US-20260125717-A1 - Bioreactor and Process for Forming Polyhydroxybutyrate Directly from Depolymerized Polyhydroxybutyrate

Abstract

A bioreactor and process are disclosed for forming polyhydroxybutyrate directly from depolymerized polyhydroxybutyrate. In two bioreactor vessels, a microorganism product, such as one or more enzymes, are combined with the polyhydroxybutyrate-containing post-consumer product materials. The microorganism can naturally secrete the one or more enzymes or can be genetically modified to secrete the enzyme. The combination of enzymes is designed to facilitate a metabolic pathway that can depolymerize PHB, convert the resulting hydroxybutyrate to hydroxybutyryl-CoA, and in turn polymerize it into PHB. Namely, a solution for the recycling of polyhydroxybutyrate to hydroxybutyrate and back to polyhydroxybutyrate.

Inventors

• Stephen Quirk

Assignees

• KIMBERLY-CLARK WORLDWIDE, INC.

Dates

Publication Date

20260507

Application Date

20250924

Claims (20)

1. 1 .- 17 . (canceled)
2. 18 . A process for recycling polyhydroxybutyrate from a polyhydroxybutyrate-containing post-consumer product comprising: within a first bioreactor vessel, contacting a post-consumer product with a thermophilic or halophilic bacteria suspension or a thermophilic or halophilic depolymerase enzyme to supply a hydroxybutyrate monomer; pumping the hydroxybutyrate monomer through a molecular weight cutoff filter, optionally followed by an ion exchange bed, to a second bioreactor vessel; and within the second bioreactor vessel, contacting the hydroxybutyrate monomer with one or more enzymes and a cofactor feed, thereby producing a recycled polyhydroxybutyrate.
3. 19 . The process of claim 18 , wherein the post-consumer product further comprises contamination that is controlled using a high salt concentration or a high temperature in the first bioreactor vessel.
4. 20 . The process of claim 18 , wherein the cofactor feed comprises Coenzyme A, nicotinamide adenine dinucleotide, Nicotinamide adenine dinucleotide phosphate, Adenosine triphosphate, Adenosine monophosphate, pyrophosphate, or a combination thereof.
5. 21 . The process of claim 18 , wherein the thermophilic bacteria suspension or the thermophilic depolymerase enzyme is a bacteria of the genera Alicyclobacillus, Amycolatopsis, Azospirillum, Deinococcus, Fervidobacterium , Gandjariella, Georgenia , Hyphomanas, Lihuaxuella, Microbulbifer , Minwuia, Rhodopseudomonas, Streptomyces, Thermanaeromonas , Thermoac tinomyces, Thermoactinospora, Thermobifida, Thermobispora , Thermocatellispora, Thermochromatium, Thermocrispum, Thermoflavimicrobium, Thermogemmatispora, Thermogemmatispora , Thermoleophilaceae, Thermomonospora , Thermostaphylospora, Thermus , or a combination thereof, or purified therefrom.
6. 22 . The process of claim 18 , wherein the halophilic bacteria suspension or the halophilic depolymerase enzyme is a bacteria of the genera Alteromonas, Arthrobacter, Azospirillum, Empedobacter, Desulfovibrio, Halobacillus , Halobacteriovorax, Haloechinothrix, Halomarina, Halomonas , Halorussus, Haloterrigena, Isoptericola, Marinobacter, Methyloligella, Micromonospora, Natronococcus, Nocardiopsis, Paracoccus, Roseivivax, Saccharomonospora, Shewanella , or a combination thereof, or purified therefrom.
7. 23 . The process of claim 18 , wherein the thermophilic bacteria suspension or the thermophilic depolymerase enzyme is a bacteria including Alicyclobacillus pomorum (WP-084453829), Amycolatopsis thermoflava (WP-123687648), Amycolatopsis thermalba (WP-094002797), Amycolatopsis rumani (WP-116109633), Azospirillum thermophilum (WP-109324320), Deinococcus actinosclerus (WP-082689076), Fervidobacterium gondwanense (SHN54810), Gandjariella thermophila (WP-137812779), Georgenia satyanarayanai (WP-146237554), Hyphomanas sp. (HA037884), Lihuaxuella thermophila (WP-089972404), Microbulbifer thermotolerans (P-197462976), Minwuia thermotolerans (WP-206420073), Rhodopseudomonas thermotolerans (WP-114356866), Rhodopseudomonas pentothenatexigens, (WP-114356866), Streptomyces thermovulgaris (WP-067396676), Thermanaeromonas toyohensis (WP-084666479), Thermoactinomyces sp. CICC 10523 (WP-198056464), Thermoactinomyces daqus (WP-033100012), Thermoactinospora sp. (NUT44302), Thermoactinospora rubra (WP-084965756), Thermobifida halotolerans (WP-068692693), Thermobifida fusca (WP-011290529), Thermobispora bispora (WP-206206594), Thermocatellispora tengchongensis , (WP-185055796), Thermochromatium 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 (WP-084659191), Thermoleophilaceae bacterium (MBA2429278), Thermomonospora echinospora (WP-160147065), Thermomonospora cellulosilytica (WP-182704610), Thermomonospora amylolytica (WP-198679325), Thermostaphylospora chromogena (WP-093263254), Thermus thermophilus (WP-197735236), Thermus aquaticus (WP-053768217), Thermus islandicus (HEO42284), or a combination thereof, or purified therefrom.
8. 24 . The process of claim 18 , wherein the halophilic bacteria suspension or the halophilic depolymerase enzyme is a bacteria including Alteromonas halophila (WP-189403400), Arthrobacter crystallopoietes (WP-005270754), Arthrobacter sp. NEB 688 (WP-173027059), Azospirillum halopraeferens (WP-029007775), Empedobacter halcabium (TXE30443), Desulfovibrio sulfodismutans (NDY59052), Halobacillus hunanensis (WP-139377117), 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 (WP-048308188), 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 (WP-041159480), Halomonas alkaliphile (WP-162218603), Halomonas sp. ZH2S (WP-160419650), Halomonas alkaliantarctica (WP-133732469), Halomonas zincidurans (WP-031384106), Halomonas chromatireducens (WP-083517585), 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 (WP-106373458), Halomonas subglaciescola (WP-079553041), Halomonas sp. HL-92 (WP-074398447), Halomonas xinjiangensis (WP-197053288), Halomonas saliphila (WP-104202516), Halomonas sp. HL-48 (WP-027336292), Halomonas qijiaojingensis (WP-189471950), Halomonas urumqiensis (WP-102588859), Halomonas lutea (WP-019020614), Halomonas lutescens (WP-188638020), Halomonas salicampi (WP-179930793), Halomonas sp. FME66 (WP-193092800), Halomonas sp. 156 (CAD5269671), Halomonas sp. L5 (WP-149329933), Halomonas nanhaiensis (WP-127060197), Halomonas titanicae (WP-144810212), Halomonas sp. SH5A2 (WP-186255949), Halomonas sp. TD01 (WP-009722522), Halomonas sp. PC (WP-127040515), Halomonas sp. RC (WP-126951333), 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 (WP-095603093), 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 (WP-168017113), 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 (WP-149782231), Haloterrigena sp. H1 (WP-138782397), Isoptericola halotolerans (WP-171781920), Marinobacter sp. X15-166B (WP-198929205), Marinobacter sp. LPB0319 (WP-2066439888), Marinobacter salaries (WP-126811858), Marinobacter sp. PJ-16 (WP-137435339), Marinobacter nanhaiticus (WP-004579452), Marinobacter bohaiensis (WP-111497193), Marinobacter sp. ANT_B65 (WP-202971753), Marinobacter sediminum (WP-203299860), Marinobacter fonticola (WP-148861082), Marinobacter sp. JB02H27 (WP-150989051), Marinobacter maritimus (WP-144775354), Marinobacter nitratireducens (WP-036130189), Marinobacter aromaticivorans (WP-100686899), Marinobacter sp. MCTG268 (WP-081899301), Marinobacter profundi (WP-099614009), Marinobacter sp. R17 (WP-123633665), Marinobacter sp. F3R11 (WP-113816648), Marinobacter lipolyticus (WP-012136507), Marinobacter sp. LV10MA510-1 (WP-098421792), Marinobacter sp. LV10R520-4 (WP-143751449), Marinobacter antarcticus (WP-072795398), Marinobacter zhejiangensis (WP-092022278), Marinobacter sp. LZ-8 (WP-138439039), Marinobacter sp. LZ-6 (WP-138437074), 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 khirikhana (WP-126168307), Shewanella halifaxensis (WP-108946642), Shewanella waksmanii (WP-028774143), Shewanella saliphila (WP-188922486), Shewanella ulleungensis (WP-188954542), Shewanella litoralis (WP-160052797), or a combination thereof, or purified therefrom.
9. 25 . The process of claim 18 , wherein the thermophilic or halophilic bacteria suspension or a thermophilic or halophilic depolymerase enzyme is either Lihuaxuella thermophila or Halomonas aquamarine or purified therefrom.
10. 26 . The process of claim 18 , wherein the thermophilic or halophilic bacteria suspension or a thermophilic or halophilic depolymerase enzyme is either salt tolerant from about 0.5 molar to about 5 molar or temperature tolerant from about 40° C. to about 120° C., or a combination thereof.
11. 27 . The process of claim 18 , wherein the thermophilic or halophilic depolymerase enzyme comprises about 12 or less Cysteine residues.
12. 28 . The process of claim 18 , wherein the thermophilic or halophilic depolymerase enzyme is produced by a genetically modified microorganism that has been genetically modified to secrete the thermophilic or halophilic depolymerase enzyme.
13. 29 . The process of claim 18 , wherein the thermophilic or halophilic depolymerase enzyme is produced by at least one type of a naturally occurring microorganism that naturally encodes the thermophilic or halophilic depolymerase enzyme.
14. 30 . The process of claim 18 , wherein the molecular weight cutoff filter is from about 3 kDa to about 30 kDa.
15. 31 . The process of claim 18 , wherein the thermophilic or halophilic depolymerase enzyme has a molecular weight of about 3 kDa or less.
16. 32 . The process of claim 18 , wherein contacting the hydroxybutyrate monomer with one or more enzymes and the cofactor feed comprises converting the hydroxybutyrate monomer to acetoacetate with a hydroxybutyrate dehydrogenase enzyme, converting acetoacetate to acetoacetate-CoA with an Acetoacetyl-CoA synthetase enzyme, reducing acetocetyl-CoA to hydroxybutyrl-CoA with an acetoacetyl-CoA reductase, and polymerizing hydroxybutyryl-CoA with a hydroxybutyrate polymerase to form polyhydroxybutyrate.
17. 33 . The process of claim 18 , wherein the post-consumer product may contain urine, menses, feces, or a combination thereof or is selected from the group comprising incontinence products, baby and childcare products, feminine care products, and family care products, or a combination thereof.
18. 34 . The process of claim 18 , wherein pumping the hydroxybutyrate monomer through a molecular weight cutoff filter to the second bioreactor vessel occurs when a measured optical density at 600 nm of the first bioreactor vessel contents is about less than 0.4 or when at least 40% of polyhydroxyalkanoate in the post-consumer product has been depolymerized to the monomer, or a combination thereof.
19. 35 . A polyhydroxybutyrate produced by the process of claim 18 .
20. 36 . A polyhydroxybutyrate produced from hydroxybutyrate liberated by the depolymerization of polyhydroxybutyrate in a multi-step enzymatic reaction comprising: in a first bioreactor vessel, contacting polyhydroxybutyrate with a depolymerase enzyme or depolymerase exhibit microorganism to supply a hydroxybutyrate monomer; and in a second bioreactor vessel, converting the hydroxybutyrate monomer to acetoacetate with a hydroxybutyrate dehydrogenase enzyme, converting acetoacetate to acetoacetate-CoA with an Acetoacetyl-CoA synthetase enzyme, reducing acetocetyl-CoA to hydroxybutyrl-CoA with an acetoacetyl-CoA reductase, and polymerizing hydroxybutyryl-CoA with a hydroxybutyrate polymerase to form polyhydroxybutyrate.

Description

RELATED APPLICATIONS The present application is a divisional of U.S. patent application Ser. No. 18/558,863 having a filing date of Nov. 3, 2023, which is the national stage entry of International Patent Application No. PCT/US2022/031284 having a filing date of May 27, 2022, and Provisional Patent Application No. 63/194,445 having a filing date of May 28, 2021, which are incorporated herein in their entirety by reference thereto. SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 22, 2025, is named KCX-2009-USDIV_SL.xml and is 13,637 bytes in size. BACKGROUND Global production of petroleum-based plastics continues to increase every year. In recent years, for instance, over 300,000,000 metric tons of petroleum-based polymers have been produced. A significant portion of the above produced polymers are used to produce single-use products, such as plastic drinking bottles, straws, packaging, and absorbent articles, including wearable absorbent articles. Most of these plastic products are discarded and do not enter the recycle stream. Particularly, absorbent articles, including personal care and child care garments, are currently made from predominantly petroleum-based plastics, such as films and nonwoven materials formed of polyethylene or polypropylene. Due to the nature of these articles, and the function they perform, it is difficult, if not impossible, to partially or completely recycle the polypropylene or polyethene materials used. It has long been hoped that biodegradable polymers produced from renewable resources (hereinafter termed “biopolymers”) would hold great promise in reducing the global accumulation of petroleum-based plastics in the environment. For example, significant research has been done on biologically derived polymers and on polymers that biodegrade in suitable environments. One such class of biopolymers are the polyhydroxyalkanoates (PHA). Much work has been accomplished on the PHA family, most notably the polyhydroxybutyrate (PHB) polymers including poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) and their copolymers. Specifically, PHB shows promise in that the polymer is derived from natural sources, can be bio-degraded by several mechanisms, and is biocompatible with human tissues. Of particular advantage, PHA family biopolymers exhibit thermoplastic properties that are very similar to some petroleum-based polymers and thus represent viable replacements for petroleum-based polymers such as polypropylene and polyethylene. Polyhydroxyalkanoates are synthesized using a variety of bacterial and archaea genera, including Halobacillus, Bacillus, Salinobacter, Flavobacterium, Chromohalobacter, Halomonas, Marinobacter, Vibrio, Pseudomonas, Halococcus, Halorhabdus, Haladaptatus, Natrialba, Haloterrigena, and Halorussus. The polyhydroxyalkanoate serves as an energy sink for these organisms. Production of polyhydroxyalkanoate polymers by the above microorganisms involves a three-step enzymatic mechanism that begins with acetyl coenzyme A. In forming PHB, the first step is catalysis of acetyl-CoA by PhaA (a β-ketothiolase) to form β-ketoacyl-CoA. This in turn is converted in a NADP-dependent reaction into R-3-hydroxyacyl-CoA by the PhaB enzyme (a-β-ketoacyl-CoA reductase). The final step, catalyzed by PhaC (a PHB synthase), is the polymerization of R-3-hydroxyacyl-CoA into PHB. Said another way, the final step of the pathway involves the polymerization of hydroxyalkanoic acid monomers into a polyhydroxyalkanoate polymer via a polyhydroxyalkanoate polymerase. Bio-synthesized polyhydroxyalkanoates accumulate in the bacterial cell as large molecular weight granules and can account for from about 60% to about 90% of the cellular dry mass. These same organisms express an extracellular and/or an intracellular PHB depolymerase (the gene phaZ) that degrades the polymer back to hydroxybutyrate (HB) and small PHB oligomers. The resulting HB is further degraded to provide a carbon and energy source for the microorganisms. From an industrial point of view HB is a dead-end in that it cannot be directly repolymerized by any known chemical process. Although it can be fully environmentally degraded, it would be economically and environmentally advantageous to be able to convert HB into a form that can readily be utilized to re-form PHB. In nature, to retrieve the energy stored in the polymer, biodegradation is accomplished by a PHA depolymerase (PHADase). Unfortunately, natural PHADase are generally not conducive to industrial processes, e.g., post-consumer recycle processes, as an enzyme that is used for any bio-industrial process must have several characteristics that typical PHADase lacks. To be broadly useful, an enzyme for use in an industrial process should be