Analysis of respiratory activity and carbon usage of a mutant of Azotobacter vinelandii impaired in poly-β-hydroxybutyrate synthesis

  • Lucero Jiménez
  • Tania Castillo
  • Celia Flores
  • Daniel Segura
  • Enrique Galindo
  • Carlos Peña
Short Communication


In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-β-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).


Azotobacter vinelandii Carbon usage Respiratory activity Poly(β-hydroxybutyrate) Alginate production 



We acknowledge the financial support from DGAPA-UNAM (Grant IT100513) and CONACyT (Grant 238535).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Anderlei T, Büchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7:157–162CrossRefPubMedGoogle Scholar
  2. 2.
    Anderlei T, Zang W, Papaspyrou M, Büchs J (2004) Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem Eng J 17:187–194CrossRefGoogle Scholar
  3. 3.
    Castillo T, Galindo E, Peña C (2013) The acetylation degree of alginates in Azotobacter vinelandii ATCC 9046 is determined by dissolved oxygen and specific growth rate: studies in glucose-limited chemostat cultivations. J Ind Microbiol Biotechnol 40:715–723CrossRefPubMedGoogle Scholar
  4. 4.
    Castillo T, Heinzle E, Peifer S, Schneider K, Peña C (2013) Oxygen supply strongly influences metabolic fluxes, production of poly(3-hydroxybutyrate) and alginate, and the degree of acetylation of alginate in Azotobacter vinelandii. Process Biochem 48(7):995–1003CrossRefGoogle Scholar
  5. 5.
    Clementi F (1997) Alginate production from Azotobacter vinelandii. Crit Rev Biotechnol 17(4):327–361CrossRefPubMedGoogle Scholar
  6. 6.
    Clarke AJ, Strating H, Blackburn NT (2000) Pathways for the O-acetylation of bacterial cell wall polysaccharides. In: Ron, J Doyle (ed), Glycomicrobiology, Plenum publishing co. ltd., New York 187–212Google Scholar
  7. 7.
    Díaz-Barrera A, Andler R, Martínez I, Peña C (2015) Poly-3-hydroxybutyrate production by Azotobacter vinelandii strains in batch cultures at different oxygen transfer rates. J Chem Technol Biotechnol. doi: 10.1002/jctb.4684 Google Scholar
  8. 8.
    Draget KI, Taylor C (2011) Chemical, physical and biological properties of alginates and biomedical implications. Food Hydrocoll 25:251–256CrossRefGoogle Scholar
  9. 9.
    Franklin MJ, Ohman DE (1996) Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation. J Bacteriol 178(8):2186–2195PubMedPubMedCentralGoogle Scholar
  10. 10.
    Galindo E, Peña C, Núñez C, Segura D, Espín G (2007) Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact 6(7)Google Scholar
  11. 11.
    Gaytan I, Peña C, Nuñez C, Córdova MS, Espín G, Galindo E (2012) Azotobacter vinelandii lacking the Na+-NQR activity: a potential source for producing alginates with improved properties and at high yield. World J Microbiol Biotechnol 28:2731–2740CrossRefPubMedGoogle Scholar
  12. 12.
    Heinzle E, Lafferty RM (1980) A kinetic model for growth and synthesis of poly-β-hydroxybutyric acid (PHB) in Alcaligenes eutrophus. Eur J App Microbiol Biotechnol 11:8–16CrossRefGoogle Scholar
  13. 13.
    Jarman TR, Pace GW (1984) Energy requirements for microbial exopolysaccharide synthesis. Arch Microbiol 137:231–235CrossRefGoogle Scholar
  14. 14.
    Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  15. 15.
    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 (13)Google Scholar
  16. 16.
    Mejía MA, Segura D, Espín G, Galindo E, Peña C (2010) Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-β-hydroxybutyrate (PHB) synthesis. J App Microbiol 108:55–61CrossRefGoogle Scholar
  17. 17.
    Noguez R, Segura D, Moreno S, Hernández A, Juárez K, Espín G (2008) Enzyme INtr, NPr and IIANtr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii. J Mol Microbiol Biotechnol 15:244–254CrossRefPubMedGoogle Scholar
  18. 18.
    Page WJ, Knosp O (1989) Hyperproduction of Poly-β-hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Microbiol 55:1334–1339PubMedPubMedCentralGoogle Scholar
  19. 19.
    Peña C, Campos N, Galindo E (1997) Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks. Appl Microbiol Biotechnol 48:510–515CrossRefGoogle Scholar
  20. 20.
    Peña C, Hernández L, Galindo E (2006) Manipulation of the acetylation degree of Azotobacter vinelandii alginate by supplementing the culture medium with 3-(N-morpholino)-propane-sulfonic acid. Lett Appl Microbiol 43:200–204CrossRefPubMedGoogle Scholar
  21. 21.
    Rehm BH, Valla S (1997) Bacterial alginates: biosynthesis and applications. Appl Microbiol Biotechnol 48:281–288CrossRefPubMedGoogle Scholar
  22. 22.
    Riley LM, Weadge JT, Baker P, Robinson H, Codée JDC, Tipton PA, Ohman D, Howell PL (2013) Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation. J Biol Chem 288:22299–22314CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Sabra W, Zeng A-P, Sabry S, Omar S, Deckwer W-D (1999) Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions. Appl Microbiol Biotechnol 52:773–780CrossRefGoogle Scholar
  24. 24.
    Sabra W, Zeng A-P, Lunsdorf H, Deckwer WD (2000) Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 66:4037–4044CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Segura D, Cruz T, Espín G (2003) Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol 179:437–443PubMedGoogle Scholar
  26. 26.
    Segura D, Guzmán J, Espín G (2003) Azotobacter vinelandii mutants that overproduce poly-β-hydroxybutyrate or alginate. Appl Microbiol Biotechnol 63:159–163CrossRefPubMedGoogle Scholar
  27. 27.
    Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinkii. Biochem J 134:225–238CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Senior PJ, Dawes EA (1971) Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem J 125:55–66CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Skjak-Braek G, Grasdalen H, Larsen B (1986) Monomer sequence and acetylation pattern by some bacterial alginates. Carbohyd Res 154:239–250CrossRefGoogle Scholar
  30. 30.
    Stevenson LH, Socolofsky MD (1966) Cyst formation and poly-3-hydroxybutyric acid accumulation in Azotobacter. J Bacteriol 91:304–310PubMedPubMedCentralGoogle Scholar
  31. 31.
    Straathman A, Windhues T, Borchard W (2004) Effects of acetylation on thermodynamic properties of seaweed alginate in sodium chloride solutions. Prog Colloid Polym Sci 127:26–30Google Scholar
  32. 32.
    Trujillo-Roldán M, Peña C, Ramírez O, Galindo E (2001) Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii. Biotechnol Prog 17:1042–1048CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  • Lucero Jiménez
    • 1
  • Tania Castillo
    • 2
  • Celia Flores
    • 1
  • Daniel Segura
    • 3
  • Enrique Galindo
    • 1
  • Carlos Peña
    • 1
  1. 1.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Centro de Investigaciones en BiotecnologíaUniversidad Autónoma del Estado de MorelosCuernavacaMexico
  3. 3.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

Personalised recommendations