Skip to main content
SpringerLink
Log in

Effect of aeration strategy on the metabolic flux of Klebsiella pneumoniae producing 1,3-propanediol in continuous cultures at different glycerol concentrations

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The microbial production of 1,3-propaneidol (1,3-PD) by Klebsiella pneumoniae in continuous fermentation was investigated under low, medium and high glycerol concentrations in the absence and presence of oxygen. The production of 1,3-PD increased with increasing glycerol concentrations, reaching a maximum (266 mmol l−1) under high glycerol concentration (760 mmol l−1) with air sparging at 0.04 vvm. The yield of 1,3-PD, however, decreased gradually with increasing glycerol concentrations, with the highest yield (0.52 mol mol−1) obtained for low glycerol concentration (270 mmol l−1) under anaerobic condition. Enzyme activity assays showed that the specific activity of glycerol dehydratase was highest (0.04 U mg−1) for culture sparged with 0.04 vvm air under high glycerol concentration. The specific activities of glycerol dehydrogenase and 1,3-propanediol oxidoreductase were also improved for all glycerol concentrations and in the presence of oxygen, implying that the dha operon was not repressed under microaerobic conditions. Analysis of metabolic fluxes showed that more carbon flux was shifted to the oxidative pathway with increasing glycerol concentrations, resulting in a reduced flux to 1,3-PD formation. However, the increases in carbon fluxes were not evenly distributed among the oxidative branches of the pathway. Furthermore, ethanol and acetic acid levels were slightly increased whereas 2,3-butanediol and lactic levels were greatly enhanced.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

C S0 :

Glycerol concentration in the feed medium mmol l−1

Cs :

Residual concentration of glycerol mmol l−1

C p :

Product concentration mmol l−1

D :

Dilution rate h−1

q p :

Formation rate of products mmol l−1

q s :

Substrate uptake rate mmol l−1

v BD :

Flux distribution of 2,3-butanediol mmol mmol−1

\( \nu_{{CO_{2} }} \) :

Flux distribution of CO2 mmol mmol−1

v EtOH :

Flux distribution of ethanol mmol mmol−1

v For :

Flux distribution of formic acid mmol mmol−1

v HAc :

Flux distribution of acetic acid mmol mmol−1

v Lac :

Flux distribution of lactic acid mmol mmol−1

v Suc :

Flux distribution of succinic acid mmol mmol−1

v Pyr :

Flux distribution of pyruvic acid mmol mmol−1

v Cit :

Flux distribution of citric acid mmol mmol−1

v Mal :

Flux distribution of malic acid mmol mmol−1

v Fum :

Flux distribution of fumaric acid mmol mmol−1

v PD :

Flux distribution of 1,3-propanediol mmol mmol−1

v s :

Consumption rate of substrate mmol mmol−1

R C :

Carbon recovery

X :

Biomass concentration g l−1

Y PD/s :

Molar yield of 1,3-propanediol mol mol−1

DCW:

Dry cell weight

1,3-PD:

1,3-Propanediol

2,3-BD:

2,3-Butanediol

3-HPA:

3-Hydroxypropanaldehyde

DHA:

Dihydroxyacetone

TCA:

Tricarboxylic acid

GDHt:

Glycerol dehydratase

GDH:

Glycerol dehydrogenase

PDOR:

1,3-Propanediol oxidoreductase

DHAK:

Dihydroxyacetone kinase

ORP:

Oxidoreduction potential

DO:

Dissolve oxygen

LG:

Low glycerol concentration

MG:

Medium glycerol concentration

HG:

High glycerol concentration

References

  1. Ahrens K, Menzel K, Zeng AP, Deckwer WD (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: III enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation. Biotechnol Bioeng 59:544–552

    Article  PubMed  CAS  Google Scholar 

  2. Barbirato F, Astruc S, Soucaille P, Camarasa C, Salmon JM, Bories A (1997) Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations. Microbiol 143:2423–2432

    Article  CAS  Google Scholar 

  3. Biebl H (1991) Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat. Appl Microbiol Biotechnol 35:701–705

    Article  CAS  Google Scholar 

  4. Biebl H, Menzel K, Zeng AP, Deckwer WD (1999) Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 52:289–297

    Article  PubMed  CAS  Google Scholar 

  5. Chen X, Zhang DJ, Qi WT, Gao SJ, Xiu ZL, Xu P (2003) Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions. Appl Microbiol Biotechnol 63:143–146

    Article  PubMed  CAS  Google Scholar 

  6. Chen X, Xiu ZL, Wang JF, Zheng DJ, Xu P (2003) Stoichiometric analysis and experimental investigation of glycerol bioconversion to 1,3-propanediol by Klebsiella pneumoniae under microaerobic conditions. Enzyme Microb Technol 33:386–394

    Article  CAS  Google Scholar 

  7. Chen Z, Liu HJ, Zhang JA, Liu DH (2009) Cell physiology and metabolic flux response of Klebsiella pneumoniae to aerobic conditions. Process Biochem 44:862–868

    Article  CAS  Google Scholar 

  8. Cheng KK, Liu DH, Sun Y, Liu WB (2004) 1,3-propnaeidol production by Klebsiella pneumoniae under different aeration strategies. Biotechnol Lett 26:911–915

    Article  PubMed  CAS  Google Scholar 

  9. Cheng KK, Liu HJ, Liu DH (2005) Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol Lett 27:19–22

    Article  PubMed  CAS  Google Scholar 

  10. Chevlier M, Lin ECC, Rodney L (1990) Hydrogen peroxide mediates the oxidative inactivation of enzymes following the switch from anaerobic to aerobic metabolism in Klebsiella pneumoniae. J Biol Chem 265:40–46

    Google Scholar 

  11. Cirde SJ, Stone L, Boruff CS (1945) Acrolein determination by means of tryptophane. Ind Eng Chem Anal Ed 17:259–262

    Article  Google Scholar 

  12. 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 Bacterial 181:2351–2357

    Google Scholar 

  13. Du CY, Yan H, Zhang YP, Li Y, Cao ZA (2006) Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae. Appl Microbiol Biotechnol 69:554–563

    Article  PubMed  CAS  Google Scholar 

  14. Forage RG, Lin ECC (1982) Dha system mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacterial 151:591–599

    CAS  Google Scholar 

  15. Gong ZH (2010) A multistage system of microbial fed-batch fermentation and its parameter identification. Math Comput Simulat 80:1903–1910

    Article  Google Scholar 

  16. Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. In: Noms JM, Ribbons DW (eds) Methods in microbiology, vol 5b. Academic, London, pp 209–344

    Google Scholar 

  17. Huang H, Gong CS, Tsao GT (2002) Production of 1,3-propanediol by Klebsiella pneumoniae. Appl Biochem Biotechnol 98:687–698

    Article  PubMed  Google Scholar 

  18. Lowery HO, Rosebrough J, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  19. Ma BB, Xu XL, Zhang GL, Wang LW, Wu M, Li C (2009) Microbial production of 1,3-propanediol by Klebsiella pneumoniae XJPD-Li under different aeration strategies. Appl Biochem Biotechnol 152:127–134

    Article  PubMed  CAS  Google Scholar 

  20. Melchiorsen CR, Jokumsen KV, Villadsen J, Johnsen MG, Israelsen H, Arnau J (2000) Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis. J Bacteriol 182:4783–4788

    Article  PubMed  CAS  Google Scholar 

  21. Menzel K, Zeng AP, Deckwer WD (1997) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microb Technol 20:82–86

    Article  CAS  Google Scholar 

  22. 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 60:617–626

    Article  PubMed  CAS  Google Scholar 

  23. 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 77:191–208

    Article  PubMed  CAS  Google Scholar 

  24. San KY, Bennett GN, Berríos-Rivera SJ, Vadali R, Sariyar B, Blackwood K (2002) Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metabolic Eng 4:182–192

    Article  CAS  Google Scholar 

  25. Sun YQ, Qi WT, Teng H, Xiu ZL, Zeng AP (2008) Mathematical modeling of glycerol fermentation by Klebsiella pneumoniae: concerning enzyme-catalytic reductive pathway and transport of glycerol and 1,3-propanediol across cell membrane. Biochem Eng J 38:22–32

    Article  CAS  Google Scholar 

  26. Wang JF, Xiu ZL, Fan SD (2001) Determination of glycerol concentration during the fermentation of glycerol to 1,3-propanediol. Ind Microbiol 31:33–35 (in Chinese)

    Google Scholar 

  27. Wang YH, Dong YS, Xiu ZL (2010) Rapid determination of organic acids in 1,3-propanediol fermentation broth with gradient elution-high performance liquid chromatography. Food Ferment Ind 265:126–130 (in Chinese)

    Google Scholar 

  28. Xiu ZL, Zeng AP (2008) Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butannediol. Appl Microbiol Biotechnol 78:917–926

    Article  PubMed  CAS  Google Scholar 

  29. Yang G, Tian JS, Li JL (2007) Fermentation of 1,3-Propanediol by a lactate deficient mutant of Klbsiella oxytoca under microaerobic conditions. Appl Microbiol Biotechnol 73:1017–1024

    Article  PubMed  CAS  Google Scholar 

  30. Zeng AP, Biebl H, Schlieker H, Deckwer WD (1993) Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme Microb Technol 15:770–779

    Article  CAS  Google Scholar 

  31. Zeng ZM, Xu YZ, Liu HJ, Guo NN, Cai ZZ, Liu DH (2008) Physiologic mechanisms of sequential products synthesis in 1,3-propaendiol fed-batch fermentation by Klebsiella pneumoniae. Biotechnol Bioeng 100:923–931

    Article  Google Scholar 

  32. Zhang QR, Teng H, Sun YQ, Xiu ZL, Zeng AP (2008) Metabolic flux and robustness analysis of glycerol metabolism in Klebsiella pneumoniae. Bioprocess Biosyst Eng 31:127–135

    Article  PubMed  CAS  Google Scholar 

  33. Zhang QR, Xiu ZL (2009) Metabolic pathway analysis of glycerol metabolism in Klebsiella pneumoniae incorporating oxygen regulatory system. Biotechnol Prog 25:103–115

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the grant from the Major State Basic Research Development Program of China (973 Program) (No.2007CB714306) and the 863 project (No. 2007AA02Z208) from the Ministry of Science and Technology of China. We thank Alan K Chang for his help with the revision of the manuscript.

Author information

Authors and Affiliations

  1. School of Life Science and Biotechnology, Dalian University of Technology, Linggong Road 2, Dalian, 116024, People’s Republic of China

    Yuanhao Wang, Hu Teng & Zhilong Xiu

Authors
  1. Yuanhao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hu Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhilong Xiu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhilong Xiu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Teng, H. & Xiu, Z. Effect of aeration strategy on the metabolic flux of Klebsiella pneumoniae producing 1,3-propanediol in continuous cultures at different glycerol concentrations. J Ind Microbiol Biotechnol 38, 705–715 (2011). https://doi.org/10.1007/s10295-010-0851-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0851-1

Keywords

Search

Navigation