Skip to main content
Log in

Development of a Cell-recycled Continuous Fermentation Process for Enhanced Production of Succinic Acid by High-yielding Mutants of Actinobacillus succinogenes

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Succinic acid (SA), a four-carbon dicarboxylic acid utilized as a platform chemical for valuable industrial products, is a major fermentation product of Actinobacillus succinogenes cells, synthetized during anaerobic metabolism. In this study, cell-recycled continuous fermentation (CRCF) was carried out in order to maximize the appreciably stable biocatalytic activity of SA-producing cells and the growth-associated mode of SA biosynthesis. Stable and long operations of CRCF could be carried out through an efficient decanter system developed in our laboratory, which could effectively separate highly dense microbial cells from the outlet stream. This allowed to overcome the wash-out phenomenon encountered at relatively low dilution rates in conventional continuous fermentation systems without cell recycling. Through careful assessment of the effects of dilution rate, composition of feeding medium, and cell recycling ratio on SA yield via the CRCF process, volumetric SA productivity could be enhanced to 3.86 g/(L·h), an approximately 5.1-fold increase compared to parallel continuous fermentation without cell recycling. A higher dilution rate and a 24% increment in SA production via increased density of active cells inside the bioreactor of the CRCF system were the probable factors inducing such a considerable enhancement in volumetric SA productivity during CRCF. Since volumetric productivity is the most important parameter determining the cost-effectiveness of a given fermentation bioprocess, it is quite evident that CRCF is a promising alternative to conventional batch or continuous fermentation without cell recycling for mass production of SA.

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

Access this article

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Delhomme, C., D. Weuster-Botz, and F. E. Kühn (2009) Succinic acid from renewable resources as a C4 building-block chemical-a review of the catalytic possibilities In aqueous media. Green Chem. 11: 13–26.

    Article  CAS  Google Scholar 

  2. Liebal, U. W., L. M. Blank, and B. E. Ebert (2018) CO2 to succinic acid — Estimating the potential of biocatalytic routes. Metab. Eng. Commun. 7: e00075.

    Article  Google Scholar 

  3. Zeikus, J. G., M. K. Jain, and P. Elankovan (1999) Biotechnology of succinic acid production and markets for derived industrial products. Appl. Microbiol. Biotechnol. 51: 545–552.

    Article  CAS  Google Scholar 

  4. Chinthapalli, R., K. Iffland, F. Aeschelmann, A. Raschka, and M. Carus (2018) Succinic acid: new bio-based building block with a huge market and environmental potential. Nova-Institute GmbH. http://news.bio-based.eu/succinic-acid-new-bio-based-building-block-with-a-huge-market-and-environmental-potential/.

  5. Markets and Markets (2019) Succinic acid market by type (bio-based succinic acid, petro-based succinic acid), end-use industry (industrial, food & beverage, coatings, pharmaceutical), and region (APAC, Europe, North America, South America, Middle East & Africa) — Forecast to 2023. https://www.marketsandmarkets.com/Market-Reports/succinic-acid-market-402.html.

  6. Cheng, K. K., G. Y. Wang, J. Zeng, and J. A. Zhang (2013) Improved succinate production by metabolic engineering. Biomed. Res. Int. 2013: 538790.

    PubMed  PubMed Central  Google Scholar 

  7. Song, H. and S. Y. Lee (2006) Production of succinic acid by bacterial fermentation. Enzyme Microb. Technol. 39: 352–361.

    Article  CAS  Google Scholar 

  8. Arifin, Y., C. Archer, S. A. Lim, L. E. Quek, H. Sugiarto, E. Marcellin, C. E. Vickers, J. O. Krömer, and L. K. Nielsen (2014) Escherichia coli W shows fast, highly oxidative sucrose metabolism and low acetate formation. Appl. Microbiol. Biotechnol. 98: 9033–9044.

    Article  CAS  Google Scholar 

  9. Lange, A., J. Becker, D. Schulze, E. Cahoreau, J. C. Portais, S. Haefner, H. Schröder, J. Krawczyk, O. Zelder, and C. Wittmann (2017) Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens. Metab. Eng. 44: 198–212.

    Article  CAS  Google Scholar 

  10. Lee, P. C., W. G. Lee, S. Y. Lee, and H. N. Chang (1999) Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochem. 35: 49–55.

    Article  CAS  Google Scholar 

  11. McKinlay, J. B., M. Laivenieks, B. D. Schindler, A. A. McKinlay, S. Siddaramappa, J. F. Challacombe, J. R. Lowry, A. Clum, A. L. Lapidus, K. B. Burkhart, V. Harkins, and C. Vieille (2010) A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production. BMC Genomics. 11: 680.

    Article  CAS  Google Scholar 

  12. Pereira, B., J. Miguel, P. Vilaça, S. Soares, I. Rocha, and S. Carneiro (2018) Reconstruction of a genome-scale metabolic model for Actinobacillus succinogenes 130Z. BMC Syst. Biol. 12: 61.

    Article  Google Scholar 

  13. McKinlay, J. B., J. G. Zeikus, and C. Vieille (2005) Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium. Appl. Environ. Microbiol. 71: 6651–6656.

    Article  CAS  Google Scholar 

  14. McKinlay, J. B., Y. Shachar-Hill, J. G. Zeikus, and C. Vieille (2007) Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers. Metab. Eng. 9: 177–192.

    Article  CAS  Google Scholar 

  15. Van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus (1997) Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167: 332–342.

    Article  CAS  Google Scholar 

  16. Wang, J., D. Qin, B. Zhang, Q. Li, S. Li, X. Zhou, L. Dong, and D. Wang (2015) Fine-tuning of ecaA and pepc gene expression increases succinic acid production in Escherichia coli. Appl. Microbiol. Biotechnol. 99: 8575–8586.

    Article  CAS  Google Scholar 

  17. Yu, J. H., L. W. Zhu, S. T. Xia, H. M. Li, Y. L. Tang, X. H. Liang, T. Chen, and Y. J. Tang (2016) Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering. Biotechnol. Bioeng. 113: 1531–1541.

    Article  CAS  Google Scholar 

  18. Zhu, L. W., L. Zhang, L. N. Wei, H. M. Li, Z. P. Yuan, T. Chen, Y. L. Tang, X. H. Liang, and Y. J. Tang (2015) Collaborative regulation of CO2 transport and fixation during succinate production in Escherichia coli. Sci. Rep. 5: 17321.

    Article  CAS  Google Scholar 

  19. Kim, P., M. Laivenieks, C. Vieille, and J. G. Zeikus (2004) Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Appl. Environ. Microbiol. 70: 1238–1241.

    Article  CAS  Google Scholar 

  20. Park, S. M., K. Eum, S. Kim, Y. S. Jeong, D. Lee, and G. T. Chun (2014) Enhanced production of succinic acid by Actinobacillus succinogenes using the production medium supplemented with recombinant carbonic anhydrases. KSBB J. 29: 155–164.

    Article  Google Scholar 

  21. Ferone, M., F. Raganati, G. Olivieri, P. Salatino, and A. Marzocchella (2017) Biosuccinic acid from lignocellulosic-based hexoses and pentoses by Actinobacillus succinogenes: Characterization of the conversion process. Appl. Biochem. Biotechnol. 183:1465–1477.

    Article  CAS  Google Scholar 

  22. Liu, Y. P., P. Zheng, Z. H. Sun, Y. Ni, J. J. Dong, and L. L. Zhu (2008) Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Bioresour. Technol. 99: 1736–1742.

    Article  CAS  Google Scholar 

  23. Bradfield M. F. A., A. Mohagheghi, D. Salvachua, H. Smith, B. A. Black, N. Dowe, G. T. Beckham, and W. Nicol (2015) Continuous succinic acid production by Actinobacillus succinogenes on xylose-enriched hydrolysate. Biotechnol. Biofuels. 8: 181.

    Article  Google Scholar 

  24. Borges, E. R. and N. Pereira (2011) Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by Actinobacillus succinogenes. J. Ind. Microbiol. Biotechnol. 38: 1001–1011.

    Article  CAS  Google Scholar 

  25. Park, S. M., Y. S. Jeong, D. Lee, S. Kim, J. Lee, and G. T. Chun (2009) Intensive strain improvement using high throughput system (HTS) and statistical medium optimization for enhanced production of succinic acid by Actinobacillus succinogenes. J. Biosci. Bioeng. 108: S132.

    Article  Google Scholar 

  26. Park, S. M. and G. T. Chun (2014) Statistical optimization of production medium for enhanced production of succinic acid produced by anaerobic fermentations of Actinobacillus succinogenes. KSBB J. 29: 165–178.

    Article  Google Scholar 

  27. Shuler, M. L. and F. Kargi (2002) Bioprocess Engineering: Basic Concepts. 2nd ed., pp. 245–262. Prentice Hall, Upper Saddle River, NJ, USA.

    Google Scholar 

  28. Doran, P. M. (2013) Bioprocess Engineering Principles. 2nd ed., pp. 798–823. Elsevier, UK.

    Google Scholar 

  29. Lee, J. W., J. Yi, T. Y. Kim, S. Choi, J. H. Ahn, H. Song, M. H. Lee, and S. Y. Lee (2016) Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens. Metab. Eng. 38: 409–417.

    Article  CAS  Google Scholar 

  30. Blank, L. M., R. L. McLaughlin, and L. K. Nielsen (2005) Stable production of hyaluronic acid in Streptococcus zooepidemicus chemostats operated at high dilution rate. Biotechnol. Bioeng. 90: 685–693.

    Article  CAS  Google Scholar 

  31. Meynial-Salles, I., S. Dorotyn, and P. Soucaille (2008) A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity. Biotechnol. Bioeng. 99: 129–135.

    Article  CAS  Google Scholar 

  32. Kim, M. I., N. J. Kim, L. Shang, Y. K. Chang, S. Y. Lee, and H. N. Chang (2009) Continuous production of succinic acid using an external membrane cell recycle system. J. Microbiol. Biotechnol. 19: 1369–1373.

    Article  CAS  Google Scholar 

  33. Lee, P. C., S. Y. Lee, and H. N. Chang (2008) Cell recycled culture of succinic acid-producing Anaerobiospirillum succiniciproducens using an internal membrane filtration system. J. Microbiol. Biotechnol. 18: 1252–1256.

    CAS  PubMed  Google Scholar 

  34. van Heerden, C. D. and W. Nicol (2013) Continuous and batch cultures of Escherichia coli KJ134 for succinic acid fermentation: metabolic flux distributions and production characteristics. Microb. Cell Fact. 12: 80.

    Article  Google Scholar 

  35. Qi, H. N., C. T. Goudar, J. D. Michaels, H. J. Henzler, G. N. Jovanovic, and K. B. Konstantinov (2003) Experimental and theoretical analysis of tubular membrane aeration for mammalian cell bioreactors. Biotechnol. Prog. 19: 1183–1189.

    Article  CAS  Google Scholar 

  36. Ryll, T., G. Dutina, A. Reyes, J. Gunson, L. Krummen, and T. Etcheverry (2000) Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: Characterization of separation efficiency and impact of perfusion on product quality. Biotechnol. Bioeng. 69: 440–449.

    Article  CAS  Google Scholar 

  37. Freeman, C. A., P. S. D. Samuel, and D. S. Kompala (2017) Compact cell settlers for perfusion cultures of microbial (and mammalian) cells. Biotechnol. Prog. 33: 913–922.

    Article  CAS  Google Scholar 

  38. Pateraki, C., M. Patsalou, A. Vlysidis, N. Kopsahelis, C. Webb, A. A. Koutinas, and M. Koutinas (2016) Actinobacillus succinogenes: advances on succinic acid production and prospects for development of integrated biorefineries. Biochem. Eng. J. 112: 285–303.

    Article  CAS  Google Scholar 

  39. Pohlscheidt, M., M. Jacobs, S. Wolf, J. Thiele, A. Jockwer, J. Gabelsberger, M. Jenzsch, H. Tebbe, and J. Burg (2013) Optimizing capacity utilization by large scale 3000 L perfusion in seed train bioreactors. Biotechnol. Prog. 29: 222–229.

    Article  CAS  Google Scholar 

  40. Stanbury, P. F., A. Whitaker, and S. J. Hall (1995) Principles of fermentation technology. 2nd ed., pp. 147–166. Elsevier, UK.

    Book  Google Scholar 

Download references

Acknowledgement

This work was carried out with the support of the 2016 Research Grant Program from the Kangwon National University.

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gie-Taek Chun.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, S.Y., Park, S.O., Yeon, J.Y. et al. Development of a Cell-recycled Continuous Fermentation Process for Enhanced Production of Succinic Acid by High-yielding Mutants of Actinobacillus succinogenes. Biotechnol Bioproc E 26, 125–136 (2021). https://doi.org/10.1007/s12257-020-0295-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-020-0295-z

Keywords

Navigation