Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Jaromir Fiala, Jan Novak, Marketa Sli ova, Martin Blaha, Hana Ei kova, Flow Cytometry  in Biotechnology, Web publication, 2003, Institute of Chemical Technology Prague, Department of Fermentation Chemistry and Bioengineering, Technicka 5, 166 28 Prague, Czech Republic

ABSTRACT

At present fermentation is monitored by indirect analyses (temperature, pH value, extract reduction, etc.), which provide only limited information on the progress of fermentation. We have applied flow cytometry in order to obtain information regarding the dynamics of microbial growth and metabolism. Flow cytometry as a rapid measuring method was used for process monitoring of the yeast Saccharomyces cerevisiae strain at the high- concentrated 16 and 20 °P wort and conventional 12 °P wort fermentation. The main results of the following parameters for observation of the fermentation process will be discussed: cell size distribution, cell granularity, viability analyses, for live and dead cell counts, cell cycle analyses and reserve material analyses (glycogen), to demonstrate the activity of the yeast. The flow cytometric data obtained show interesting changes in cell morphology and physiology as a function of process conditions, and can be used for optimalization of pitching yeast (preparation in optimum condition for fermentation) and prediction of biotechnological process. At present fermentation is monitored by indirect analyses (temperature, pH value, extract reduction, etc.), which provide only limited information on the progress of fermentation. We have applied flow cytometry in order to obtain information regarding the dynamics of microbial growth and metabolism. Flow cytometry as a rapid measuring method was used for process monitoring of the yeast Saccharomyces cerevisiae strain at the high- concentrated 16 and 20 °P wort and conventional 12 °P wort fermentation. The main results of the following parameters for observation of the fermentation process will be discussed: cell size distribution, cell granularity, viability analyses, for live and dead cell counts, cell cycle analyses and reserve material analyses (glycogen), to demonstrate the activity of the yeast. The flow cytometric data obtained show interesting changes in cell morphology and physiology as a function of process conditions, and can be used for optimalization of pitching yeast (preparation in optimum condition for fermentation) and prediction of biotechnological process.

FLOW CYTOMETRY

Flow cytometry is a rapid technique originally developed for analysing mammalian cells. The technique permits analysis of single cells in a suspension, for example for DNA content, immunogenic properties, protein content, intracellular pH and enzyme activities. Despite the many advantages of flow cytometry, implementation of the technique in microbiological laboratories is still limited

PRINCIP

Flow cytometric studies of bacteria and yeasts are based on staining with a fluorochrome. The stained cells in suspension are injected into the centre of a fast-moving carrier fluid called sheath fluid. Caused by hydrodynamic focusing, the cells are centred in the middle and arrive one by one at the measuring area of the flow chamber where a light source of a defined wavelength is focused on the flow cell. The stained cells are excited at a particular wavelength and emit light at a longer wavelength. The individual cells traverse the focus of the light beam very rapidly, minimizing the risk of two cells being calculated as one. The emitted light is separated from the excitating light by optical filters and mirrors permitting only light of a certain wavelength to reach the photomultiplier, which measures the emitted light dividing it into channels according to the fluorescence intensity. The analysis is often presented as a histogram showing the number of cells versus fluorescence intensity. A narrow histogram indicates that the population is homogenous with regard to the parameter analysed.

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CONCLUSIONS

Flow cytometry is a useful analytical technique for obtaining biological data during fermentation. The different biological data obtained show interesting changes in cell biology as a function of process conditions.

One major advantage is the speed of sample handling, which allows a fast increase in the number of isolates that can be screened. Furthermore, the large amounts of data on individual cells will prove useful in the detection of small changes in samples (cell size, cell granularity, sterol contents, glycogen contents etc.) not normally detected by conventional analyses.

Data of flow cytometry analysis and its relation to properties of yeast cells are now evaluated and results will be published later.

LITERATURE

1. Steward, G. G. (1999) High gravity brewing. Brewers' Guardian, 31.

2. Younis, O. S. and Steward, G. G. (1999) Effect of malt wort, very-high-gravity malt wort, and very-high-gravity adjunct wort on volatile production in Saccharomyces cerevisiae. American Society of Brewing Chemists, 39.

3. Steward, G. G. (2001) Fermentation of high gravity worts. Its influence on yeast metabolism and morphology. Journal of The Institute of Brewing, 73.

4. Hutter, K. J. (2001) Yeast management and process control by flow cytometric analysis. Journal of The Institute of Brewing, 72.

5. Muller, S., Losche, A. and Bley, T. (1992) Flow-cytometric investigation of sterol content and proliferation activity of yeast. Acta Biotechnol. 12,5, 365.

6. Porro, D., Smeraldi, C., Ranzi, B. M., Martegani, E. and Albertina, L. (1994) Analysis of respiratory activity in growing budding yeast by flow cytometry. ECB6: Proceedings of the 6th European Congress on Biotechnology, 577.

7. Bendová, O., Kahler, M.: Pivovarské kvasinky, SNTL Praha 1981.

8. Pátková, J., Šmogrovièová, D.: Kvasný Prùm., 47, 2001, s.7.

9. Dobrý, J.: Zmìny obsahovì významných látek piva a aktivity kvasinek pøi technologii HGB,Diplomová práce, VŠCHT, Praha, 2001

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