Applied Biochemistry and Biotechnology

, Volume 176, Issue 5, pp 1315–1334 | Cite as

Influence of Feeding and Controlled Dissolved Oxygen Level on the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Copolymer by Cupriavidus sp. USMAA2-4 and Its Characterization

  • K. Shantini
  • A. R. M. Yahya
  • A. A. Amirul


Copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been the center of attention in the bio-industrial fields, as it possesses superior mechanical properties compared to poly(3-hydroxybutyrate) [P(3HB)]. The usage of oleic acid and 1-pentanol was exploited as the carbon source for the production of P(3HB-co-3HV) copolymer by using a locally isolated strain Cupriavidus sp. USMAA2-4. In this study, the productivity of polyhydroxyalkanoate (PHA) was improved by varying the frequency of feeding in fed-batch culture. The highest productivity (0.48 g/L/h) that represents 200 % increment was obtained by feeding the carbon source and nitrogen source three times and also by considering the oxygen uptake rate (OUR) and oxygen transfer rate (OTR). A significantly higher P(3HB-co-3HV) concentration of 25.7 g/L and PHA content of 66 wt% were obtained. The 3-hydroxyvalerate (3HV) monomer composition obtained was 24 mol% with the growth of 13.3 g/L. The different frequency of feeding carried out has produced a blend copolymer and has broadened the monomer distribution. In addition, increase in number of granules was also observed as the frequency of feeding increases. In general, the most glaring increment in productivity offer advantage for industrial P(3HB-co-3HV) production, and it is crucial in developing cost-effective processes for commercialization.


Biopolymer 1-Pentanol Oleic acid Productivity P(3HB-co-3HV) Fed-batch 



The authors acknowledge the USM Science Fellowship awarded to Shantini [RU(1001/441/CIPS/AUPE001)] that has resulted in this article.

Compliance with Ethical Standards

All the co-authors have seen and agreed with the contents of the manuscript, and there is no financial interest to report. We certify that the submission is the original work by us and is not under review in any other publication. We also would like to justify that we do not have any conflict of interest to declare and this study does not involve the usage of animals.


  1. 1.
    Chien, C. C., Chen, C. C., Choi, M. H., Kung, S. S., & Wei, Y. H. (2007). Production of poly [beta]-hydroxybutyrate (PHB) by Vibrio spp. isolated from marine environment. Journal of Biotechnology, 132, 259–263.CrossRefGoogle Scholar
  2. 2.
    Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M., & Shah, S. (2007). Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants – A review. Biotechnology Advances, 25, 148–175.CrossRefGoogle Scholar
  3. 3.
    Akiyama, M., Taima, Y., & Doi, Y. (1992). Production of poly(3-hydroxyalkanoates) by a bacterium of the genus Alcaligenes utilizing long-chain fatty acids. Applied Microbiology and Biotechnology, 37, 698–701.CrossRefGoogle Scholar
  4. 4.
    Ayub, N. D., Pettinari, M. J., Méndez, B. S., & López, N. I. (2007). The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island. Plasmid, 58, 240–248.CrossRefGoogle Scholar
  5. 5.
    Iwata, T., Tsunoda, K., Aoyagi, Y., Kusaka, S., Yonezawa, N., & Doi, Y. (2003). Mechanical properties of uniaxially cold-drawn films of poly ([R]-3-hydroxybutyrate). Polymer Degradation and Stability, 79, 217–224.CrossRefGoogle Scholar
  6. 6.
    Sudesh, K., Abe, H., & Doi, Y. (2000). Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Progress in Polymer Science, 25, 1503–1555.CrossRefGoogle Scholar
  7. 7.
    Kim, D. Y., Park, D. S., Kwon, S. B., Chung, M. G., Bae, K. S., Park, H. Y., & Rhee, Y. H. (2009). Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co polyesters with a high molar fraction of 3-hydroxyvalerate by an insect symbiotic Burkholderia sp. IS-01. The Journal of Microbiology, 47, 651–656.CrossRefGoogle Scholar
  8. 8.
    Cerrone, F., Duane, G., Casey, E., Davis, R., Belton, I., Kenny, S. T., Guzik, M. W., Woods, T., Babu, R. P., & O’Connor, K. (2014). Fed batch strategies using butyrate for high cell density cultivation of Pseudomonas putida and its use as a biocatalyst. Applied Microbiology and Biotechnology, 98(22), 9217–9228.CrossRefGoogle Scholar
  9. 9.
    Khanna, S., & Srivastava, A. K. (2005). Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry, 40, 607–619.CrossRefGoogle Scholar
  10. 10.
    Lee, S. Y., & Choi, J. I. (1998). Effects of fermentation performance by Alcaligenes latus. Polymer Degradation and Stability, 59, 387–393.CrossRefGoogle Scholar
  11. 11.
    Chen, G. Q., Zhang, G., Park, S. J., & Lee, S. Y. (2001). Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Applied Microbiology and Biotechnology, 57, 50–55.CrossRefGoogle Scholar
  12. 12.
    Amirul, A. A., Yahya, A. R. M., Sudesh, K., Azizan, M. N. M., & Majid, M. I. A. (2009). Isolation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) producer from Malaysian environment using γ-butyrolactone as carbon source. World Journal Of Microbiology and Biotechnology, 25, 1199–1206.CrossRefGoogle Scholar
  13. 13.
    Ochoa, F. G., & Gomez, E. (2009). Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview. Biotechnology Advances, 27, 153–176.CrossRefGoogle Scholar
  14. 14.
    Majid, M. I. A. (1988). PhD thesis, University of Bath.Google Scholar
  15. 15.
    Chee, J.-W., Amirul, A. A., Majid, M. I. A., & Mansor, S. M. (2008). Factors influencing the release of Mitragyna speciosa crude extracts from biodegradable P(3HB-co-4HB). International Journal of Pharmaceutics, 361, 1–6.CrossRefGoogle Scholar
  16. 16.
    Mancini, S. D., & Zanin, M. (1999). Recyclability of Pet from virgin resin. Materials Research, 2, 33–38.CrossRefGoogle Scholar
  17. 17.
    Amirul, A. A., Syairah, S. N., Yahya, A. R. M., Azizan, M. N. M., & Majid, M. I. A. (2008). Synthesis of biodegradable polyesters by Gram negative bacterium isolated from Malaysian environment. World Journal of Microbiology and Biotechnology, 24, 1327–1332.CrossRefGoogle Scholar
  18. 18.
    Loo, C. Y., Lee, W. H., Tsuge, T., Doi, Y., & Sudesh, K. (2005). Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnology Letters, 27, 1405–1410.CrossRefGoogle Scholar
  19. 19.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275.Google Scholar
  20. 20.
    Jeong, H., Park, J., & Kim, H. (2013). Determination of NH4 + in environmental water with interfering substances using the modified Nessler method. Journal of Chemistry, Article ID 359217.Google Scholar
  21. 21.
    Solorzano, L. (1969). Determination of ammonia in natural waters by the phenolhypochlorite method. Limnology and Oceonography, 14(5), 799–801.CrossRefGoogle Scholar
  22. 22.
    Braunegg, G., Sonnleitner, B., & Lafferty, R. M. (1978). A rapid gas chromatography method for determination of the poly-β-hydroxybutyric acid in microbial biomass. European Journal of Applied Microbiology and Biotechnology, 6, 29–37.CrossRefGoogle Scholar
  23. 23.
    Lenczak, J. L., Schmidell, W., & Aragao, G. M. F. (2013). High cell density strategies for polyhydroxyalkanoate production: a review. Journal of Industrial Microbiology and Biotechnology, 40(3-4), 275–286.CrossRefGoogle Scholar
  24. 24.
    Yamane, T., Chen, X., & Ueda, S. (1996). Growth-associated production of poly(3-hydroxyvalerate) from n-pentanol by a methylotrophic bacterium, Paracoccus denitrificans. Applied and Environmental Microbiology, 62, 380–384.Google Scholar
  25. 25.
    Shang, L., Jiang, M., & Chang, H. N. (2003). Poly(3-hydroxybutyrate) synthesis in fed batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations. Biotechnology Letters, 25, 1415–1419.CrossRefGoogle Scholar
  26. 26.
    Lee, S. Y., Choi, J. I., & Wong, H. H. (1999). Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review. International Journal of Biological Macromolecules, 25, 31–36.CrossRefGoogle Scholar
  27. 27.
    Madden, L. A., & Anderson, A. J. (1998). Synthesis and characterization of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxybutyrate) polymer mixtures production in high-density fed –batch. Macromolecules, 31, 5660–5667.CrossRefGoogle Scholar
  28. 28.
    Shang, L., Yim, S. C., Park, H. G., & Chang, H. N. (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. Biotechnology Progress, 20, 140–144.CrossRefGoogle Scholar
  29. 29.
    Choi, J. I., & Lee, S. Y. (1999). High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant. Escherichia coli Applied Environmental and Microbiology, 65, 4363–4368.Google Scholar
  30. 30.
    Majid, M. I. A., Akmal, D. H., Few, L. L., Agustien, A., Toh, M. S., Samian, M. R., Najimudin, N., & Azizan, M. N. (1999). Production of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Erwinia sp. USMI-20. International Journal of Biological Macromolecules, 25, 95–104.CrossRefGoogle Scholar
  31. 31.
    Ma, C.K., Chua, H., Yu, P.H.U., & Hong, K. (2000). Optimal production of polyhydroxyalkanoates in activated sludge biomass. Applied Biochemistry and Biotechnology, 84-86, 981-989.Google Scholar
  32. 32.
    Garcia, I. L., Lopez, J. A., Dorado, M. P., Kopsahelis, N., Alexandri, M., Papanikolaou, S., Villar, M. A., & Koutinas, A. A. (2013). Evaluation of by-products from the biodiesel industry as fermentation feedstock for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Cupriavidus necator. Bioresource Technology, 130, 16–22.CrossRefGoogle Scholar
  33. 33.
    Quangliano, J. C., & Miyazaki, S. S. (1997). Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-ß-hydroxybutyrate obtained from Azotobacter chroococcum 6B. Applied Microbiology and Biotechnology, 48, 662–664.CrossRefGoogle Scholar
  34. 34.
    Calik, P., Yilgor, P., Ayhan, P., & Demir, A. S. (2004). Oxygen transfer effects on recombinant benzaldehyde lyase production. Chemical Engineering Science, 59, 5075–5083.CrossRefGoogle Scholar
  35. 35.
    Zafar, M., Kumar, S., & Dhiman, A. K. (2012). Modeling and optimization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from cane molasses by Azohydromonas lata MTCC 2311 in a stirred-tank reactor: effect of agitation and aeration regimes. Journal of Industrial Microbiology and Biotechnology, 39, 987–1001.CrossRefGoogle Scholar
  36. 36.
    Third, K. A., Newland, M., & Ruwisch, R. C. (2003). The effect of dissolved oxygen on PHB accumulation in activated sludge cultures. Biotechnology and Bioengineering, 82, 238–250.CrossRefGoogle Scholar
  37. 37.
    Choi, J. C., Shin, H. D., & Lee, Y. H. (2002). Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed batch culture of recombinant. Eschericia Coli Enzyme and Microbial Technology, 32, 178–185.CrossRefGoogle Scholar
  38. 38.
    Nyman, A. K. (2010). Master thesis.Google Scholar
  39. 39.
    Kamiya, N., Yamamoto, Y., Inoue, Y., & Chujo, R. (1989). Microstructure of bacterially synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules, 22, 1676–1682.CrossRefGoogle Scholar
  40. 40.
    Ivanova, G., Serafim, L. S., Lemos, P. C., Ramos, A. M., Reis, M. A. M., & Cabrita, E. J. (2009). Influence of feeding strategies of mixed microbial cultures on the chemical composition and microstructure of copolyesters P(3HB-co-3HV) analyzed by NMR and statistical analysis. Magnetic Resonance in Chemistry, 47, 497–504.CrossRefGoogle Scholar
  41. 41.
    Luo, S., Grubb, D. T., & Netravali, A. N. (2002). The effect of molecular weight on the lamellar structure, thermal and mechanical properties of poly(hydroxybutyrate-co- hydroxyvalerates). Polymer, 43, 4159–4166.CrossRefGoogle Scholar
  42. 42.
    Scandola, M., Ceccoralli, G., & Doi, Y. (1990). Viscoelastic relaxations and thermal properties of bacterial poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). International Journal of Biological Macromolecules, 12, 112–117.CrossRefGoogle Scholar
  43. 43.
    Doi, Y. (1990). Microbial polyesters. Ch 3. New York: VCH Publishers.Google Scholar
  44. 44.
    You, J. W., Chiu, H. J., Shu, W. J., & Don, T. M. (2003). Influence of hydroxyvalerate content on the crystallization kinetics of poly(hydroxybutyrate-co-hydroxyvalerate). Journal of Polymer Research, 10, 47–54.CrossRefGoogle Scholar
  45. 45.
    Zhang, H. F., Ma, L., Wang, Z. H., & Chen, G. Q. (2009). Biosynthesis and characterization of 3-hydroxyalkanoate terpolyesters with adjustable properties by Aeromonas hydrophila. Biotechnology and Bioengineering, 104(3), 582–589.CrossRefGoogle Scholar
  46. 46.
    Kunioka, M., Tamaki, A., & Doi, Y. (1989). Crystalline and thermal properties of bacterial copolyesters: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Macromolecules, 22, 694–697.CrossRefGoogle Scholar
  47. 47.
    Galego, N., Rozsa, C., Sanchez, R., Fung, J., Vazquez, A., & Tomas, J. S. (2000). Characterization and application of poly(ß-hydroxyalkanoates) family as composite Biomaterial. Polymer Testing, 19, 485–49.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • K. Shantini
    • 1
  • A. R. M. Yahya
    • 1
  • A. A. Amirul
    • 1
    • 2
    • 3
  1. 1.School of Biological SciencesUniversiti Sains MalaysiaMindenMalaysia
  2. 2.Malaysian Institute of Pharmaceuticals and NutraceuticalsMOSTIPenangMalaysia
  3. 3.Centre for Chemical BiologyUniversiti Sains MalaysiaPenangMalaysia

Personalised recommendations