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Rapid flow cytometric method for viability determination of solventogenic clostridia

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Abstract

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone–butanol–ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.

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Abbreviations

ABE:

Acetone–butanol–ethanol

BOX:

Bis-(1,3-dibutylbarbituric acid)trimethine oxonol

FDA:

Carboxyfluorescein diacetate

EtBr:

Ethidium bromide

FC:

Flow cytometry

cFDA:

Fluorescein diacetate

PI:

Propidium iodide

RH 123:

Rhodamine 123

VBNC:

Viable but nonculturable cells

References

  • Adam P, Sobek O, Scott CS (2007) Analysis of cerebrospinal fluid cell populations with monoclonal antibodies. Folia Microbiol 52:529–534

    Article  CAS  Google Scholar 

  • Branská B, Linhová M, Patáková P, Paulová P, Melzoch K (2011) Fluorescence analysis as a tool for determination of viability of microorganisms. Chem List 105:586–593 (in Czech)

    Google Scholar 

  • Cronin UP, Wilkinson MG (2010) The potential of flow cytometry in the study of Bacillus cereus. J Appl Microbiol 108:1–16

    Article  PubMed  CAS  Google Scholar 

  • Ferencko L, Cote MA, Rotman B (2004) Esterase activity as a novel parameter of spore germination in Bacillus anthracis. Biochem Biophys Res Commun 319:854–858

    Article  PubMed  CAS  Google Scholar 

  • Hewit ChJ, Nebe-von-Caron G (2004) The application of multi-parameter flow cytometry to monitor individual microbial cell physiological state. Adv Biochem Eng Biotechnol 89:197–223

    Google Scholar 

  • Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microb Infect 2:1523–1535

    Article  CAS  Google Scholar 

  • Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KK (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101:209–228

    Article  PubMed  CAS  Google Scholar 

  • Linhová M, Patáková P, Lipovský J, Fribert P, Paulová L, Rychtera M, Melzoch K (2010) Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum. Folia Microbiol 55:340–344

    Article  Google Scholar 

  • Nebe-von-Caron G, Stephens P, Barley RA (1998) Assessment of bacterial viability status by flow cytometry and single cell sorting. J Appl Microbiol 84:988–998

    Article  Google Scholar 

  • Ohye DF, Murrell WG (1973) Exosporium and spore coat formation in Bacillus cereus T. J Bacteriol 115:1179–1190

    PubMed  CAS  Google Scholar 

  • Papadimitriou K, Pratsinis H, Nebe-von-Caron G, Kletsas D, Tsakalidou E (2006) Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes. Int J Food Microbiol 111:197–205

    Article  PubMed  CAS  Google Scholar 

  • Patáková P, Lipovský J, Čížková H, Fořtová J, Rychtera M, Melzoch K (2009) Exploitation of food feedstock and waste for production of biobutanol. Czech J Food Sci 27:276–283

    Google Scholar 

  • Patáková P, Toure SSM, Šimáček P, Čížková H, Lipovský J, Linhová M, Fribert P, Rychtera M, Melzoch K (2011) Exploitation of sugar beet for biobutanol production. Sugar Sugar Beet J 127:46–49 (in Czech)

    Google Scholar 

  • Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley-Liss, New York

    Book  Google Scholar 

  • Tracy BP, Gaida SM, Papoutsakis ET (2008) Development and application of flow-cytometric techniques for analyzing and sorting endospore-forming clostridia. Appl Environ Microbiol 74:7497–7506

    Article  PubMed  CAS  Google Scholar 

  • Walker PD, Thomson RO, Baillie A (1967) Fine structure of clostridia with special reference to the location of antigens and enzymes. J Appl Microbiol 30:444–449

    Article  CAS  Google Scholar 

  • Want A, Hancocks H, Thomas CR, Stocks SM, Nebe-von-Caron G, Hewitt ChJ (2011) Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures. Biotechnol Lett 33:1395–1405

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

This research was supported by projects No. FR-TI1/218 of the Ministry of Industry and Trade of the Czech Republic; No. MSM6046137305 and by the specific university research project MSMT No. 21/2011 of the Ministry of Education, Youth and Sport of the Czech Republic; and No. TEO1020080 of Technology Agency of the Czech Republic (Copetence Centres).

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Authors and Affiliations

  1. Department of Biotechnology, Institute of Chemical Technology Prague, Technická 5, 166 28, Prague, Czech Republic

    Michaela Linhová, Barbora Branská, Petra Patáková, Jakub Lipovský, Petr Fribert, Mojmír Rychtera & Karel Melzoch

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  1. Michaela Linhová
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  2. Barbora Branská
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  3. Petra Patáková
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  4. Jakub Lipovský
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  5. Petr Fribert
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  6. Mojmír Rychtera
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  7. Karel Melzoch
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Correspondence to Michaela Linhová.

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Linhová, M., Branská, B., Patáková, P. et al. Rapid flow cytometric method for viability determination of solventogenic clostridia. Folia Microbiol 57, 307–311 (2012). https://doi.org/10.1007/s12223-012-0131-8

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  • DOI: https://doi.org/10.1007/s12223-012-0131-8

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