Skip to main content
SpringerLink
Log in

Using Flow Cytometry to Evaluate the Stress Physiological Response of the Yeast Saccharomyces carlsbergensis ATCC 6269 to the Presence of 5-Hydroxymethylfurfural During Ethanol Fermentations

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Lignocellulosic materials have been considered low-cost effective substrates for bioethanol production. However, lignocellulosic pretreatment releases toxic compounds such as 5-hydroxymethylfurfural (HMF) that is known to inhibit the yeast growth and ethanol production. In this work, flow cytometry was used to monitor the physiological response of the yeast Saccharomyces carlsbergensis ATCC 6269 in the presence of different initial HMF concentrations within the range of 0–15 g/L, in terms of cell membrane integrity, potential, and intracellular lipids. It was observed that the HMF presence affected more significantly the yeast growth than the ethanol production. At 15 g/L HMF, the yeast growth and fermentation ability were completely inhibited. The cell membrane integrity and potential decreased as the initial HMF concentration increased. At the end of the fermentation process with 10 g/L HMF, the yeast culture contained 45 % of cells with depolarized plasma membrane, 52 % of cells with permeabilized plasma membrane, and 53 % of cells with increasing reactive oxygen species (ROS) levels. Using the Nile Red stain, it was observed that intracellular polar lipids were more affected by the initial HMF concentration than the neutral lipids, probably due to the extensive membrane damage.

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

Similar content being viewed by others

Abbreviations

μ :

Specific growth rate

DHR:

Dihydrorhodamine 123

DiOC6(3):

3,3-Dihexylocarbocyanine iodide

FC:

Flow cytometry

HMF:

5-Hydroxymethylfurfural

NR:

Nile Red

PI:

Propidium iodide

ROS:

Reactive oxygen species

X max :

Maximum biomass concentration

Y X/glu :

Biomass yield

YEtOH/glu :

Ethanol yield

References

  1. Zheng-yun, W., Yu, D., Li, T., Yue-hing, L., Yi-jie, Z., & Wen-xue, Z. (2010). Investigating the effects of two lignocellulose degradation by-products (furfural and acetic acid) on ethanol fermentations by six ethanologenic yeast strains. African Journal of Biotechnology, 9, 8661–8666.

    Google Scholar 

  2. Almeida, J. R. M., Modig, T., Petersson, A., Hähn-Hägerdal, B., & Gorwa-Grauslund, M. F. (2007). Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. Journal of Chemical Technology and Biotecnology, 23, 40–349.

    Google Scholar 

  3. Taherzadeh, M. J., Gustafsson, L., Niklasson, C., & Lidé, G. (2000). Physiological effects of 5-hydrpxymethylfurfural on Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 53, 701–708.

    Article  CAS  Google Scholar 

  4. Oliva, J. M., Sáez, F., Ballesteros, I., González, A., Negro, M. J., Manzanares, P., & Ballesteros, M. (2003). Effect of lignocellulosic degradation compounds from steam explosion pretreatment on ethanol fermentation by thermotolerant yeast Kluyveromyces marxianus. Applied Biochemistry Biotechnology, 105-108, 141–153.

    Article  CAS  Google Scholar 

  5. Liu, Z. L., Slininger, P. J., Dien, B. S., Berhow, M. A., Kurtzman, C. P., & Gorsich, S. W. (2004). Adaptative response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. Journal of Industrial Microbiology and Biotechnology, 31, 345–352.

    Article  CAS  Google Scholar 

  6. Bellido, C., Bolado, S., Coca, M., Lucas, S., González-Benito, G., & García-Cubero, M. T. (2011). Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresource Technology, 102, 10868–10874.

    Article  CAS  Google Scholar 

  7. Freitas, C., Nunes, E., Passarinho, P. C., Reis, A., & Lopes da Silva, T. (2013). Use of multi-parameter flow cytometry as tool to monitor the impact of formic acid on Saccharomyces carlsbergensis batch ethanol fermentations. Applied Biochemistry and Biotechnology, 169, 2038–2048.

    Article  CAS  Google Scholar 

  8. Freitas, C., Nunes, E., Passarinho, P. C., Reis, A., & Lopes da Silva, T. (2012). Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry (2012). Applied Biochemistry and Biotechnology, 168, 1501–1015.

    Article  CAS  Google Scholar 

  9. Lopes da Silva, T., Feijão, D., & Reis, A. (2010). Using multi-parameter flow cytometry to monitor the yeast Rhodotorula glutinis CCMI 145 batch growth and oil production towards biodiesel. Applied Biochemistry and Biotechnology, 162, 2166–2176.

    Article  Google Scholar 

  10. Modig, T., Liden, G., & Taherzadeh, M. J. (2002). Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochemical Journal, 363, 769–776.

    Article  CAS  Google Scholar 

  11. Lee, H., Cho, D. H., Kim, Y. H., Shin, S. J., Kim, S. W., Han, S. O., Lee, J., Kim, S. W., & Park, C. (2011). Tolerance of Saccharomyces cerevisiae K35 to lignocellulose-derived inhibitory compounds. Biotechnology and Bioprocess Engineering, 16, 755–760.

    Article  CAS  Google Scholar 

  12. Zaldivar, J., Martinez, A., & Ingram, L. O. (1999). Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnology and Bioengineering, 65, 24–33.

    Article  CAS  Google Scholar 

  13. Allen, S. A., Clark, W., McCaffery, J. M., Cai, Z., Lanctot, A., Slininger, P. J., Liu, Z. L., & Gorsich, S. W. (2010). Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnology for Biofuels, 3, 2–10.

    Article  Google Scholar 

  14. Muller, S., & Losche, A. (2004). Population profiles of a commercial yeast strain in the course of brewing. Journal of Food Engineering, 63, 375–381.

    Article  Google Scholar 

  15. Chen, X., Li, Z., Zhang, X., Hu, F., Ryu, D. D. Y., & Bao, J. (2009). Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Applied Biochemistry and Biotechnology, 159, 591–604.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratório Nacional de Energia e Geologia, I.P, Unidade de Bioenergia, Estrada do Paço do Lumiar, 22, 1649-038, Lisbon, Portugal

    Teresa Lopes da Silva, Cátia Baptista, Alberto Reis & Paula C. Passarinho

Authors
  1. Teresa Lopes da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cátia Baptista
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alberto Reis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paula C. Passarinho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Teresa Lopes da Silva.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lopes da Silva, T., Baptista, C., Reis, A. et al. Using Flow Cytometry to Evaluate the Stress Physiological Response of the Yeast Saccharomyces carlsbergensis ATCC 6269 to the Presence of 5-Hydroxymethylfurfural During Ethanol Fermentations. Appl Biochem Biotechnol 181, 1096–1107 (2017). https://doi.org/10.1007/s12010-016-2271-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2271-9

Keywords

Search

Navigation