Synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with high 4HB composition and PHA content using 1,4-butanediol and 1,6-hexanediol for medical application


Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] with high 4HB monomer harbors enhanced biocompatibility and mechanical properties, which are useful as implantable and absorbable biomaterial in medical and pharmaceutical fields. Transformant Cupriavidus sp. USMAA1020 with an additional PHA synthase gene, phaC was found to produce P(3HB-co-4HB) with 86 mol% of 4HB monomer composition and high PHA content of 69 wt% in shake flask cultivation using mixed substrate of 1,6-hexanediol and 1,4-butanediol. Single-stage cultivation in 3 L fermentation has confirmed the ability of this strain to produce high 4HB monomer composition of 95 mol% in large scale fermentation with 75 wt% PHA content and high PHA concentration of 18.7 g/L. Interestingly, this strain was capable of surviving higher carbon concentration (1.05 wt% C) than the wild-type strain (0.69 wt% C). The present study results in P(3HB-co-4HB) with 1.3 and 2.3-fold PHA content and concentration respectively, with the ability to accumulate 2.1-fold 4HB monomer composition higher compared to the wild-type strain. A higher specific growth rate of 0.123 h−1 accompanied by high product yield, Yp/x of 3.8 times more than the wild-type strain were also obtained. Image from TEM showed cells with PHA content of 75 wt%, whichwere occupied with significant PHA granule. This copolymer possesses an average molecular weight (M w) and a polydispersity index of 156 kDa and 3.5 respectively with a tensile strength, elongation at break and Young’s modulus of 22.9 MPa, 463.2% and 187.3 MPa respectively. This polymer has a glass transition temperature (T g) and melting temperature (T m) of −48.9 °C and 61.9 °C respectively.

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  1. 1.

    Sudesh K, Iwata T (2008) Sustainability of biobased and biodegradable plastics. Clean - Soil, Air, Water 36(5–6):433–442

    CAS  Article  Google Scholar 

  2. 2.

    Grande D, Ramier J, Versace DL, Renard E, Langlois V (2017) Design of functionalized biodegradable PHA-based electrospun scaffolds meant for tissue engineering applications. New Biotechnol 37:129–137

    CAS  Article  Google Scholar 

  3. 3.

    Winnacker M, Rieger B (2017) Copolymers of polyhydroxyalkanoates and polyethylene glycols: recent advancements with biological and medical significance. Polym Int 66(4):497–503

    CAS  Article  Google Scholar 

  4. 4.

    Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54(4):450–472

    CAS  Google Scholar 

  5. 5.

    Amoli RI, Baei MS, Pirouz F (2013) Polyhydroxyalkanoate production by cultivating Hydrogenophaga pseudoflava in fed batch culture. Middle-East J Sci Res 14(8):1035–1039

    Google Scholar 

  6. 6.

    Choi J, Lee SY (1999) Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl Microbiol Biotechnol 51(1):13–21

    CAS  Article  Google Scholar 

  7. 7.

    Lee WH, Azizan MNM, Sudesh K (2004) Effects of culture conditions on the composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) synthesized by Comamonas acidovorans. Polym Degrad Stab 84(1):129–134

    CAS  Article  Google Scholar 

  8. 8.

    Saito Y, Nakamura S, Hiramitsu M, Doi Y (1996) Microbial synthesis and properties of poly(3-ydroxybutyrate-co-4-hydroxybutyrate). Polym Int 39:169–174

    CAS  Article  Google Scholar 

  9. 9.

    Lefebvre G, Rocher M, Braunegg G (1997) Effects of low dissolved-oxygen concentrations on poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Alcaligenes eutrophus. Appl Environ Microbiol 63(3):827–833

    CAS  Google Scholar 

  10. 10.

    Wang B, Sharma-Shivappa RR, Olson JW, Khan SA (2012) Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies. Bioprocess Biosyst Eng 35(9):1591–1602

    CAS  Article  Google Scholar 

  11. 11.

    Nelson T, Kaufman E, Kline J, Sokoloff L (1981) The extraneural distribution of γ-hydroxybutyrate. J Neurochem 37(5):1345–1348

    CAS  Article  Google Scholar 

  12. 12.

    Williams SF, Martin DP (1996) Applications of PHAs in Medicine and Pharmacy. Biopolymers Online:1–38

  13. 13.

    Martin DP, Williams SF (2003) Medical applications of poly-4-hydroxybutyrate: a strong flexible absorbable biomaterial. Biochem Eng J 16(2):97–105

    CAS  Article  Google Scholar 

  14. 14.

    Vigneswari S, Murugaiyah V, Kaur G, Abdul Khalil HPS, Amirul AA (2016) Simultaneous dual syringe electrospinning system using benign solvent to fabricate nanofibrous P(3HB-co-4HB)/collagen peptides construct as potential leave-on wound dressing. Mater Sci Eng C 66:147–155

    CAS  Article  Google Scholar 

  15. 15.

    Huong KH, Mohd Yahya AR, Amirul AA (2013) Pronounced synergistic influence of mixed substrate cultivation on single step copolymer P(3HB-co-4HB) biosynthesis with a wide range of 4HB monomer composition. J Chem Technol Biotechnol 89(7):1023–1029

    Article  Google Scholar 

  16. 16.

    Huong KH, Kannusamy S, Lim SYH, Amirul AA (2015) Biosynthetic enhancement of single-stage poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production by manipulating the substrate mixtures. J Ind Microbiol Biotechnol 42(9):1291–1297

    CAS  Article  Google Scholar 

  17. 17.

    Kimura H, Ohura T, Matsumoto T, Ikarashi T (2008) Effective biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with high 4HB fractions by Wautersia eutropha in the presence of α-amino acids. Polym Int 57:149–157

    CAS  Article  Google Scholar 

  18. 18.

    Zhou XY, Yuan XX, Shi ZY, Meng DC, Jiang WJ, Wu LP, Chen JC, Chen GQ (2012) Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli. Microb Cell Factories 11(1):54

    CAS  Article  Google Scholar 

  19. 19.

    Syafiq IM, Huong KH, Shantini K, Vigneswari S, Aziz NA, Amirul AAA, Bhubalan K (2017) Synthesis of high 4-hydroxybutyrate copolymer by Cupriavidus sp. transformants using one-stage cultivation and mixed precursor substrates strategy. Enzym Microb Technol 98(November):1–8

    CAS  Article  Google Scholar 

  20. 20.

    Amirul AA, Yahya ARM, Sudesh K, Azizan MNM, Majid MIA (2008) Biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer by Cupriavidus sp. USMAA1020 isolated from Lake Kulim, Malaysia. Bioresour Technol 99(11):4903–4909

    CAS  Article  Google Scholar 

  21. 21.

    Majid MIA, Ismail J, Few LL, Tan CF (2002) The degradation kinetics of poly(3-hydroxybutyrate) under non-aqueous and aqueous conditions. Eur Polym J 38(4):837–839

    CAS  Article  Google Scholar 

  22. 22.

    Chai HL, Ahmad R, Yahya ARM, Majid MIA, Amirul AA (2009) Microbial synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer by Cupriavidus sp. USMAA2-4 through a two step cultivation process. Afr J Biotechnol 8(17):4189–4196

    CAS  Google Scholar 

  23. 23.

    Iqbal NM, Amirul AA (2013) Synthesis of P(3HB-co-4HB) copolymer with target-specific 4HB molar fractions using combinations of carbon substrates. Journal of Chemical Technology & Biotechnology 89(3):407–418

    Article  Google Scholar 

  24. 24.

    Babel W, Brinkmann U, Muller RH (1993) The auxiliary substrate concept - an approach for overcoming limits of microbial performances. Acta. Biotechnology 13(3):211–242

    CAS  Google Scholar 

  25. 25.

    Huong, K. H., Teh, C. H., & Abdullah, A. A. A. (2017). Microbial-based synthesis of highly elastomeric biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thermoplastic. International Journal of Biological Macromolecules

  26. 26.

    Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25(10):1503–1555

    CAS  Article  Google Scholar 

  27. 27.

    Steinbüchel A, Lütke-Eversloh T (2003) Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 16(2):81–96

    Article  Google Scholar 

  28. 28.

    Aldor IS, Keasling JD (2003) Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 14:475–483

    CAS  Article  Google Scholar 

  29. 29.

    Hema R, Amirul AA (2012) Yellow-pigmented Cupriavidus sp., a novel bacterium capable of utilizing glycerine pitch for the sustainable production of P(3HB-co-4HB). J Chem Technol Biotechnol 88(6):1030–1038

    Google Scholar 

  30. 30.

    Vigneswari S, Nik LA, Majid MI a, Amirul AA (2009) Improved production of poly(3-hydroxybutyrate-co-4-hydroxbutyrate) copolymer using a combination of 1,4-butanediol and γ-butyrolactone. World J Microbiol Biotechnol 26(4):743–746

    Article  Google Scholar 

  31. 31.

    Zhu L, Zhu Y, Zhang Y, Li Y (2012) Engineering the robustness of industrial microbes through synthetic biology. Trends Microbiol 20(2):94–101

    CAS  Article  Google Scholar 

  32. 32.

    Lau N-S, Sudesh K (2012) Revelation of the ability of Burkholderia sp. USM (JCM 15050) PHA synthase to polymerize 4-hydroxybutyrate monomer. AMB Express 2:41–43

    Article  Google Scholar 

  33. 33.

    Grabow N, Schmohl K, Khosravi A, Philipp M, Scharfschwerdt M, Graf B, Schmitz KP, Steinhoff G (2004) Mechanical and structural properties of a novel hybrid heart valve scaffold for tissue engineering. Artif Organs 28(11):971–979

    CAS  Article  Google Scholar 

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The authors acknowledge the research grants provided by the Ministry of Science, Technology and Innovation (02-05-23-SF0023) and also the USM Research University Grant (1001/PBIOLOGI/811304) that has resulted in this article.

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Correspondence to A. A. Amirul.

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Norhafini, H., Thinagaran, L., Shantini, K. et al. Synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with high 4HB composition and PHA content using 1,4-butanediol and 1,6-hexanediol for medical application. J Polym Res 24, 189 (2017).

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  • P(3HB-co-4HB)
  • 4HB monomer
  • Mixed substrate
  • PHA synthase
  • Transformant Cupriavidus sp. USMAA1020