Applied Biochemistry and Biotechnology

, Volume 187, Issue 3, pp 782–799 | Cite as

Continuous Succinic Acid Fermentation by Actinobacillus Succinogenes: Assessment of Growth and Succinic Acid Production Kinetics

  • Mariateresa FeroneEmail author
  • Francesca Raganati
  • Giuseppe Olivieri
  • Piero Salatino
  • Antonio Marzocchella


Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. The paper reports the characterization of the growth kinetics of Actinobacillus succinogenes DSM 22257 using glucose as carbon source. Tests were carried out in a continuous bioreactor operated under controlled pH. Under steady-state conditions, the conversion process was characterized in terms of concentration of glucose, cells, acids, and pH. The effects of acid—succinic, acetic, and formic—concentration in the medium on fermentation performance were investigated. The fermentation was interpreted according to several models characterized by substrate and product inhibition. The selected kinetic model of biomass growth and of metabolite production described the microorganism growth rate under a broad interval of operating conditions. Under the investigated operating conditions, results pointed out that: no substrate inhibition was observed; acetic acid did not inhibit the cell growth and succinic acid production.


Biorefinery Succinic acid Actinobacillus succinogenes Kinetics Product inhibition 


Smax, Pmax

Critical concentrations of substrate or products (g/L)


Concentration of acetic, formic and succinic acid, glucose (g/L)


Specific growth rate (h-1)


Maximum specific growth rate (h-1)


Dilution rate (h-1)


Substrate saturation constant (g/L)


Substrate inhibition coefficient (g/L)


Products inhibition coefficient (g/L)


Exponent of inhibitory products


ATP yield (gDM/molATP)

\( {Y}_{\mathrm{ATP}}^{\mathrm{MAX}} \)

Maximum ATP yield (gDM/molATP)


Mass ratio between biomass and succinic acid (gDM/g)


Ratio between succinic acid and ATP moles (g/molATP)


Cell concentration (gDM/L)


Mean square deviation


Correlation coefficient


Funding Information

The study was supported by the Ministero dell’Istruzione, delll’Università e della Ricerca project “Development of green technologies for production of BIOchemicals and their use in preparation and industrial application of POLImeric materials from agricultural biomasses cultivated in a sustainable way in Campania Region – BIOPOLIS” PON03PE_00107_1/1.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Nghiem, N. P., Davison, B. H., Suttle, B. E., & Richardson, G. R. (1997). Production of succinic acid by Anaerobiospirillum succiniciproducens. Applied Biochemistry and Biotechnology, 63–65(1), 565–576. Scholar
  2. 2.
    Zeikus, J. G., Jain, M. K., & Elankovan, P. (1999). Biotechnology of succinic acid production and markets for derived industrial products. Applied Microbiology and Biotechnology, 51(5), 545–552. Scholar
  3. 3.
    Guettler, M. V., Rumler, D., & Jain, M. K. (1999). Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. International Journal of Systematic Bacteriology, 49, 207–216.CrossRefGoogle Scholar
  4. 4.
    Samuelov, N. S., Lamed, R., Lowe, S., & Zeikus, J. G. (1991). Influence of C02-HC03- levels and pH on growth, succinate production, and enzyme activities of Anaerobiospirillum succiniciproducens. Applied and Environmental Microbiology, 57(10), 3013–3019.Google Scholar
  5. 5.
    Lee, P., Lee, S., Hong, S., & Chang, H. (2002). Isolation and characterization of a new succinic acid-producing bacterium, Mannheimia succiniciproducens MBEL55E, from bovine rumen. Applied Microbiology and Biotechnology, 58(5), 663–668. Scholar
  6. 6.
    Salvachúa, D., Smith, H., St. John, P. C., Mohagheghi, A., Peterson, D. J., Black, B. A., Dowe, N., & Beckham, G. T. (2016). Succinic acid production from lignocellulosic hydrolysate by Basfia succiniciproducens. Bioresource Technology, 214, 558–566. Scholar
  7. 7.
    Isar, J., Agarwal, L., Saran, S., & Saxena, R. K. (2006). Succinic acid production from Bacteroides fragilis: process optimization and scale up in a bioreactor. Anaerobe, 12(5-6), 231–237. Scholar
  8. 8.
    Jantama, K., Zhang, X., Moore, J. C., Shanmugam, K. T., Svoronos, S. A., & Ingram, L. O. (2008). Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C. Biotechnology and Bioengineering, 101(5), 881–893. Scholar
  9. 9.
    Guettler, I. M. V. (1996). Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms. Michigan Biotechnology Institute, USA, US patent 5,504,004, (55618).Google Scholar
  10. 10.
    Ferone, M., Ercole, A., Olivieri, G., Salatino, P., & Marzocchella, A. (2018). Continuous succinic acid fermentation by Actinobacillus succinogenes in a packed bed reactor. Biotechnology for Biofuels, 11.
  11. 11.
    Carvalho, M., Matos, M., Roca, C., & Reis, M. A. M. (2014). Succinic acid production from glycerol by Actinobacillus succinogenes using dimethylsulfoxide as electron acceptor. New Biotechnology, 31(1), 133–139. Scholar
  12. 12.
    Sun, Z., Li, M., Qi, Q., Gao, C., & Lin, C. S. K. (2014). Mixed food waste as renewable feedstock in succinic acid fermentation. Applied Biochemistry and Biotechnology, 174(5), 1822–1833. Scholar
  13. 13.
    Du, C., Lin, S. K. C., Koutinas, A., Wang, R., & Webb, C. (2007). Succinic acid production from wheat using a biorefining strategy. Applied Microbiology and Biotechnology, 76(6), 1263–1270. Scholar
  14. 14.
    Zheng, P., Zhang, K., Yan, Q., Xu, Y., & Sun, Z. (2013). Enhanced succinic acid production by Actinobacillus succinogenes after genome shuffling. Journal of Industrial Microbiology and Biotechnology, 40(8), 831–840. Scholar
  15. 15.
    Liu, Y.-P., Zheng, P., Sun, Z.-H., Ni, Y., Dong, J.-J., & Wei, P. (2008). Strategies of pH control and glucose-fed batch fermentation for production of succinic acid by Actinobacillus succinogenes CGMCC1593. Journal of Chemical Technology and Biotechnology, 83(5), 722–729. Scholar
  16. 16.
    Bretz, K., & Kabasci, S. (2012). Feed-control development for succinic acid production with Anaerobiospirillum succiniciproducens. Biotechnology and Bioengineering, 109(5), 1187–1192. Scholar
  17. 17.
    Nandasana, A. D., & Kumar, S. (2008). Kinetic modeling of lactic acid production from molasses using Enterococcus faecalis RKY1. Biochemical Engineering Journal, 38(3), 277–284. Scholar
  18. 18.
    Qureshi, N., Annous, B. A., Ezeji, T. C., Karcher, P., & Maddox, I. S. (2005). Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates. Microbial Cell Factories, 24(4), 1–21. Scholar
  19. 19.
    Maharaj, K., Bradfield, M. F. A., & Nicol, W. (2014). Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility, stability and productivity. Applied Microbiology and Biotechnology, 98(17), 7379–7386. Scholar
  20. 20.
    Corona-González, R. I., Miramontes-Murillo, R., Arriola-Guevara, E., Guatemala-Morales, G., Toriz, G., & Pelayo-Ortiz, C. (2014). Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid. Bioresource Technology, 164, 113–118. Scholar
  21. 21.
    Chen, P., Cheng, Z., Hua, X., & Zheng, P. (2017). Construction of fibrous bed bioreactor for enhanced succinic acid production using wastewater of dextran fermentation. Bioprocess and Biosystems Engineering, 40, 1859–1866. Scholar
  22. 22.
    López-Garzón, C. S., van der Wielen, L. A. M., & Straathof, A. J. J. (2014). Green upgrading of succinate using dimethyl carbonate for a better integration with fermentative production. Chemical Engineering Journal, 235, 52–60. Scholar
  23. 23.
    Lee, P. C., Lee, S. Y., & Chang, H. N. (2010). Kinetic study on succinic acid and acetic acid formation during continuous cultures of Anaerobiospirillum succiniciproducens grown on glycerol. Bioprocess and Biosystems Engineering, 33(4), 465–471. Scholar
  24. 24.
    Lin, S. K. C., Du, C., Koutinas, A., Wang, R., & Webb, C. (2008). Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes. Biochemical Engineering Journal, 41(2), 128–135. Scholar
  25. 25.
    Vlysidis, A., Binns, M., Webb, C., & Theodoropoulos, C. (2011). Glycerol utilisation for the production of chemicals: Conversion to succinic acid, a combined experimental and computational study. Biochemical Engineering Journal, 58–59, 1–11. Scholar
  26. 26.
    Brink, H. G., & Nicol, W. (2014). Succinic acid production with Actinobacillus succinogenes: rate and yield analysis of chemostat and biofilm cultures. Microbial Cell Factories, 13(111), 111. Scholar
  27. 27.
    Russo, M. E., Maffettone, P. L., Marzocchella, A., & Salatino, P. (2008). Bifurcational and dynamical analysis of a continuous biofilm reactor. Journal of Biotechnology, 135(3), 295–303. Scholar
  28. 28.
    Ferone, M., Raganati, F., Olivieri, G., Salatino, P., & Marzocchella, A. (2017). Biosuccinic acid from lignocellulosic-based hexoses and pentoses by Actinobacillus succinogenes: characterization of the conversion process. Applied Biochemistry and Biotechnology, 183(4), 1465–1477. Scholar
  29. 29.
    Bauchop, T., & Elsden, R. S. (1960). The growth of microorganisms in relation to their metabolism and energy supply. Journal of General Microbiology, 23, 457–469.Google Scholar
  30. 30.
    Meyer, C. L., & Papoutsakis, E. T. (1989). Continuous and biomass recycle fermentations of Clostridium acetobutylicum. Part 1. Bioprocess Engineering, 4, 1–10.Google Scholar
  31. 31.
    S. J. Pirt, (1965) The Maintenance Energy of Bacteria in Growing Cultures. Proceedings of the Royal Society B: Biological Sciences 163(991), 224–231Google Scholar
  32. 32.
    S. J. Pirt, (1982) Maintenance energy: a general model for energy-limited and energy-sufficient growth. Archives of Microbiology 133(4), 300–302Google Scholar
  33. 33.
    Bradfield, M. F. A., & Nicol, W. (2016). The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes. Applied Microbiology and Biotechnology, 100(22), 9641–9652.
  34. 34.
    Michael W.W. Adams, (1990) The structure and mechanism of iron-hydrogenases. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1020(2), 115-145Google Scholar
  35. 35.
    Urbance, S. E., Pometto, A. L., DiSpirito, A. A., & Denli, Y. (2004). Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors. Applied Microbiology and Biotechnology, 65, 664–670.
  36. 36.
    van Heerden, C. D., & Nicol, W. (2013). Continuous succinic acid fermentation by Actinobacillus succinogenes. Biochemical Engineering Journal, 73, 5–11.
  37. 37.
    Bradfield, M. F. A., & Nicol, W. (2014). Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation. Biochemical Engineering Journal, 85, 1–7.
  38. 38.
    Palmqvist, E., & Hahn-Hägerdal, B. (2000). Fermentation of lignocellulosic hydrolysates. I: Inhibition and detoxification. Bioresource Technology, 74(1), 17–24.
  39. 39.
    Li, Q., Wang, D., Wu, Y., Yang, M., Li, W., Xing, J., & Su, Z. (2010). Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130ZT. Journal of Microbiology, 48, 290–296.
  40. 40.
    Pateraki, C., Almqvist, H., Ladakis, D., Lidén, G., Koutinas, A. A., & Vlysidis, A. (2016). Modelling succinic acid fermentation using a xylose based substrate. Biochemical Engineering Journal, 114, 26–41.

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Dipartimento di Ingegneria Chimicadei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico IINaplesItaly

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