessel 10 and fed 4 to a second vessel 120, where it can be metabolized by the microorganism 9 grown within the second vessel 120.

[00104] In such an embodiment, the HA monomer stream 4 can be processed before being used as a carbon source by microorganisms 9 in the second container 120 in a metabolic process. For example, the HA monomer can be separated from other components of a stream 4 by filtration. In one aspect, the HA monomer can be separated by using a size exclusion filter (such as a filter of Petition 870240024923, dated 22 / 03 / 2024, p. 61 / 96 / 68 molecular weight cutoff), having a size of about 30 kD or less, such as about 25 kD or less, such as about 20 kD or less, such as about 15 kD or less, such as about 10 kD or less, such as about 5 kD or less, such as about 4.5 kD or less, such as about 4 kD or less, such as about 3.5 kD or less, such as about 3 kD or less, such as about 2 kD or more, such as about 3 kD or more, or any intervals or values ​​between them.

[00105] For example, a filter can be appropriately sized to retain any remaining pieces of post-consumer product and any remaining PHA, as well as any remaining PHADase. Retaining PHADase can be useful as it can prevent the depolymerization of PHA produced in the second container 120. Furthermore, removing larger residual particles before the formation of new polymer can be used to control the environment within the second container 120.

[00106] In one embodiment, stream 4 of a depolymerization component can be subjected to an ion exchange filter (such as a Dowex ion exchange bed). This can be particularly beneficial when the enzyme used in the depolymerization component differs with respect to preferred environment compared to the microorganism used to metabolically produce the new polymer. By way of example, in those embodiments where a halophilic enzyme is used in a depolymerization component (e.g., when the environment inside the first vessel 110 includes a high salt concentration). Treatment of stream 4 by using an ion exchange filter can remove the salt from stream 4 and provide a salt-free HA monomer to the second vessel 120.

[00107] Similar to a single-vessel system, a two-vessel system can allow the microorganism culture to grow until the HA carbon source is depleted and microorganism growth and PHA production cease, after which the microorganism can be removed 112 from Petition 870240024923, dated 03 / 22 / 2024, page 62 / 96 / 68 container 120 and the post-processing 30 carried out including, for example, polymer extraction, polymer purification, residue removal, etc. to produce a PHA 14 product suitable for use in the formation of a new product.

[00108] This disclosure can be better understood with reference to the following examples. Example 1 Chemical products

[00109] All general chemicals, media components, and granular PHB were obtained from Millipore-Sigma, Inc. Molecular biology reagents, including competent cells, PCR reagents, and all protein purification reagents, were purchased from New England Biolabs, Inc. Bacterial growth Lysobacter enzymogenes (ATCC 55439) was cultured in Trypticase Soy Broth (per liter: 17.0 g tryptone, 3.0 g soybean, 2.5 g dextrose, 5.0 g NaCl, 2.5 g K2HPO4; pH 7.3) at 30°C. Overnight cultures were harvested by centrifugation at 10,000 x g for 15 minutes, resuspended in phosphate-buffered saline (PBS), and recentrifuged to remove all medium components. This was performed twice. The final bacterial sediment was resuspended to a final concentration of 1.0 x 10⁸ CFU / mL in PBS before use. For PHB depolymerization or polymerization reactions, washed cells were transferred to flasks containing M9 medium supplemented with 1x MEM amino acids, 1.0 mM glutamine, 1x M9 salts, 1x MEM vitamins, and 10% (w / v) hydroxybutyrate. Growth occurred at 30°C. PHB Depolymerase Expression Construct

[00111] The amino acid sequence of L. enzymogenes depolymerase PHB (WP_074867011) was used as a recombinant protein source. Petition 870240024923, dated 03 / 22 / 2024, page 63 / 96 / 68 overexpressed. The recombinant L. enzymogenes PHBDase is composed of 570 amino acids and has a mass of 59.1 kDa (including N-ter glycine). The enzyme has a pI of 4.75 and contains eight cysteine ​​residues. The protein sequence is provided in FIG. 1 (SEQ ID NO: 1). The 11 tryptophan residues transmit a significant fluorescent signal that is useful for stability and unfolding studies. Protein purification was straightforward using a T7 expression system, and homogeneous enzyme was produced with a yield of 23.4 mg / L.

[00112] The first 35 amino acids (MSAVRSLHRSAPRAARWLSLSVLLAGVCCAAPAFA - SEQ ID NO: 2) constituted a signal sequence, and this sequence was removed from the construct. A histidine expression sequence and a TEV protease cleavage signal sequence: MHHHHHHGSENLYFQG (SEQ ID NO: 3) were attached to the amino-terminal portion of the enzyme sequence. After cleavage, the recombinant proteins had an N-ter sequence that began with a glycine residue. This new amino acid sequence was reverse-translated into DNA and codon-optimized for expression in E. coli using the Gene Designer program from ATUM, Inc. The gene was assembled using standard PCR techniques from ATUM, Inc. and cloned into the p454-MR expression vector (ampr, medium-strength ribosomal binding site). Insertion was verified by DNA sequencing after construct. Expression and purification of PHB Depolymerase

[00113] The expression plasmid was used to transform chemically competent Oragami2-(DE3) bacteria. Individual colonies were selected from LB-Amp plates and used for expression screening. The colonies were cultured at 37°C for 12 hours in LB medium supplemented with 100 μg / mL ampicillin. This culture was used to inoculate fresh LB-AMP vials at a 1:100 inoculum. These cultures were grown at Petition 870240024923, dated 03 / 22 / 2024, pages 64 / 96 / 68 The cells were incubated at 37°C until OD595 = 0.4 (typically 4 hours), at which point isopropyl eD-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Growth was continued for 12 hours. Cells were harvested by centrifugation at 10,000 x g for 15 minutes and frozen at -80°C until use (minimum freezing time was 24 hours). Cells were thawed on ice and resuspended in Buffer A (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9) (typically 1 mL per gram of cells). Cells were disrupted by two passes through a French press, followed by centrifugation at 30,000 x g for 30 minutes. The crude extract was mixed with an equal volume of loaded His-Bind resin paste, and the mixture was poured into a 5 cm x 4.9 cc column. The column was washed with 10 volumes of column wash buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9) at a flow rate of 0.2 mL / min.The enzyme was eluted from the column by adding 3 column volumes of 0.5 M NaCl, 20 mM Tris-HCl, 1.0 M imidazole, pH 7.9. Fractions were collected (1.0 mL). Enzyme-containing fractions were pooled after SDS-PAGE analysis. The pooled fractions were applied to a 70 cm x 4.9 cc Sephadex G-75 column (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Homogeneous protein-containing fractions were pooled (after SDS-PAGE inspection) and concentrated to 5 mg / mL using Centricon filters. The enzyme was stored frozen at -20°C until use. The histidine-labeled region was removed from the enzymes using TEV protease. The protein was diluted to 1.0 mg / mL in 10 mM Tris-HCl, pH 7.5, 25 mM NaCl. 100 U of TEV protease were added per mg of enzyme (approximate ratio of 1:100 (w / w)). The reaction was allowed to proceed for 16 hours at 4°C. The mixture was passed over a nickel-labeled column. A column volume of eluent was collected representing the purified, unlabeled enzyme. PHB depolymerase assay

[00114] An assay was used to measure β-hydroxybutyrate Petition 870240024923, dated 03 / 22 / 2024, pp. 65 / 96 60 / 68 directly using the Sigma-Aldrich MAK272 hydroxybutyrate assay kit. HB was measured fluorometrically (λ6Χ = 535 nm, λem = 587 nm). Aliquots (10 μE) were removed from the PHB depolymerase reaction at various times, mixed with 50 μE of the supplied HB assay buffer, and pipetted into one well of a 96-well flat-bottom black plate. The plate was incubated at room temperature in the dark for 30 minutes. Fluorescence emission intensity was measured using a Molecular Dynamics SpectraMax M5. Fluorescence readings were converted to HB concentration by comparison with a standard curve constructed from known concentrations of pure hydroxybutyrate. All kinetic parameters are calculated according to Segei (1993). PHB Film Formation

[00115] PHB films were formed by solvent casting from heated chloroform, as described by (Anbukarasu and Sauvageau, 2015). PHB granules were mixed with chloroform to a final concentration of 50 mg / mL and the mixture was heated to 70°C (covered) with stirring for one hour (the time to completely dissolve the PHB granules). The solution was poured into glass Petri dishes to a depth of approximately 2 mm and the solvent was evaporated at 25°C. The samples were aged for five days (1.0 atm, 25°C) and then vacuum dried for 3 hours to remove any remaining chloroform. The final films were removed from the Petri dish and cut into 2 cm x 2 cm squares. PHB extraction from bacterial cells

[00116] PHB was extracted from the cells using a modified procedure (Mostafa et al., 2020). The bacterial pellets were dried at 70°C for two hours, and the dried cells were washed with acetone and ethanol for 20 minutes and treated with 50 mL of 30% sodium hypochlorite and 50 mL of chloroform. This mixture was incubated for 1 hour at 37°C in a shaker at 150°C. Petition 870240024923, dated 03 / 22 / 2024, page 66 / 96 / 68 rpm and then centrifuged at 10,000 xg for 45 minutes. The supernatant was decanted and the chloroform was evaporated under a stream of nitrogen at 40°C. The total mass of PHB was determined by weighing (after subtracting a simulated control reaction not containing PHB) and is expressed in g / L. Alternatively, the method of Arikawa et al (2017) was employed. In this method, the final bacterial sediment was sonicated in the presence of 0.5% SDS (the typical volume was 20 mL per liter of culture). The insoluble PHB was collected by centrifugation at 15,000 x g for 30 minutes, washed in water, recentrifuged, washed in ethanol, recentrifuged, and the final sediment was air-dried as above and weighed. Depolymerization / Polymerization Method A

[00117] Purified PHB depolymerase was added to supplemented M9 medium (less HB) at a final concentration of 10 mg / mL along with various amounts of PHB film. The flask was kept at 30°C with agitation at 250 rpm. Timed aliquots were removed and subjected to the described HB assay. Agitation was continued until all the PHB film was depolymerized (by visual inspection) or the measured HB concentration stagnated. The flask was boiled for ten minutes to completely inactivate the enzyme, followed by cooling to 30°C. This flask was then inoculated with a fresh culture of L. enzymogenes to a final concentration of 1.0 x 107 CFU / mL. Growth was initiated at 30°C with agitation at 250 rpm, and the number of cells was monitored by removing timed aliquots from the flask and measuring the optical density at 600 nm. After the start of the stationary phase, the cells were pelleted and PHB was extracted.During the PHB formation phase (approximately 48-72 hours), the extracellular HB concentration was measured by removing timed aliquots from the vial and testing the HB concentration as described. Petition 870240024923, dated 03 / 22 / 2024, pages 67 / 96 / 68

[00118] As illustrated in FIG. 2, purified L. enzimogenes PHBDase was efficient in degrading PHB films. Within three hours, the entire PHB film was converted to HB as shown in FIG. 2. This HB, in turn, was efficiently used to support the growth of L. enzimogenes when used as the sole carbon source, as shown in FIG. 3 where open circles indicate the reduction of HB in the flask and closed circles indicate bacterial growth. As indicated, throughout the 72-hour growth experiment, the number of bacterial cells increased (as measured by optical density at 600 nm) and the concentration of HB in the flask decreased. This method clearly showed that exogenously added HB could be used as the sole carbon source by L. enzimogenes. Bacterial growth only stabilized when the HB concentration was undetectable in the assay. Method B

[00119] Purified PHB depolymerase was added to supplemented M9 medium (less HB) at a final concentration of 10 mg / mL plus various amounts of PHB film. Washed L. enzimogenes cells were immediately added at a final concentration of 1.0 x 107 CFU / mL. The flask was allowed to continue growing for a further 72 hours. The cells were pelleted by centrifugation at 15,000 xg for 15 minutes and PHB was extracted from the pellet. During the PHB formation phase, the extracellular HB concentration was measured by removing timed aliquots from the flask and testing the HB concentration as described above.

[00120] As shown in FIG. 4, when culturing L. enzimogenes in the presence of PHB and PHBDase, growth (closed circles) began after a short interval, a period during which part of the PHB film was converted to HB by the purified L. enzimogenes PHBDase enzyme. As growth continued, the enzyme hydrolyzed more PHB film, Petition 870240024923, dated 03 / 22 / 2024, p. 68 / 96 / 68 releasing HB into the medium (open circles). This can be seen as an early increase in the fluorescence emission signal in FIG. 4. As indicated, HB accumulation peaked at approximately six hours, when there was an increase in bacterial growth (measured as optical density at 600 nm). Growth was linear until approximately nine hours, when the growth rate slowed to a lower level of linearity during the remainder of the examination period. This demonstrated that the purified enzyme was stable in the presence of a growing bacterial culture throughout the growth period and produced metabolically active HB. Method C

[00121] Supplemented M9 medium (minus HB) was inoculated with L. enzymogenes cells to a final concentration of 1.0 x 107 CFU / mL. Various amounts of PHB film were added to the flask. The flasks were kept at 30°C with agitation at 250 rpm and cell counts were monitored by withdrawing timed aliquots from the flask and measuring the optical density at 600 nm. These same aliquots were subjected to the described HB assay. The flask was allowed to continue growing for 48–72 hours. The cells were pelleted by centrifugation at 15,000 xg for 15 minutes and the PHB was extracted from the pellet.

[00122] As indicated in FIG. 5, HB did not need to be added exogenously to a growing bacterial culture, nor did a purified enzyme need to be added to the bacterial culture to convert a PHB-containing product into HB and to further convert that HB into metabolic material that drives the formation of new PHB accumulation in the bacterial cell. As indicated, L. enzymogenes could be used in a single-vessel reaction to biochemically drive the overall depolymerization / polymerization reactions. When the PHB film and bacteria were cultured together, there was an interval of approximately six hours before Petition 870240024923, dated 03 / 22 / 2024, pp. 69 / 96 / 68, stated that HB could be detected in the culture (open circles). Bacterial growth (closed circles) beyond the inoculum was not detectable for approximately 12 hours, until there was a certain degree of HB accumulation in the medium. At that point, linear growth began and continued throughout the examination period until approximately 60 hours. The HB concentration reached a maximum approximately 34 hours after inoculation and decreased until hour 60, where it stabilized at a low (but stable) level.

[00123] This approach was also examined using a second bacterial species (C2 method), Pseudomonas fluorescens, as the depolymerizing bacterial species. In this case, both bacterial genera were added at a final concentration of 5.0 x 10⁶ CFU / mL at time zero. Timed aliquots from the flask were removed to measure total bacterial density (OD at 600 nm) and to test the HB concentration as described above. The cells were pelleted by centrifugation at 15,000 x g for 15 minutes and PHB was extracted from the pellet.

[00124] P. fluorescens secretes a particularly kinetically fast PHBDase. The use of such a species could accelerate the rate of a process. As indicated in FIG. 6, measurable HB (closed circles) was observed in the co-culture within the first hour and reached a maximum plateau between two and 10 hours, at which point the HB concentration decreased linearly until hour 50, when it then slowly became unmeasurable. There was a significantly shorter lag period for the onset of L. enzymogenes growth (two hours; closed circles) compared to the data in FIG. 5. This was attributed solely to the faster availability of significant levels of HB. The growth of L. enzymogenes was biphasic, the first period of faster growth between two and eight hours, followed by a slower growth rate during the remainder of the experimental period. Method D Petition 870240024923, dated 03 / 22 / 2024, pp. 70 / 96 / 68

[00125] Intermediate-phase E. coli Origami-2 expression cells containing an expression plasmid encoding Pseudomonas geniculata PHBDase (with an added gram-negative signal sequence) were added at a final concentration of 1.0 x 10⁷ CFU / mL to M9 medium (less HB) supplemented with 0.5 mM IPTG. The flask also contained various amounts of PHB film. This flask was immediately inoculated with 1.0 x 10⁷ CFU / mL washed L. enzimogenes cells. The flask was maintained at 30°C with agitation at 250 rpm for 48–72 hours. The cells were pelleted by centrifugation at 15,000 x g for 15 minutes and PHB was extracted from the pellet. During the PHB formation phase, the extracellular HB concentration was measured by removing timed aliquots from the flask and testing the HB concentration as described above.

[00126] In this method, the enzyme was secreted after being produced in a bacterial expression system in the growth medium along with the L. enzimogenes culture. The P. geniculate PHBDase was cloned into a T7 expression plasmid and the construct was transformed into an expression-competent strain of E. coli. In the presence of IPTG, the bacteria begin to overproduce the enzyme and secrete it into the medium. This initiated the depolymerization of the PHB film in the flask and the subsequent growth of the L. enzimogenes culture. The expression systems were highly efficient, such that the observed latency period was less than 30 minutes before HB was measurable in the fluorescent assay. These results are shown in FIG. 7. Selection pressure was maintained in E. coli by adding ampicillin to the medium (the vector contained the resistance gene amp).

[00127] At the end of each method, the bacterial cells were collected by centrifugation and frozen at -20°C until all experiments were completed. PHB was extracted from the cell mass. Petition 870240024923, dated 03 / 22 / 2024, pp. 71 / 96 / 68 as described in Methods. A control extraction was processed containing a similar mass of L. enzimogenes bacteria that were cultured in a rich medium (and therefore should be devoid of intracellular PHB). At the end of the reaction, the total amount of material remaining in the reaction tubes was weighed and the mass of the control extraction was subtracted from that value. Typically, the mass in the control extraction was only 0.02g.

[00128] The resulting extraction data are shown in FIG. 8. As shown, PHB yields were similar for all methods in this work. The control experiment (Con) represented a similar growth mass of L. enzimogenes in Tryptic Soy Broth medium with glucose as a carbon source. It should be noted that no effort was made to optimize any of the methods beyond what has been described, and with such optimization, as would be evident to anyone skilled in the art, increased PHB yields could be achieved.

[00129] The nature of the extracted material proved to be PHB by observing that it could be completely converted into measurable HB using the standard assay. This is shown in Figure 9, which shows the HP formation curve for each of the methods (Method A, closed circles; Method B, open circles; Method C, closed squares; Method C2, open squares; Method D, open triangles). For an equal mass input and the same amount of enzyme added, the depolymerization kinetics were almost identical in all methods examined. This illustrates that any of the methods, as well as a combination of methods, could be successfully used for a circular recycling process. Example 2

[00130] A variety of different bacteria were acquired from the American Type Culture Collection and were propagated and cultured on supplemented M9 bacteria, as described in Example 1. One Petition 870240024923, dated 03 / 22 / 2024, p. 72 / 96 / 68. Depolymerization / polymerization as described according to Method C above was performed using each bacterium. At the end of the growth period, the cells were harvested and the internal PHB was isolated and measured as described above. Table 4 below shows the amount of new PHB synthesized by each of the bacteria examined. Table 4 Reference Organism ATCC PHB Concentration (g / L) Acidovorax facilis 55745 0.24 Bacillus thuringiensis 39756 0.52 Dyella japonica BAA-939 0.50 Escherichia coli BAA-769 0.00 Halomonas aquamarina 35134 0.55 Lysobacter antibioticus 29480 0.41 Lysobacter enzymogenes 29487 0.65 Lysobacter gummosus 39472 0.32 Pseudomonas aeruginosa 10145 0.35 Pseudomonas fluorescens 13525 0.56 Rhodanobacter denitrificans BAA-1447 0.48 Shewanella frididmarina 700550 0.80 Thermobifida fusca 27730 0.65 Thermus thermophilus 27634 0.40 Xanthomonas vesicatoria 35937 0.35

[00131] As can be seen, there was significant variation in the PHB recovered both as a function of genus and by different species within a genus. For example, the PHB recovery from Acidovorax (the lowest-yielding producer) was 3.3 times lower than the yield recovered from Shewanella (the highest-yielding producer). Furthermore, within a single genus there were species differences. For example, in the genus Lysobacter, there was a 2-fold difference in PHB recovered between the best and worst species. Species-level differences were also observed in the genus Pseudomonas. Variations depending on growth conditions can also be expected. Thus, a balance can be found between identifying a genus that is compatible with the requirements of a process (e.g., high or elevated temperature, pressure, salt concentration, etc.) and then identifying a species within that genus that maximizes yield or may be... Petition 870240024923, dated 03 / 22 / 2024, page 73 / 96 / 68 optimized to maximize yield.

[00132] These and other modifications and variations of the present invention may be carried out by those skilled in the art without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Furthermore, it should be understood that facets of the various aspects may be interchanged in whole or in part. In addition, persons of ordinary skill in the art will note that the description presented is for illustrative purposes only and should not be construed as a limitation of the invention, which is described in more detail in the appended claims. Petition 870240024923, dated 03 / 22 / 2024, pp. 74 / 96

Claims

1 / 4 CLAIMS 1. A method for treating a post-consumer product, characterized in that it comprises: placing a post-consumer product in contact with a polyhydroxyalkanoate depolymerase (PHADase), the post-consumer product including a first polyhydroxyalkanoate, the PHADase catalyzing the depolymerization of the first polyhydroxyalkanoate and releasing a hydroxyalkanoate monomer from the first polyhydroxyalkanoate;cultivate a first microorganism in the presence of the hydroxyalkanoate monomer, the first microorganism being able to metabolize the hydroxyalkanoate monomer as a source of metabolic carbon, the first microorganism being able to produce a second polyhydroxyalkanoate, wherein the cultivation is carried out under a condition that encourages the production of the second polyhydroxyalkanoate by the first microorganism, wherein the first microorganism is selected from the genus Lysobacter, wherein the condition that encourages the production of the second polyhydroxyalkanoate by the first microorganism includes one of the following: limiting metabolic carbon sources, with the exception of the presence of the hydroxyalkanoate monomer at a concentration equal to or less than 2 millimolars, deprivation of nitrogen-containing nutrients, or deprivation of phosphate-containing nutrients.

2. Method according to claim 1, characterized in that the first polyhydroxyalkanoate is a first polyhydroxybutyrate.

3. Method according to claim 1, characterized in that PHADase is a purified enzyme.

4. Method according to claim 3, characterized by the fact that the PHADase is expressed by the first microorganism or is a modified enzyme that includes one or more amino acid modifications compared to a PHADase expressed by the first microorganism, or in which the PHADase is expressed by a second microorganism.

5. Method according to claim 1, characterized in that PHADase is expressed by a second microorganism, the method including culturing the second microorganism in the presence of the post-consumer product and thus contacting the post-consumer product with PHADase.

6. Method according to claim 5, characterized in that the first microorganism and the second microorganism are the same.

7. Method according to claim 5, characterized in that the second microorganism is a genetically modified microorganism.

8. Method according to claim 1, characterized in that the first microorganism is brought into contact with the hydroxyalkanoate monomer after the depolymerization of the first polyhydroxyalkanoate has been completed.

9. Method according to claim 1, characterized in that the first microorganism is brought into contact with the hydroxyalkanoate monomer together with the depolymerization of the first polyhydroxyalkanoate.

10. Method according to claim 1, characterized in that at least one of the contact step and the culture step occurs under extreme conditions, where the polyhydroxyalkanoate depolymerase comprises an extremozyme.

11. Method according to claim 10, characterized in that the extreme condition comprises one or more of a salt concentration of 0.5 M or higher, a temperature of 40°C or higher or a temperature of 10°C or less, a pressure of 0.5 MPa or higher, a pH of 1 to 5.5 or a pH of 7.5 to 11.5, in the presence of ionizing radiation of 1000 Gy or higher, or any combination thereof.

12. Method according to claim 1, characterized in that the condition that encourages the production of the second polyhydroxyalkanoate by the first microorganism comprises one or more of the following: a metabolic carbon source other than the hydroxyalkanoate monomer at a concentration equal to or less than 2 millimolars; deprivation of nitrogen-containing nutrients; deprivation of phosphate-containing nutrients; an environmental condition at or near the limit of the environmental condition in which the microorganism survives.

13. Method according to claim 1, characterized in that the condition that encourages the production of the second polyhydroxyalkanoate by the first microorganism comprises providing the hydroxyalkanoate monomer as the sole metabolic carbon source in the culture.

14. Method according to claim 1, characterized in that the condition that stimulates the production of the second polyhydroxyalkanoate by the first microorganism comprises a metabolic carbon source other than the hydroxyalkanoate monomer present at a concentration of 2 millimolars or less.

15. Method according to claim 1, characterized in that the first microorganism is selected from Lysobacter aestuarii, Lysobacter antibioticus, Lysobacter bugurensis, Lysobacter capsica, Lysobacter lacus, Lysobacter lycopersici, Lysobacter maris, Lysobacter niastensis, Lysobacter profundi, Lysobacter sp. A03, Lysobacter sp. cf310, Lysobacter sp. H21R20, Lysobacter sp. H21R4, Lysobacter sp. H23M41, Lysobacter sp. R19, Lysobacter sp. Root604, Lysobacter sp. Petition 870250031625, dated 17 / 04 / 2025, p. 13 / 20 4 / 4 Root690, Lysobacter sp. Root916, Lysobacter sp. Root983, Lysobacter sp.TY2-98, Lysobacter spongiae, Lysobacter spongiicola, Lysobacter alkalisoli, Lysobacter arseniciresistens, Lysobacter daejeonensis, Lysobacter dokdonensis, Lysobacter gilvus, Lysobacter gummosus, Lysobacter maris, Lysobacter oculi, Lysobacter panacisoli, Lysobacter penaei, Lysobacter prati, Lysobacter psychrotolerans, Lysobacter pythonis, Lysobacter ruishenii, Lysobacter segetis, Lysobacter silvestris, Lysobacter silvisoli, Lysobacter soli, Lysobacter sp. 17J7-1, Lysobacter sp. Alg18-2.2, Lysobacter sp. Cm-3-T8, Lysobacter sp. H23M47, Lysobacter sp. HDW10, Lysobacter sp. II4, Lysobacter sp. N42, Lysobacter sp. OAE881, Lysobacter sp. Root494, Lysobacter sp. URHA0019, Lysobacter sp. WF-2, Lysobacter sp. yr284, Lysobacter tabacisoli, Lysobacter telluris, Lysobacter tolerans, Lysobacter xinjiangensis.

16. System for carrying out the method as defined in claim 1, characterized in that the system includes a single container within which the contact step and the culture step occur, or in that the system includes a first container within which the contact step occurs and includes a second container within which the culture step occurs. Petition 870250031625, dated 04 / 17 / 2025, p. 14 / 20

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