Poly(3-hydroxybutyrate) accumulation by Azotobacter vinelandii under different oxygen transfer strategies

  • Alvaro Díaz-BarreraEmail author
  • Viviana Urtuvia
  • Claudio Padilla-Córdova
  • Carlos Peña
Fermentation, Cell Culture and Bioengineering - Original Paper


Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6 mmol L−1 h−1) and specific oxygen uptake rate (11.6 mmol g−1 h−1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6 w w−1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.


Azotobacter vinelandii Oxygen transfer Poly(3-hydroxybutyrate) 



Financial support came from the FONDECYT Grant 1170896 and Project PCCI40039. The present work was supported by the postdoctoral fellowship of the VRIEA-PUCV.


  1. 1.
    Chen GQ, Jiang XR (2018) Engineering microorganisms for improving polyhydroxyalkanoates biosynthesis. Curr Opin Biotechnol 53:20–25CrossRefGoogle Scholar
  2. 2.
    Wang J, Yu J (2001) Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions. J Ind Microbiol Biotechnol 26:121–126CrossRefGoogle Scholar
  3. 3.
    Díaz-Barrera A, Andler R, Martínez I, Peña C (2016) Poly-3-hydroxybutyrate production by Azotobacter vinelandii strains in batch cultures at different oxygen transfer rates. J Chem Technol Biotechnol 91:1063–1071CrossRefGoogle Scholar
  4. 4.
    Millán M, Segura D, Galindo E, Peña C (2016) Molecular mass of poly-3-hydroxybutyrate (P3HB) produced by Azotobacter vinelandii is determined by the ratio of synthesis and degradation under fixed dissolved oxygen tension. Process Biochem 51:950–958CrossRefGoogle Scholar
  5. 5.
    Peña C, López S, García A, Espín G, Romo-Uribe A, Segura D (2014) Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN). Ann Microbiol 64:39–47CrossRefGoogle Scholar
  6. 6.
    Galindo E, Peña C, Núñez C, Segura D, Espín G (2007) Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoates production by Azotobacter vinelandii. Microb Cell Fact 6(7):1–16Google Scholar
  7. 7.
    Khanna S, Srivastava AK (2005) Recent advances in microbial polyhydroxyalkanoates. Process Biochem 40:607–619CrossRefGoogle Scholar
  8. 8.
    Peralta-Gil M, Segura D, Guzmán J, Servín-González L, Espín G (2002) Expression of the Azotobacter vinelandii poly-β-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and it dependent on the transcriptional activator PhbR. J Bacteriol 184(20):5672–5677CrossRefGoogle Scholar
  9. 9.
    Segura D, Guzmán J, Espín G (2003) Azotobacter vinelandii mutants that overproduce poly-β-hydroxybutyrate or alginate. Appl Microbiol Biotechnol 63:159–163CrossRefGoogle Scholar
  10. 10.
    Díaz-Barrera A, Soto E, Altamirano C (2012) Alginate production and alg8 gene expression by Azotobacter vinelandii in continuous cultures. J Ind Microbiol Biotechnol 39:613–621CrossRefGoogle Scholar
  11. 11.
    Lozano E, Galindo E, Peña C (2011) Oxygen transfer rate during the production of alginate by Azotobacter vinelandii under oxygen-limited and non oxygen-limited conditions. Microb Cell Fact 10:13CrossRefGoogle Scholar
  12. 12.
    García A, Ferrer P, Albiol J, Castillo T, Segura D, Peña C (2018) Metabolic flux analysis and the NAD(P)H/NAD(P)+ ratios in chemostat cultures of Azotobacter vinelandii. Microb Cell Fact 17:10CrossRefGoogle Scholar
  13. 13.
    Díaz-Barrera A, Silva P, Ávalos R, Acevedo F (2009) Alginate molecular mass produced by Azotobacter vinelandii in response to changes of the O2 transfer rate in chemostat cultures. Biotechnol Lett 31:825–829CrossRefGoogle Scholar
  14. 14.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428CrossRefGoogle Scholar
  15. 15.
    Anderlei T, Büchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7(2):157–162CrossRefGoogle Scholar
  16. 16.
    Díaz-Barrera A, Peña C, Galindo E (2007) The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii. Appl Microbiol Biotechnol 76(4):903–910CrossRefGoogle Scholar
  17. 17.
    García A, Segura D, Espín G, Galindo E, Castillo T, Peña C (2014) High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process. Biochem Eng J 82:117–123CrossRefGoogle Scholar
  18. 18.
    Millán M, Salazar M, Segura D, Castillo T, Díaz-Barrera A, Peña C (2017) Molecular mass of poly-3-hydroxybutyrate (P3HB) produced by Azotobacter vinelandii is influenced by the polymer content in the inoculum. J Biotech 259:50–55CrossRefGoogle Scholar
  19. 19.
    Koller M, Bona R, Chiellini E, Grillo Fernandes E, Horvat P, Kutschera C, Hesse P, Braunegg G (2008) Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora. Bioresour Technol 99:4854–4863CrossRefGoogle Scholar
  20. 20.
    Faccin DJL, Rech R, Secchi AR, Cardozo NSM, Ayub MAZ (2013) Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium. Process Biochem 48:420–425CrossRefGoogle Scholar
  21. 21.
    Ouyang P, Wang H, Hajnal I, Wu Q, Guo Y, Chen GQ (2018) Increasing oxygen availability for improving poly(3-hydroxybutyrate) production by Halomonas. Metab Eng 45:20–31CrossRefGoogle Scholar
  22. 22.
    Castillo T, Heinzle E, Peifer S, Schneider K, Peña C (2013) Oxygen supply strongly influences metabolic fluxes, the production of poly(3-hydroxybutyrate) and alginate, and the degree of acetylation of alginate in Azotobacter vinelandii. Process Biochem 48(7):995–1003CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  1. 1.Escuela de Ingeniería BioquímicaPontificia Universidad Católica de ValparaísoValparaísoChile
  2. 2.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

Personalised recommendations