pH and base counterion affect succinate production in dual-phase Escherichia coli fermentations

Original Paper

Abstract

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO2. A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using 13C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)2 was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)2, the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g.

Keywords

Succinic acid CO2 Calcium 

Notes

Acknowledgments

Financial support from the US Department of Energy (DE-FG26-04NT42126) is gratefully acknowledged. We acknowledge Drs. G.P. Wylie, T.M. Andacht and D.R. Phillips for NMR and MS analyses. We also acknowledge S.A. Lee for technical assistance and Y. Zhu for helpful discussion.

References

  1. 1.
    Agarwal L, Isar J, Meghwanshi GK, Saxena RK (2006) A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli. J Appl Microbiol 100:1348–1354. doi: 10.1111/j.1365-2672.2006.02894.x PubMedCrossRefGoogle Scholar
  2. 2.
    Booth IR (1985) Regulation of cytoplasmic pH in bacteria. Microbiol Rev 49:359–378PubMedGoogle Scholar
  3. 3.
    Chatterjee R, Millard CS, Champion K, Clark DP, Donnelly MI (2001) Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl Environ Microbiol 67:148–154. doi: 10.1128/AEM.67.1.148-154.2001 PubMedCrossRefGoogle Scholar
  4. 4.
    Corwin LM, Fanning GR (1968) Studies of parameters affecting the allosteric nature of phosphoenolpyruvate carboxylase of Escherichia coli. J Biol Chem 243:3517–3525PubMedGoogle Scholar
  5. 5.
    Donnelly MI, Millard CS, Clark DP, Chen MJ, Rathke JW (1998) A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol. Appl Biochem Biotechnol 70–72:187–198. doi: 10.1007/BF02920135 CrossRefGoogle Scholar
  6. 6.
    Eiteman MA, Chastain MJ (1997) Optimization of the ion-exchange analysis of organic acids from fermentation. Anal Chim Acta 338:69–75. doi: 10.1016/S0003-2670(96)00426-6 CrossRefGoogle Scholar
  7. 7.
    Frahm B, Blank HC, Cornand P, Oelssner W, Guth U, Lane P, Munack A, Johannsen K, Portner R (2002) Determination of dissolved CO2 concentration and CO2 production rate of mammalian cell suspension culture based on off-gas measurement. J Biotechnol 99:133–148. doi: 10.1016/S0168-1656(02)00180-3 PubMedCrossRefGoogle Scholar
  8. 8.
    Gokarn RR, Eiteman MA, Altman E (2000) Metabolic analysis of Escherichia coli in the presence and absence of carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase. Appl Environ Microbiol 66:1844–1850. doi: 10.1128/AEM.66.5.1844-1850.2000 PubMedCrossRefGoogle Scholar
  9. 9.
    Gouesbet G, Abaibou H, Wu LF, Mandrand-Berthelot MA, Blanco C (1993) Osmotic repression of anaerobic metabolic systems in Escherichia coli. J Bacteriol 175:214–221PubMedGoogle Scholar
  10. 10.
    Guettler MV, Jain MK, Soni BK (1998) Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms. US patent 5,723,322, 3 March 1998Google Scholar
  11. 11.
    Guettler MV, Rumler D, Jain MK (1999) Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. Int J Syst Bacteriol 49:207–216PubMedCrossRefGoogle Scholar
  12. 12.
    Isar J, Agarwal L, Saran S, Saxena RK (2006) Succinic acid production from Bacteroides fragilis: process optimization and scale up in a bioreactor. Anaerobe 12:231–237. doi: 10.1016/j.anaerobe.2006.07.001 PubMedCrossRefGoogle Scholar
  13. 13.
    Lee PC, Lee WG, Kwon S, Lee SY, Chang HN (2000) Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey. Appl Microbiol Biotechnol 54:23–27. doi: 10.1007/s002530000331 PubMedCrossRefGoogle Scholar
  14. 14.
    Lee PC, Lee WG, Lee SY, Chang HN (2001) Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. Biotechnol Bioeng 72:41–48. doi: 10.1002/1097-0290(20010105)72:1<41::AID-BIT6>3.0.CO;2-N PubMedCrossRefGoogle Scholar
  15. 15.
    Lee SJ, Song H, Lee SY (2006) Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production. Appl Environ Microbiol 72:1939–1948. doi: 10.1128/AEM.72.3.1939-1948.2006 PubMedCrossRefGoogle Scholar
  16. 16.
    Lin H, Bennett GN, San KY (2005) Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate. Biotechnol Bioeng 89:148–156. doi: 10.1002/bit.20298 PubMedCrossRefGoogle Scholar
  17. 17.
    Lin H, Bennett GN, San KY (2005) Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab Eng 7:116–127. doi: 10.1016/j.ymben.2004.10.003 PubMedCrossRefGoogle Scholar
  18. 18.
    MacKintosh C, Nimmo HG (1988) Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308. Biochem J 250:25–31PubMedGoogle Scholar
  19. 19.
    Miczynski ZN (1886) Uber die Bestimmung der Loslichkeit einiger Sauren und Salze der Oxalsaurereihe in Wasser bei verschiedenen Temperaturen. Monatsh Chem 7:255–272. doi: 10.1007/BF01516575 CrossRefGoogle Scholar
  20. 20.
    O’Leary MH (1982) Phosphoenolpyruvate carboxylase: an enzymologist’s view. Annu Rev Plant Physiol 33:297–315. doi: 10.1146/annurev.pp.33.060182.001501 CrossRefGoogle Scholar
  21. 21.
    Olsen K, Budde B, Siegumfeldt H, Rechinger K, Jakobsen M, Ingmer H (2002) Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy. Appl Environ Microbiol 68:4145–4147. doi: 10.1128/AEM.68.8.4145-4147.2002 PubMedCrossRefGoogle Scholar
  22. 22.
    Rosman KJR, Taylor PDP (1998) Isotopic compositions of the elements. Pure Appl Chem 70:217–230. doi: 10.1351/pac199870010217 CrossRefGoogle Scholar
  23. 23.
    Sakuma T, Yamada N, Saito H, Kakegawa T, Kobayashi H (1998) pH dependence of the sodium ion extrusion systems in Escherichia coli. Biochim Biophys Acta 1363:231–237. doi: 10.1016/S0005-2728(97)00102-3 PubMedCrossRefGoogle Scholar
  24. 24.
    Samuelov N, Lamed R, Lowe S, Zeikus JG (1991) Influence of CO2–HCO3- levels and pH on growth, succinate production, and enzyme activities of Anaerobiospirillum succiniciproducens. Appl Environ Microbiol 57:3013–3019PubMedGoogle Scholar
  25. 25.
    Sanchez AM, Bennett GN, San KY (2005) Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant. Biotechnol Prog 21:358–365. doi: 10.1021/bp049676e PubMedCrossRefGoogle Scholar
  26. 26.
    Sanchez AM, Bennett GN, San KY (2006) Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains. Metab Eng 8:209–226. doi: 10.1016/j.ymben.2005.11.004 PubMedCrossRefGoogle Scholar
  27. 27.
    Savinell JM, Palsson BO (1992) Network analysis of intermediary metabolism using linear optimization. I. Development of mathematical formalism. J Theor Biol 154:421–454. doi: 10.1016/S0022-5193(05)80161-4 PubMedCrossRefGoogle Scholar
  28. 28.
    Schmidt K, Carlsen M, Nielsen J, Villadsen J (1997) Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices. Biotechnol Bioeng 55:831–840. doi: 10.1002/(SICI)1097-0290(19970920)55:6<831::AID-BIT2>3.0.CO;2-H PubMedCrossRefGoogle Scholar
  29. 29.
    Stumm W, Morgan J (1996) Dissolved carbon dioxide. In: Aquatic chemistry, John Wiley, New York, p 192Google Scholar
  30. 30.
    Trchounian A, Kobayashi H (1999) Fermenting Escherichia coli is able to grow in media of high osmolarity, but is sensitive to the presence of sodium ion. Curr Microbiol 39:109–114. doi: 10.1007/s002849900429 PubMedCrossRefGoogle Scholar
  31. 31.
    Urbance S, Pometto AIII, Dispirito A, Denli Y (2004) Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors. Appl Microbiol Biotechnol 65:664–670. doi: 10.1007/s00253-004-1634-2 PubMedCrossRefGoogle Scholar
  32. 32.
    Van der Werf MJ, Guettler MV, Jain MK, Zeikus JG (1997) Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130z. Arch Microbiol 167:332–342. doi: 10.1007/s002030050452 PubMedCrossRefGoogle Scholar
  33. 33.
    Van Dien SJ, Strovas T, Lidstroml ME (2003) Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry. Biotechnol Bioeng 84:45–55. doi: 10.1002/bit.10745 PubMedCrossRefGoogle Scholar
  34. 34.
    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 Biotechnol 28:325–332. doi: 10.1038/sj.jim.7000250 PubMedCrossRefGoogle Scholar
  35. 35.
    Vemuri GN, Eiteman MA, Altman E (2002) Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 68:1715–1727. doi: 10.1128/AEM.68.4.1715-1727.2002 PubMedCrossRefGoogle Scholar
  36. 36.
    Wang Q, Chen X, Yang Y, Zhao X (2006) Genome-scale in silico aided metabolic analysis and flux comparisons of Escherichia coli to improve succinate production. Appl Microbiol Biotechnol 73:887–894. doi: 10.1007/s00253-006-0535-y PubMedCrossRefGoogle Scholar
  37. 37.
    Zeikus JG, Jain MK, Elankovan P (1999) Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol 51:545–552. doi: 10.1007/s002530051431 CrossRefGoogle Scholar
  38. 38.
    Zhu Y, Eiteman MA, DeWitt K, Altman E (2007) Homolactate fermentation by metabolically engineered Escherichia coli strains. Appl Environ Microbiol 73:456–464. doi: 10.1128/AEM.02022-06 PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  1. 1.Center for Molecular BioEngineeringUniversity of GeorgiaAthensUSA

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