Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in Bacillus aryabhattai and cytotoxicity evaluation of PHBV/poly(ethylene glycol) blends

  • 39 Accesses

Abstract

The study described poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation in Bacillus aryabhattai PHB10 for the first time and evaluated the polymer induced cytotoxicity in-vitro with PHBV/poly(ethylene glycol) (PEG) blends. The B. aryabhattai strain produced 2.8 g/L PHBV, equivalent to 71.15% of cell dry mass in a medium supplemented with propionic acid, after 48 h incubation. The optimum temperature and pH for the copolymer accumulation was 31 °C and 7, respectively. The gas chromatography–mass spectrometry and nuclear magnetic resonance analyses confirmed the polymer obtained as PHBV. The differential scanning calorimetry analysis revealed that the melting point of the material as 90 °C and its thermal stability up to 220 °C. The average molecular weight (Mn) and polydispersity index of the sample was estimated by gel permeation chromatography analysis and observed as 128.508 kDa and 2.82, respectively. The PHBV showed tensile strength of 10.3 MPa and elongation at break of 13.3%. The PHBV and their blends with PEG were tested for cytotoxicity on human keratinocytes (HaCaT cells) and the cells incubated with PHBV/PEG2kDa blends were 99% viable, whereas with the PHBV alone showed comparatively higher cytotoxicity. The significant improvement in the cell viability of PHBV/PEG2kDa blends indicates its potential as a candidate for skin graft applications.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Abd-El-Haleem DA (2009) Biosynthesis of polyhydroxyalkanotes in wild type yeasts. Pol J Microbiol 58(1):37–41

  2. Andreeben B, Lange AB, Robenek H, Steinbüchel A (2010) Conversion of glycerol to poly(3-hydroxypropionate) in recombinant Escherichia coli. Appl Environ Microbiol 76(2):622–626.2

  3. Aramvash A, Hajizadeh-Turchi S, Moazzeni-zavareh F, Gholami-Banadkuki N, Malek-sabet N, Akbari-Shahabi Z (2016) Effective enhancement of hydroxyvalerate content of PHBV in Cupriavidus necator and its characterization. Int J Biol Macromol 87:397–404

  4. ASTM D882-12 (2012) Standard Test Method for Tensile Properties of Thin Plastic Sheeting, ASTM International, West Conshohocken, PA. www.astm.org

  5. Avella M, Martuscelli E, Raimo M (2000) Properties of blends and composites based on poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-hydroxyvalerate) (PHBV) copolymers. J Mater Sci 35:523–545

  6. Berger E, Ramsay BA, Ramsay JA, Chavarie C, Braunegg G (1989) PHB recovery by hypochlorite digestion of non-PHB biomass. Biotechnol Tech 3(4):227–232

  7. Bhuwal AK, Singh G, Aggarwal NK, Goyal V, Yadav A (2014) Poly-β-hydroxybutyrate production and management of cardboard industry effluent by new Bacillus sp. NA10. Bioresour Bioprocess. https://doi.org/10.1186/s40643-014-0009-5

  8. Castellano D, Sanchis A, Blanes M et al (2017) Electrospun poly (hydroxybutyrate) scaffolds promote engraftment of human skin equivalents via macrophage M2 polarization and angiogenesis. J Tissue Eng Regen Med. https://doi.org/10.1002/term.2420

  9. Catoni SE, Trindade KN, Gomes CA, Schneider AL, Pezzin A, Soldi V (2013) Influence of poly (ethylene glycol)-(PEG) on the properties of influence of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)-PHBV. Polímeros 23(3):320–325

  10. Chan RT, Marçal H, Russell RA, Holden PJ, Foster LJ (2011) Application of polyethylene glycol to promote cellular biocompatibility of polyhydroxybutyrate films. Int J Polym Sci. https://doi.org/10.1155/2011/473045

  11. Chen Q, Wang Q, Wei G, Liang Q, Qi Q (2011) Production in Escherichia coli of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources. Appl Environ Microbiol 77(14):4886–4893

  12. Cheng G, Cai Z, Wang L (2003) Biocompatibility and biodegradation of poly (hydroxybutyrate)/poly (ethylene glycol) blend films. J Mater Sci Mater Med 14(12):1073–1078

  13. Gunaratne LMWK, Shanks RA, Amarasinghe G (2004) Thermal history effects on crystallisation and melting of poly (3-hydroxybutyrate). Thermochim Acta 423(1):127–135

  14. Güngörmedi G, Demirbilek M, Mutlu MB, Denkbaş EB, Çabuk A (2014) Polyhydroxybutyrate and hydroxyvalerate production by Bacillus megaterium strain A1 isolated from hydrocarbon-contaminated soil. J Appl Polym Sci. https://doi.org/10.1002/app.40530

  15. Hu X, Liu H, Gao Z, Su T, Wang Z, Yang L (2017) Blending modification of PHBV/PBS/PEG and its biodegradation. Polym Plast Technol Eng 56(10):1128–1135

  16. Jiang L, Zhang J (2013) Biodegradable polymers and polymer blends. In: Ebnesajjad S (ed) Handbook of biopolymers and biodegradable plastics. William Andrew Publishing, Boston, pp 109–128

  17. Jost V, Miesbauer O (2018) Effect of different biopolymers and polymers on the mechanical and permeation properties of extruded PHBV cast films. J Appl Polym Sci 135(15):46153

  18. Juengert JR, Bresan S, Jendrossek D (2018) Determination of polyhydroxybutyrate (PHB) content in Ralstonia eutropha using gas chromatography and Nile Red staining. Bio-protocol. https://doi.org/10.21769/BioProtoc.2748

  19. Kuciel S, Mazur K, Jakubowska P (2019) Novel biorenewable composites based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with natural fillers. J Polym Environ 27(4):803–815

  20. Kumar PKA, Shamala TR, Kshama L, Prakash MH, Joshi GJ, Chandrashekar A, Kumari KSL, Divyashree MS (2006) Bacterial synthesis of poly (hydroxybutyrate-co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers. J Appl Microbiol 103(1):204–209

  21. Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36

  22. Lee S, Ka JO, Song HG (2012) Growth promotion of Xanthium italicum by application of rhizobacterial isolates of Bacillus aryabhattai in microcosm soil. J Microbiol 50(1):45–49

  23. Lemoigne M (1926) Products of dehydration and of polymerizationof β-hydroxybutyric acid. Bull Soc Chem Biol 8:770–782

  24. Li T, Ye J, Shen R, Zong Y, Zhao X, Lou C, Chen GQ (2016) Semi-rational approach for ultra-high poly(3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening. ACS Synth Biol 5(11):1308–1317

  25. Little CJ, Bawolin NK, Chen X (2011) Mechanical properties of natural cartilage and tissue-engineered constructs. Tissue Eng Rev 17:213–227

  26. Liu Q, Zhang H, Deng B, Zhao X (2014) Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate): structure, property, and fiber. Int J Polym Sci. https://doi.org/10.1155/2014/374368

  27. Lomas AJ, Webb WR, Han J, Chen GQ, Sun X, Zhang Z, El Haj AJ, Forsyth NR (2013) Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications. Tissue Eng Part C Methods 19(8):577–585

  28. Masood F, Hasan F, Ahmed S, Hameed A (2012) Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus FA11 isolated from TNT-contaminated soil. Ann Microbiol 62(4):1377–1384

  29. Modi S, Koelling K, Vodovotz Y (2013) Assessing the mechanical, phase inversion and rheological properties of poly-[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) blended with poly-(l-lactic acid) (PLA). Eur Polym J 49:3681–3690

  30. Monnier A, Rombouts C, Kouider D, About I, Fessi H, Sheibat-Othman N (2016) Preparation and characterization of biodegradable polyhydroxybutyrate-co-hydroxyvalerate/polyethylene glycol-based microspheres. Int J Pharm 513(1–2):49–61

  31. Moorkoth D, Nampoothiri KM (2016) Production and characterization of poly (3-hydroxy butyrate-co-3 hydroxyvalerate)(PHBV) by a novel halotolerant mangrove isolate. Bioresour Technol 201:253–260

  32. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63

  33. Myung J, Flanagan JC, Waymouth RM, Criddle CS (2017) Expanding the range of polyhydroxyalkanoates synthesized by methanotrophic bacteria through the utilization of omega-hydroxyalkanoate co-substrates. AMB Expres 7(1):118

  34. Napathorn SC (2014) Biocompatibilities and biodegradation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)s produced by a model metabolic reaction-based system. BMC Microbiol 14:285

  35. Pailan S, Gupta D, Apte S, Krishnamurthi S, Saha P (2015) Degradation of organophosphate insecticide by a novel Bacillus aryabhattai strain SanPS1, isolated from soil of agricultural field in Burdwan, West Bengal, India. Int Biodeterior Biodegrad 103:191–195

  36. Parker RE (1979) Continuous distribution: tests of significance. In: Parker RE (ed) Introductory statistics for biology, 2nd edn. Cambridge University Press, London, pp 18–42

  37. Pillai AB, Kumar AJ, Kumarapillai H (2018) Enhanced production of poly (3-hydroxybutyrate) in recombinant Escherichia coli and EDTA-microwave-assisted cell lysis for polymer recovery. AMB Express 8(1):142

  38. Pillai AB, Kumar AJ, Thulasi K, Kumarapillai H (2017a) Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai. Braz J Microbiol 48(3):451–460

  39. Pillai AB, Kumar AJ, Thulasi K, Reghunathan D, Prasannakumar M, Kumarapillai H (2017b) Draft genome sequence of Bacillus aryabhattai strain PHB10, a poly (3-hydroxybutyrate)-accumulating bacterium isolated from domestic sewerage. Genome Announc 5(41):e01072–e1117

  40. Qi Q, Rehm BH (2001) Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase. Microbiology 147(12):3353–3358

  41. Riekes MK, Junior LR, Pereira RN, Borba PA, Fernandes D, Stulzer HK (2013) Development and evaluation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and polycaprolactone microparticles of nimodipine. Curr Pharm Des 19:7264–7270

  42. Rodrigues JA, Parra DF, Lugão AB (2005) Crystallization on films of PHB/PEG blends: evaluation by DSC. J Therm Anal Calorim 79(2):379–381

  43. Salgaonkar BB, Mani K, Braganca JM (2013) Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16. J Appl Microbiol 114(5):1347–1356

  44. Seo MD, Kang TJ, Lee CH, Lee AY, Noh M (2012) HaCaT keratinocytes and primary epidermal keratinocytes have different transcriptional profiles of cornified envelope-associated genes to T helper cell cytokines. Biomol Ther 20(2):171

  45. Shabna A, Saranya V, Malathi J, Shenbagarathai R, Madhavan HN (2014) Indigenously produced polyhydroxyalkanoate dased co-polymer as cellular supportive biomaterial. J Biomed Mater Res A 102:3470–3476

  46. Shang L, Yim SC, Park HG, Chang HN (2004) Sequential feeding of glucose and valerate in a fed-batch culture of Ralstonia eutropha for production of poly(hydroxybutyrate-co-hydroxyvalerate) with high 3-hydroxyvalerate fraction. Biotechnol Prog 20:140–144

  47. Shi H, Shiraishi M, Shimizu K (1997) Metabolic flux analysis for biosynthesis of poly(β-hydroxybutyric acid) in Alcaligenes eutrophus from various carbon sources. J Ferment Bioeng 84(6):579–587

  48. Shivaji S, Chaturvedi P, Begum Z et al (2009) Janibacter hoylei sp. nov., Bacillus isronensis sp. nov. and Bacillus aryabhattai sp. nov., isolated from cryotubes used for collecting air from the upper atmosphere. Int J Syst Evol Microbiol 59(12):2977–2986

  49. Smith JR, Lamprou DA (2014) Polymer coatings for biomedical applications: a review. Trans IMF. https://doi.org/10.1179/0020296713Z.000000000157

  50. Su WF (2013) Polymer size and polymer solutions. Principles of polymer design and synthesis. Lecture notes in chemistry. Springer, Berlin, pp 9–26

  51. Sundaramurthi D, Krishnan UM, Sethuraman S (2013) Biocompatibility of poly (3-hydroxybutyrate-co3-hydroxyvalerate)(PHBV) nanofibers for skin tissue engineering. J Biomed Nanotechnol 9(8):1383–1392

  52. Van-Thuoc D, Huu-Phong T, Thi-Binh N, Thi-Tho N, Minh-Lam D, Quillaguamán J (2012) Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in Northern Vietnam. Microbiologyopen 1(4):395–406

  53. Vilos C, Morales FA, Solar PA et al (2013) Paclitaxel-PHBV nanoparticles and their toxicity to endometrial and primary ovarian cancer cells. Biomaterials 34:4098–4108

  54. Wang YY, Lü LX, Shi JC, Wang HF, Xiao ZD, Huangn NP (2011) Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility. Biomacromol 12(3):551–559

  55. Wang Y, Chen R, Cai J, Liu Z, Zheng Y, Wang H, Li Q, He N (2013) Biosynthesis and thermal properties of PHBV produced from levulinic acid by Ralstonia eutropha. PLoS ONE 8(4):e60318

  56. Wu J, Xue K, Li H, Sun J, Liu K (2013) Improvement of PHBV scaffolds with bioglass for cartilage tissue engineering. PLoS ONE 8(8):e71563

  57. Xiang H, Wang S, Wang R, Zhou Z, Peng C, Zhu M (2013) Synthesis and characterization of an environmentally friendly PHBV/PEG copolymer network as a phase change material. Sci China Chem 56(6):716–723

  58. Yan Y, Zhang L, Yu MY et al (2016) The genome of Bacillus aryabhattai T61 reveals its adaptation to Tibetan Plateau environment. Genes Genom 38(3):293–301

Download references

Acknowledgements

The authors are grateful to Prof. M. Radhakrishna Pillai, Director, RGCB for providing the facilities; the Kerala Forest Research Institute, Peechi for GC–MS analysis and the SAIF at STIC-Kochi for the NMR and TGA facilities.

Funding

This work was supported by funding from the Department of Biotechnology (DBT), Government of India as an intramural grant. ABP received a research fellowship from the Council of Scientific and Industrial Research (CSIR), India (SRF; 09/716(0149)/2012-EMR-I).

Author information

ABP and HK conceived and designed the experiments; ABP and AJK did the experiments; ABP, AJK and HK analyzed the data and wrote this manuscript. All authors read and approved the final manuscript.

Correspondence to Harikrishnan Kumarapillai.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Balakrishna Pillai, A., Jaya Kumar, A. & Kumarapillai, H. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in Bacillus aryabhattai and cytotoxicity evaluation of PHBV/poly(ethylene glycol) blends. 3 Biotech 10, 32 (2020). https://doi.org/10.1007/s13205-019-2017-9

Download citation

Keywords

  • Bacillus aryabhattai PHB10
  • Cytotoxicity
  • Gas chromatography–mass spectrometry
  • HaCaT cells
  • Poly(ethylene glycol)
  • Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)