This work presents a comprehensive analysis of the biodegradation of polyhydroxybutyrate (PHB) and chemically modified PHB with different chemical and crystal structures in a soil environment. A polymer modification reaction was performed during preparation of the chemically modified PHB films, utilizing 2,5-dimethyl-2,5-di(tert-butylperoxy)-hexane as a free-radical initiator and maleic anhydride. Films of neat PHB and chemically modified PHB were prepared by extrusion and thermocompression. The biological agent employed was natural mixed microflora in the form of garden soil. The course and extent of biodegradation of the films was investigated by applying various techniques, as follows: a respirometry test to determine the production of carbon dioxide through microbial degradation; scanning electron microscopy (SEM); optical microscopy; fluorescence microscopy; differential scanning calorimetry (DSC); and X-ray diffraction (XRD). Next-generation sequencing was carried out to study the microbial community involved in biodegradation of the films. Findings from the respirometry test indicated that biodegradation of the extruded and chemically modified PHB followed a multistage (2-3) course, which varied according to the spatial distribution of amorphous and crystalline regions and their spherulitic morphology. SEM and polarized optical microscopy (POM) confirmed that the rate of biodegradation depended on the availability of the amorphous phase in the interspherulitic region and the width of the interlamellar region in the first stage, while dependence on the size of spherulites and thickness of spherulitic lamellae was evident in the second stage. X-ray diffraction revealed that orthorhombic α-form crystals with helical chain conformation degraded concurrently with β-form crystals with planar zigzag conformation. The nucleation of PHB crystals after 90 days of biodegradation was identified by DSC and POM, a phenomenon which impeded biodegradation. Fluorescence microscopy evidenced that the crystal structure of PHB affected the physiological behavior of soil microorganisms in contact with the surfaces of the films.

Department of Environmental Protection Engineering Faculty of Technology Tomas Bata University in Zlín Nad Ovčírnou 3685 760 01 Zlín Czech Republic

Department of Physics and Material Engineering Faculty of Technology Tomas Bata University in Zlín Vavrečkova 5669 760 01 Zlin Czech Republic

Department of Polymer Engineering Faculty of Technology Tomas Bata University in Zlín Vavrečkova 5669 760 01 Zlín Czech Republic

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Emadian S. M.; Onay T. T.; Demirel B. Biodegradation of bioplastics in natural environments. Waste Manage. 2017, 59, 526–536. 10.1016/j.wasman.2016.10.006. PubMed DOI

Nishida H.; Tokiwa Y. Effects of higher-order structure of poly (3-hydroxybutyrate) on its biodegradation. II. Effects of crystal structure on microbial degradation. J. Environ. Polym. Degrad. 1993, 1 (1), 65–80. 10.1007/BF01457654. DOI

Kliem S.; Kreutzbruck M.; Bonten C. Review on the biological degradation of polymers in various environments. Materials 2020, 13 (20), 4586.10.3390/ma13204586. PubMed DOI PMC

Manfra L.; Marengo V.; Libralato G.; Costantini M.; De Falco F.; Cocca M. Biodegradable polymers: A real opportunity to solve marine plastic pollution?. J. Hazard. Mater. 2021, 416, 12576310.1016/j.jhazmat.2021.125763. PubMed DOI

Bahl S.; Dolma J.; Jyot Singh J.; Sehgal S. Biodegradation of plastics: A state of the art review. Mater. Today: Proc. 2021, 39, 31–34. 10.1016/j.matpr.2020.06.096. DOI

Kumagai Y.; Kanesawa Y.; Doi Y. Enzymatic degradation of microbial poly (3-hydroxybutyrate) films. Makromol. Chem. 1992, 193 (1), 53–57. 10.1002/macp.1992.021930105. DOI

Tomasi G.; Scandola M.; Briese B. H.; Jendrossek D. Enzymatic degradation of bacterial poly (3-hydroxybutyrate) by a depolymerase from Pseudomonas lemoignei. Macromolecules 1996, 29 (2), 507–513. 10.1021/ma951067n. DOI

Abe H.; Doi Y.; Aoki H.; Akehata T. Solid-state structures and enzymatic degradabilities for melt-crystallized films of copolymers of (R)-3-hydroxybutyric acid with different hydroxyalkanoic acids. Macromolecules 1998, 31 (6), 1791–1797. 10.1021/ma971559v. DOI

Gan Z.; Kuwabara K.; Abe H.; Iwata T.; Doi Y. The role of polymorphic crystal structure and morphology in enzymatic degradation of melt-crystallized poly (butylene adipate) films. Polym. Degrad. Stab. 2005, 87 (1), 191–199. 10.1016/j.polymdegradstab.2004.08.007. DOI

Iwata T.; Aoyagi Y.; Tanaka T.; Fujita M.; Takeuchi A.; Suzuki Y.; Uesugi K. Microbeam X-ray diffraction and enzymatic degradation of poly [(R)-3-hydroxybutyrate] fibers with two kinds of molecular conformations. Macromolecules 2006, 39 (17), 5789–5795. 10.1021/ma060908v. DOI

Iwata T.; Doi Y.; Tanaka T.; Akehata T.; Shiromo M.; Teramachi S. Enzymatic degradation and adsorption on poly [(R)-3-hydroxybutyrate] single crystals with two types of extracellular PHB depolymerases from Comamonas acidovorans YM1609 and Alcaligenes faecalis T1. Macromolecules 1997, 30 (18), 5290–5296. 10.1021/ma970491g. DOI

Przybysz-Romatowska M.; Haponiuk J.; Formela K. Reactive extrusion of biodegradable aliphatic polyesters in the presence of free-radical-initiators: A review. Polym. Degrad. Stab. 2020, 182, 10938310.1016/j.polymdegradstab.2020.109383. DOI

Chen C.; Peng S.; Fei B.; Zhuang Y.; Dong L.; Feng Z.; Chen S.; Xia H. Synthesis and characterization of maleated poly (3-hydroxybutyrate). J. Appl. Polym. Sci. 2003, 88 (3), 659–668. 10.1002/app.11771. DOI

Wei L.; McDonald A. G. Peroxide induced cross-linking by reactive melt processing of two biopolyesters: Poly(3-hydroxybutyrate) and poly(l-lactic acid) to improve their melting processability. J. Appl. Polym. Sci. 2015, 132 (13), 41724.10.1002/app.41724. DOI

Dong W.; Ma P.; Wang S.; Chen M.; Cai X.; Zhang Y. Effect of partial crosslinking on morphology and properties of the poly (β-hydroxybutyrate)/poly (d, l-lactic acid) blends. Polym. Degrad. Stab. 2013, 98 (9), 1549–1555. 10.1016/j.polymdegradstab.2013.06.033. DOI

Šerá J.; Serbruyns L.; De Wilde B.; Koutný M. Accelerated biodegradation testing of slowly degradable polyesters in soil. Polym. Degrad. Stab. 2020, 171, 10903110.1016/j.polymdegradstab.2019.109031. DOI

Plastics – Determination of the ultimate aerobic biodegradability of plastic materials in soil by measuring the oxygen demand in a respirometer or the amount of carbon dioxide evolved. ISO 17556, International Organization for Standardization, 2012.

Julinova M.; Slavik R.; Kalendova A.; Smida P.; Kratina J. Biodeterioration of plasticized PVC/montmorillonite nanocomposites in aerobic soil environment. Iran. Polym. J. 2014, 23 (7), 547–557. 10.1007/s13726-014-0249-4. DOI

Plastic – Determination of tensile properties. ISO 527-1,3, International Organization for Standardization, 2020.

Quispe M. M.; Lopez O. V.; Boina D. A.; Stumbé J. F.; Villar M. A. Glycerol-based additives of poly (3-hydroxybutyrate) films. Polym. Test. 2021, 93, 10700510.1016/j.polymertesting.2020.107005. DOI

Wei L.; McDonald A. G.; Stark N. M. Grafting of bacterial polyhydroxybutyrate (PHB) onto cellulose via in situ reactive extrusion with dicumyl peroxide. Biomacromolecules 2015, 16 (3), 1040–1049. 10.1021/acs.biomac.5b00049. PubMed DOI

Illumina. 16S Metagenomic sequencing library preparation: Preparing 16S ribosomal RNA gene amplicons for the illumina MiSeq system; Illumina, 2013.

Callahan B. J.; McMurdie P. J.; Rosen M. J.; Han A. W.; Johnson A. J. A.; Holmes S. P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods. 2016, 13 (7), 581–583. 10.1038/nmeth.3869. PubMed DOI PMC

McMurdie P. J.; Holmes S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013, 8 (4), e6121710.1371/journal.pone.0061217. PubMed DOI PMC

Gu Z.; Eils R.; Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 2016, 32 (18), 2847–2849. 10.1093/bioinformatics/btw313. PubMed DOI

Quast C.; Pruesse E.; Yilmaz P.; Gerken J.; Schweer T.; Yarza P.; Peplies J.; Glöckner F. O. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012, 41 (D1), D590–D596. 10.1093/nar/gks1219. PubMed DOI PMC

Ma P.; Cai X.; Lou X.; Dong W.; Chen M.; Lemstra P. J. Styrene-assisted melt free-radical grafting of maleic anhydride onto poly (β-hydroxybutyrate). Polym. Degrad. Stab. 2014, 100, 93–100. 10.1016/j.polymdegradstab.2013.12.005. DOI

Xu J.; Guo B. H.; Yang R.; Wu Q.; Chen G. Q.; Zhang Z. M. In situ FTIR study on melting and crystallization of polyhydroxyalkanoates. Polymer 2002, 43 (25), 6893–6899. 10.1016/S0032-3861(02)00615-8. DOI

Hong S. G.; Lin Y. C.; Lin C. H. Improvement of the thermal stability of polyhydroxybutyrates by grafting with maleic anhydride by different methods: differential scanning calorimetry, thermogravimetric analysis and gel permeation chromatography. J. Appl. Polym. Sci. 2008, 110 (5), 2718–2726. 10.1002/app.28782. DOI

Lugito G.; Woo E. M.; Chuang W. T. Interior lamellar assembly and optical birefringence in poly (trimethylene terephthalate) spherulites: Mechanisms from past to present. Crystals 2017, 7 (2), 56.10.3390/cryst7020056. DOI

Chuang W. T.; Hong P. D.; Chuah H. H. Effects of crystallization behavior on morphological change in poly (trimethylene terephthalate) spherulites. Polymer 2004, 45 (7), 2413–2425. 10.1016/j.polymer.2004.01.048. DOI

Chen H. B.; Chen L.; Zhang Y.; Zhang J. J.; Wang Y. Z. Morphology and interference color in spherulite of poly (trimethylene terephthalate) copolyester with bulky linking pendent group. Phys. Chem. Chem. Phys. 2011, 13 (23), 11067–11075. 10.1039/c0cp02176h. PubMed DOI

Yun J. H.; Kuboyama K.; Chiba T.; Ougizawa T. Crystallization temperature dependence of interference color and morphology in poly (trimethylene terephthalate) spherulite. Polymer 2006, 47 (13), 4831–4838. 10.1016/j.polymer.2006.04.031. DOI

Yun J. H.; Kuboyama K.; Ougizawa T. High birefringence of poly (trimethylene terephthalate) spherulite. Polymer 2006, 47 (5), 1715–1721. 10.1016/j.polymer.2005.12.067. DOI

Woo E. M.; Lugito G. Origins of periodic bands in polymer spherulites. Eur. Polym. J. 2015, 71, 27–60. 10.1016/j.eurpolymj.2015.07.045. DOI

Hosier I. L.; Bassett D. C. A study of the morphologies and growth kinetics of three monodisperse n-alkanes: C122H246. C162H326 and C246H494. Polymer 2000, 41 (25), 8801–8812. 10.1016/S0032-3861(00)00223-8. DOI

Najafi N.; Heuzey M. C.; Carreau P. J. Crystallization behavior and morphology of polylactide and PLA/clay nanocomposites in the presence of chain extenders. Polym. Eng. Sci. 2013, 53 (5), 1053–1064. 10.1002/pen.23355. DOI

Iglesias-Montes M. L.; Soccio M.; Luzi F.; Puglia D.; Gazzano M.; Lotti N.; Manfredi L. B.; Cyras V. P. Evaluation of the factors affecting the disintegration under a composting process of poly (lactic acid)/poly (3-hydroxybutyrate)(PLA/PHB) blends. Polymers 2021, 13 (18), 3171.10.3390/polym13183171. PubMed DOI PMC

Tarazona N. A.; Machatschek R.; Lendlein A. Unraveling the interplay between abiotic hydrolytic degradation and crystallization of bacterial polyesters comprising short and medium side-chain-length polyhydroxyalkanoates. Biomacromolecules 2020, 21 (2), 761–771. 10.1021/acs.biomac.9b01458. PubMed DOI

Bonartsev A. P.; Boskhomodgiev A. P.; Iordanskii A. L.; Bonartseva G. A.; Rebrov A. V.; Makhina T. K.; Myshkina V. L.; Yakovlev S. A.; Filatova E. A.; Ivanov E. A.; Bagrov D. V.; Zaikov G. E. Hydrolytic degradation of poly(3-hydroxybutyrate), polylactide and their derivatives: Kinetics, crystallinity, and surface morphology. Mol. Cryst. Liq. Cryst. 2012, 556 (1), 288–300. 10.1080/15421406.2012.635982. DOI

Pei R.; Tarek-Bahgat N.; Van Loosdrecht M. C. M.; Kleerebezem R.; Werker A. G. Influence of environmental conditions on accumulated polyhydroxybutyrate in municipal activated sludge. Water Res. 2023, 232, 11965310.1016/j.watres.2023.119653. PubMed DOI

Prapruddivongs C.; Apichartsitporn M.; Wongpreedee T. Effect of silica resources on the biodegradation behavior of poly (lactic acid) and chemical crosslinked poly (lactic acid) composites. Polym. Test. 2018, 71, 87–94. 10.1016/j.polymertesting.2018.08.026. DOI

Abou-Zeid D. M.; Müller R. J.; Deckwer W. D. Biodegradation of aliphatic homopolyesters and aliphatic– aromatic copolyesters by anaerobic microorganisms. Biomacromolecules 2004, 5 (5), 1687–1697. 10.1021/bm0499334. PubMed DOI

García-Depraect O.; Lebrero R.; Rodriguez-Vega S.; Bordel S.; Santos-Beneit F.; Martínez-Mendoza L. J.; Aragão Borner R.; Börner T.; Muñoz R. Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: Kinetics, carbon fate and particle size effect. Bioresour. Technol. 2022, 344, 12626510.1016/j.biortech.2021.126265. PubMed DOI

Gangurde N. S.; Patil Y. P.; Jain R.; Sayyed R. Z. Poly-β-hydroxybutyrate biodegradation by mixed culture population vis-à-vis single culture population under varying environmental conditions: a new approach. Indian J. Exp. Biol. 2017, 55, 311–320.

Woolnough C. A.; Yee L. H.; Charlton T.; Foster L. J. R. Environmental degradation and biofouling of ‘green’plastics including short and medium chain length polyhydroxyalkanoates. Polym. Int. 2010, 59 (5), 658–667. 10.1002/pi.2746. DOI

Mergaert J.; Anderson C.; Wouters A.; Swings J.; Kersters K. Biodegradation of polyhydroxyalkanoates. FEMS Microbiol. Lett. 1992, 103, 317–321. 10.1111/j.1574-6968.1992.tb05853.x. PubMed DOI

Boyandin A. N.; Prudnikova S. V.; Filipenko M. L.; Khrapov E. A.; Vasil’ev A. D.; Volova T. G. Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms. Appl. Biochem. Microbiol. 2012, 48, 28–36. 10.1134/S0003683812010024. PubMed DOI

Avella M.; Rota G. L.; Martuscelli E.; Raimo M.; Sadocco P.; Elegir G.; Riva R. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and wheat straw fibre composites: thermal, mechanical properties and biodegradation behaviour. J. Mater. Sci. 2000, 35, 829–836. 10.1023/A:1004773603516. DOI

Mousavioun P.; George G. A.; Doherty W. O. Environmental degradation of lignin/poly (hydroxybutyrate) blends. Polym. Degrad. Stab. 2012, 97 (7), 1114–1122. 10.1016/j.polymdegradstab.2012.04.004. DOI

Cho J. Y.; Park S. L.; Lee H. J.; Kim S. H.; Suh M. J.; Ham S.; Bhatia S. K.; Gurav R.; Park S. H.; Park K.; Yoo D.; Yang Y. H. Polyhydroxyalkanoates (PHAs) degradation by the newly isolated marine Bacillus sp. JY14. Chemosphere 2021, 283, 13117210.1016/j.chemosphere.2021.131172. PubMed DOI

Park S. L.; Cho J. Y.; Kim S. H.; Lee H.; Kim S. H.; Suh M. J.; Ham S.; Bhatia S. K.; Gurav R.; Park S.; Park K.; Kim Y.; Yang Y. Novel polyhydroxybutyrate-degrading activity of the Microbulbifer Genus as confirmed by Microbulbifer sp. SOL03 from the marine environment. J. Microbiol. Biotechnol. 2022, 32, 27–36. 10.4014/jmb.2109.09005. PubMed DOI PMC

Grassie N.; Murray E. J.; Holmes P. A. The thermal degradation of poly (-(D)-β-hydroxybutyric acid): part 2-changes in molecular weight. Polym. Degrad. Stab. 1984, 6 (2), 95–103. 10.1016/0141-3910(84)90075-2. DOI

Pospisilova A.; Melcova V.; Figalla S.; Mencik P.; Prikryl R. Techniques for increasing the thermal stability of poly [(R)-3-hydroxybutyrate] recovered by digestion methods. Polym. Degrad. Stab. 2021, 193, 10972710.1016/j.polymdegradstab.2021.109727. DOI

Reddy M. M.; Deighton M.; Gupta R. K.; Bhattacharya S. N.; Parthasarathy R. Biodegradation of oxo-biodegradable polyethylene. J. Appl. Polym. Sci. 2009, 111 (3), 1426–1432. 10.1002/app.29073. DOI

Matsumura S.; Kurita H.; Shimokobe H. Anaerobic biodegradability of polyvinyl alcohol. Biotechnol. Lett. 1993, 15, 749–754. 10.1007/BF01080150. DOI

Morse M. C.; Liao Q.; Criddle C. S.; Frank C. W. Anaerobic biodegradation of the microbial copolymer poly (3-hydroxybutyrate-co-3-hydroxyhexanoate): Effects of comonomer content, processing history, and semi-crystalline morphology. Polymer 2011, 52 (2), 547–556. 10.1016/j.polymer.2010.11.024. DOI

Rudnik E.; Briassoulis D. Comparative biodegradation in soil behaviour of two biodegradable polymers based on renewable resources. J. Polym. Environ. 2011, 19 (1), 18–39. 10.1007/s10924-010-0243-7. DOI

Tsuji H.; Suzuyoshi K. Environmental degradation of biodegradable polyesters 1. Poly (ε-caprolactone), poly [(R)-3-hydroxybutyrate], and poly (L-lactide) films in controlled static seawater. Polym. Degrad. Stab. 2002, 75 (2), 347–355. 10.1016/S0141-3910(01)00240-3. DOI

Gallet G.; Lempiäinen R.; Karlsson S. Characterisation by solid phase microextraction–gas chromatography–mass spectrometry of matrix changes of poly (l-lactide) exposed to outdoor soil environment. Polym. Degrad. Stab. 2000, 71 (1), 147–151. 10.1016/S0141-3910(00)00165-8. DOI

Gazzano M.; Tomasi G.; Scandola M. X-ray investigation on melt-crystallized bacterial poly (3-hydroxybutyrate). Macromol. Chem. Phys. 1997, 198 (1), 71–80. 10.1002/macp.1997.021980106. DOI

Zhang J.; Kasuya K.; Hikima T.; Takata M.; Takemura A.; Iwata T. Mechanical properties, structure analysis and enzymatic degradation of uniaxially cold-drawn films of poly [(R)-3-hydroxybutyrate-co-4-hydroxybutyrate]. Polym. Degrad. Stab. 2011, 96 (12), 2130–2138. 10.1016/j.polymdegradstab.2011.09.011. DOI

Roohi; Zaheer M. R.; Kuddus M. PHB (poly-β-hydroxybutyrate) and its enzymatic degradation. Polym. Adv. Technol. 2018, 29 (1), 30–40. 10.1002/pat.4126. DOI

Trainer M. A.; Charles T. C. The role of PHB metabolism in the symbiosis of rhizobia with legumes. Appl. Microbiol. Biotechnol. 2006, 71 (4), 377–386. 10.1007/s00253-006-0354-1. PubMed DOI

Ratcliff W. C.; Kadam S. V.; Denison R. F. Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia. FEMS Microbiol. Ecol. 2008, 65 (3), 391–399. 10.1111/j.1574-6941.2008.00544.x. PubMed DOI

Catone M. V.; Ruiz J. A.; Castellanos M.; Segura D.; Espin G.; Lopez N. I. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes. PloS One. 2014, 9 (6), e9887310.1371/journal.pone.0098873. PubMed DOI PMC

Wu M.; Li G.; Huang H.; Chen S.; Luo Y.; Zhang W.; Li K.; Zhou J.; Ma T. The simultaneous production of sphingan Ss and poly (R-3-hydroxybutyrate) in Sphingomonas sanxanigenens NX02. Int. J. Biol. Macromol. 2016, 82, 361–368. 10.1016/j.ijbiomac.2015.09.071. PubMed DOI