Applied Microbiology and Biotechnology

, Volume 69, Issue 5, pp 554–563 | Cite as

Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae

  • C. Du
  • H. Yan
  • Y. Zhang
  • Y. Li
  • Z. CaoEmail author
Applied Microbial and Cell Physiology


Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different ORP levels: 10, −140, −190 and −240 mV. From the results, the ORP level of −190 mV was preferable, which resulted in fast cell growth and high 1,3-propanediol concentration. The NAD+/NADH ratio was determined at different ORP levels, and a critical NAD+/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD+/NADH>4) and relatively reductive environment (NAD+/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio of NAD+/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant ORP fermentation at −190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production.


Fermentation NADH Metabolic Flux Anaerobic Fermentation Acetic Acid Production 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by China Basic Research and Development Project (973) No. 2003CB716007. We thank Prof. Jilun Li for supplying the microorganisms.


  1. Abbad-Andaloussi S, Guedon E, Spiesser E, Petitdemange H (1996) Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431. Lett Appl Microbiol 22:311–314Google Scholar
  2. Abbad-Andaloussi S, Amine J, Gerard P, Petitdemange H (1998) Effect of glucose on glycerol metabolism by Clostridium butyricum DSM 5431. J Appl Microbiol 84:515–522CrossRefPubMedGoogle Scholar
  3. Adams MWW, Stiefel EI (1998) Biochemistry–biological hydrogen production: not so elementary. Science 282:1842–1843CrossRefPubMedGoogle Scholar
  4. Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea JL, Goma G, Molina-Jouve C, Guillouet SE (2004) Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process. Appl Microbiol Biotechnol 63:537–542CrossRefPubMedGoogle Scholar
  5. Barbirato F, Astruc S, Soucaille P, Camarasa C, Salmon J-M, Bories A (1997) Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations. Microbiology 143:2423–2432PubMedCrossRefGoogle Scholar
  6. Barbirato F, Himmi HE, Conte T, Bories A (1998) 1,3-Propanediol production by fermentation: an interesting way to valorize glycerin from the ester and ethanol industries. Ind Crops Prod 7:281–289CrossRefGoogle Scholar
  7. Berovic M (1999) Scale-up of citric acid fermentation by redox potential control. Biotechnol Bioeng 64:552–557CrossRefPubMedGoogle Scholar
  8. Biebl H, Menzel H, Zeng AP, Deckwer WD (1999) Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 52:289–297CrossRefPubMedGoogle Scholar
  9. Bjornsson L, Murto M, Mattiasson B (2000) Evaluation of parameters for monitoring an anaerobic co-digestion process. Appl Microbiol Biotechnol 54:844–849CrossRefPubMedGoogle Scholar
  10. Boenigk R, Bowien S, Gottschalk G (1993) Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii. Appl Microbiol Biotechnol 38:453–457CrossRefGoogle Scholar
  11. Bourel G, Hinnini S, Divies C, Garmyn D (2003) The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas. J Appl Microbiol 94:280–288CrossRefPubMedGoogle Scholar
  12. Chen X, Xiu ZL, Wang JF, Zhang DJ, Xu P (2003a) Stoichiometric analysis and experimental investigation of glycerol bioconversion to 1,3-propanediol by Klebsiella pneumoniae under microaerobic conditions. Enzyme Microb Technol 33:386–394CrossRefGoogle Scholar
  13. Chen X, Zhang DJ, Qi WT, Gao SJ, Xiu ZL, Xu P (2003b) Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions. Appl Microbiol Biotechnol 63:143–146CrossRefPubMedGoogle Scholar
  14. Cheng KK, Liu DH, Sun Y, Liu WB (2004) 1,3-Propanediol production by Klebsiella pneumoniae under different aeration strategies. Biotechnol Lett 26:911–915CrossRefPubMedGoogle Scholar
  15. Costenoble R, Valadi H, Gustafsson L, Niklasson C, Franzen CJ (2000) Microaerobic glycerol formation in Saccharomyces cerevisiae. Yeast 16:1483–1495CrossRefPubMedGoogle Scholar
  16. Daniel R, Gottschalk G (1992) Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii DHA regulon. FEMS Microbiol Lett 100:281–286CrossRefGoogle Scholar
  17. de Graef MR, Alexeeva S, Snoep JL, de Mattos MJT (1999) The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J Bacteriol 181:2351–2357PubMedGoogle Scholar
  18. Elliott SJ, Leger C, Pershad HR, Hirst J, Heffron K, Ginet N, Blasco F, Rothery RA, Weiner JH, Armstrong FA (2002) Detection and interpretation of redox potential optima in the catalytic activity of enzymes. Biochim Biophys Acta 1555:54–59PubMedCrossRefGoogle Scholar
  19. Han SK, Shin HS (2004) Biohydrogen production by anaerobic fermentation of food waste. Int J Hydrogen Energy 29:569–577CrossRefGoogle Scholar
  20. Hartlep M, Hussmann W, Prayitno N, Meynial-Salles I, Zeng AP (2002) Study of two-stage processes for the microbial production of 1,3-propanediol from glucose. Appl Microbiol Biotechnol 60:60–66CrossRefPubMedGoogle Scholar
  21. Homann T, Tag C, Biebl H, Deckwer WD, Schink B (1990) Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl Microbiol Biotechnol 33:121–126CrossRefGoogle Scholar
  22. Jimenez AM, Borja R, Alonso V, Martin A (1997) Influence of aerobic pretreatment with Penicillium decumbens on the anaerobic digestion of beet molasses alcoholic fermentation wastewater in suspended and immobilized cell bioreactors. J Chem Technol Biotechnol 69:193–202CrossRefGoogle Scholar
  23. Kastner JR, Eiteman MA, Lee SA (2003) Effect of redox potential on stationary-phase xylitol fermentations using Candida tropicalis. Appl Microbiol Biotechnol 63:96–100CrossRefPubMedGoogle Scholar
  24. Li JL, Burris RH (1983) Influence of pN2 and pD2 on HD formation by various nitrogenases. Biochemistry 22:4472–4480CrossRefPubMedGoogle Scholar
  25. Mazumdar S, Springs SL, McLendon GL (2003) Effect of redox potential of the heme on the peroxidase activity of cytochrome b562. Biophys Chem 105:263–268CrossRefPubMedGoogle Scholar
  26. Menzel K, Zeng AP, Deckwer WD (1997a) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microb Technol 20:82–86CrossRefGoogle Scholar
  27. Menzel K, Zeng AP, Deckwer WD (1997b) Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol 56:135–142CrossRefPubMedGoogle Scholar
  28. Menzel K, Ahrens K, Zeng AP, Deckwer WD (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. enzymes and fluxes of pyruvate metabolism. Biotechnol Bioeng 60:617–626CrossRefPubMedGoogle Scholar
  29. Nemeth A, Kupcsulik B, Sevella B (2003) 1,3-propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026. World J Microbiol Biotechnol 19:659–663CrossRefGoogle Scholar
  30. Ouvry A, Wache Y, Tourdot-Marechal R, Divies C, Cachon R (2002) Effects of oxidoreduction potential combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum. FEMS Microbiol Lett 214:257–261CrossRefPubMedGoogle Scholar
  31. Papanikolaou S, Ruiz-Sanchez P, Pariset B, Blanchard F, Fick M (2000) High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. J Biotechnol 77:191–208CrossRefPubMedGoogle Scholar
  32. Papanikolaou S, Fick M, Aggelis G (2004) The effect of raw glycerol concentration on the production of 1,3-propanediol by Clostridium butyricum. J Chem Technol Biotechnol 79:1189–1196CrossRefGoogle Scholar
  33. Riondet C, Cachon R, Wache Y, Alcaraz G, Divies C (1999) Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential. Eur J Biochem 262:595–599CrossRefPubMedGoogle Scholar
  34. Riondet C, Cachon R, Wache Y, Alcaraz G, Divies C (2000) Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli. J Bacteriol 182:620–626CrossRefPubMedGoogle Scholar
  35. Sridhar J, Eiteman MA (1999) Influence of redox potential on product distribution in Clostridium thermosuccinogenes. Appl Biochem Biotechnol 82:91–101CrossRefGoogle Scholar
  36. Sridhar J, Eiteman MA (2001) Metabolic flux analysis of Clostridium thermosuccinogenes—effects of pH and culture redox potential. Appl Biochem Biotechnol 94:51–69CrossRefPubMedGoogle Scholar
  37. Tong I, Cameron D (1992) Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing genes from Klebsiella pneumoniae dha regulon genes. Appl Biochem Biotechnol 34–35:149–159Google Scholar
  38. Vemuri GN, Eiteman MA, Altman E (2002) Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions. J Ind Microbiol Biotech 28:325–332CrossRefGoogle Scholar
  39. Xiu ZL, Zeng AP, Deckwer WD (1998) Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition. Biotechnol Bioeng 57:251–261CrossRefPubMedGoogle Scholar
  40. Xiu ZL, Song BH, Wang ZT, Sun LH, Feng EM, Zeng AP (2004) Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in one- and two-stage anaerobic cultures. Biochem Eng J 19:189–197CrossRefGoogle Scholar
  41. Yang D, Li C, Du CY, Zhang YP, Cao ZA (2003) Production of 1,3-propanediol by Klebsiella pneumoniae in two stages two substrates fermentation. Chin J Process Eng 3:269–273Google Scholar
  42. Zeng AP, Biebl H, Deckwer WD (1993) Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme Microb Technol 15:770–779CrossRefGoogle Scholar
  43. Zheng XJ, Yu HQ (2004) Roles of pH in biologic production of hydrogen and volatile fatty acids from glucose by enriched anaerobic cultures. Appl Biochem Biotechnol 112:79–90CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Institute of Biochemical Engineering, Department of Chemical EngineeringTsinghua UniversityBeijingChina
  2. 2.Key Laboratory of Industrial BiotechnologySouthern Yangtze UniversityJiangsuChina

Personalised recommendations