Applied Microbiology and Biotechnology

, Volume 76, Issue 4, pp 903–910 | Cite as

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii

  • A. Díaz-Barrera
  • C. Peña
  • E. Galindo
Applied Microbial and Cell Physiology


The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTRmax) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTRmax decreased. The molecular mass obtained at 3.0 mmol l−1 h−1 was 7.0 times higher (1,560 kDa) than at 9.0 mmol l−1 h−1 (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.


Oxygen transfer rate Alginate Molecular mass Oxygen limitation 



This work was financed in part by DGAPA-UNAM (grant IN 231305-2). A. Díaz-Barrera thanks DGEP-UNAM for his PhD scholarship and to the Pontificia Universidad Católica de Valparaíso (complementary scholarship). The technical assistance of David Castañeda in some of the experiments is gratefully acknowledged. The authors thank A. Linares for computer support.


  1. Anderlei T, Büchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7:157–162CrossRefGoogle Scholar
  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. Boiardi J (1994) Metabolic cost of nitrogen incorporation by N2-fixing Azotobacter vinelandii is affected by the culture pH. Biotechnol Lett 16:1195–1198CrossRefGoogle Scholar
  4. Brivonese A, Sutherland I-W (1989) Polymer production by a mucoid strain of Azotobacter vinelandii in batch culture. Appl Microbiol Biotechnol 30:97–102CrossRefGoogle Scholar
  5. Byun T, Zeng A-P, Deckwer W-D (1994) Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate. Bioprocess Eng 11:167–175Google Scholar
  6. Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S-O (2005) Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 27:399–406CrossRefGoogle Scholar
  7. Charoenrat T, Ketudat-Cairns M, Jahic M, Veide A, Enfors S-O (2006) Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process. Biochem Eng J 30:205–211CrossRefGoogle Scholar
  8. Chen J-Y, Wen CM, Chen T-L (1999) Effect of oxygen transfer on lipase production by Acinetobacter radioresistens. Biotechnol Bioeng 62:311–316CrossRefGoogle Scholar
  9. Galindo E, Peña C, Núñez C, Segura D, Espin G (2007) Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact 6 7:1–16Google Scholar
  10. Herbst H, Schumpe A, Deckwer W-D (1992) Xanthan production in stirred tank fermenters: oxygen transfer and scale-up. Chem Eng Technol 15:425–434CrossRefGoogle Scholar
  11. Jarman T, Pace G (1984) Energy requirements of microbial exopolysaccharide synthesis. Arch Microbiol 137:231–235CrossRefGoogle Scholar
  12. Kaplan A (1969) The determination of urea, ammonia and urease. Methods Biochem Anal 17:311–324CrossRefGoogle Scholar
  13. Karr D, Waters J, Emerich D (1983) Analysis of poly-β-hydroxybutyrate in Rhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection. Appl Environ Microbiol 46:1339–1344Google Scholar
  14. Khatri N, Hoffmann F (2006) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93:871–879CrossRefGoogle Scholar
  15. Kuhla J, Oelze J (1988) Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii. J Bacteriol 170:5325–5329Google Scholar
  16. Leitão J-H, Sá-Corriea I (1997) Oxygen-depend upregulation of transcription of alginates genes algA, algC and algD in Pseudomonas aeruginosa. Res Microbiol 148:37–43CrossRefGoogle Scholar
  17. Maier U, Büchs J (2001) Characterisation of the gas-liquid mass transfer in shaking bioreactors. Biochem Eng J 7:99–106CrossRefGoogle Scholar
  18. Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428CrossRefGoogle Scholar
  19. Oelze J (2000) Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence? FEMS Microbiol Rev 24:321–333CrossRefGoogle Scholar
  20. Page W, Tindale A, Chandra M, Kwon E (2001) Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-β-hydroxybutyrate. Microbiol 147:483–490Google Scholar
  21. Parente E, Crudele M, Aquino M, Clementi F (1998) Alginate production by Azotobacter vinelandii DSM576 in batch fermentation. J Ind Microb Biotechnol 20:171–176CrossRefGoogle Scholar
  22. 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
  23. Peña C, Trujillo-Roldán M, Galindo E (2000) Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii. Enzyme Microb Technol 27:390–398CrossRefGoogle Scholar
  24. Peña C, Peter C, Büchs J, Galindo E (2007) Evolution of the specific power consumption and oxygen transfer rate in alginate producing cultures of Azotobacter vinelandii conducted in shake flasks. Biochem Eng J DOI  10.1016/j.bej.2007.02.019
  25. Post E, Kleiner D, Oelze J (1983) Whole cell respiration and nitrogenase activities in Azotobacter vinelandii growing in oxygen controlled continuous culture. Arch Microbiol 134:68–72CrossRefGoogle Scholar
  26. Remminghorst U, Rehm B (2006a) Bacterial alginates: from biosynthesis to applications. Biotechnol Lett 28:1701–1712CrossRefGoogle Scholar
  27. Remminghorst U, Rehm B (2006b) In vitro alginate polymerization and the functional role of alg8 in alginate production by Pseudomonas aeruginosa. Appl Environ Microbiol 72:298–305CrossRefGoogle Scholar
  28. Reyes C, Peña C, Galindo E (2003) Reproducing shake flasks performance in stirred fermentors: production of alginates by Azotobacter vinelandii. J Biotechnol 105:189–198CrossRefGoogle Scholar
  29. Richard A, Margaritis A (2003) Rheology, oxygen transfer and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis. Biotechnol Bioeng 82:299–305CrossRefGoogle Scholar
  30. 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
  31. Sabra W, Zeng A-P, Lunsdorf H, Deckwer W-D (2000) Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 66:4037–4044CrossRefGoogle Scholar
  32. Sabra W, Zeng A-P, Deckwer W-D (2001) Bacterial alginate: physiology, product quality and process aspects. Appl Microbiol Biotechnol 56:315–325CrossRefGoogle Scholar
  33. Sahoo D, Agarwal G (2002) Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I2B. Biotechnol Bioeng 78:545–555CrossRefGoogle Scholar
  34. Trujillo-Roldán M, Peña C, Galindo E (2003) Components in the inoculum determine the kinetics of Azotobacter vinelandii cultures and the molecular weight of its alginate. Biotechnol Lett 25:1251–1254CrossRefGoogle Scholar
  35. Trujillo-Roldán M, Moreno S, Espín G, Galindo E (2004) The roles of oxygen and alginate-lyase in determining the molecular weight of alginate produced by Azotobacter vinelandii. Appl Microbiol Biotechnol 63:742–747CrossRefGoogle Scholar
  36. Varma A, Boesch B, Palsson B (1993) Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates. Appl Environ Microbiol 59:2465–2473Google Scholar
  37. Zeng A-P, Byun T, Posten C, Deckwer W-D (1994) Use of the respiratory quotient as a control parameter for optimum oxygen supply and scale-up of 2,3-butanediol production under microaerobic conditions. Biotechnol Bioeng 19:1107–1114CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

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