As to perform ABE fermentation means to handle clostridial population in different stages of the life cycle (see Fig. 1), determination of share of metabolically active i. e. vital cells in the population, is very important. Based on testing of various fluorescent viability probes with different principles of functioning, bisoxonol (BOX) was chosen as a convenient dye for

C. pasteurianum viability determination (Linhova et al., 2010b). BOX stains depolarized cells with destroyed membrane potential i. e. nonviable cells. When the cells were fixed by 5 min boiling, whole population was labelled (Fig.3b) but in case of growing population (Fig.3a) most of cells remained non-stained. After optimization of staining conditions, flow cytometry was used for determination of culture viability (see Fig.4).

Population of viable cells in the left dot-plot diagram can be seen under the gate (in lower half of the diagram). In upper half of the left diagram, there are rests of cells after spores germination and sporulating cells, the share of which does not exceed 15%.

Then the method was used for viability determination during batch cultivation (see Fig.5). Bioreactor was inoculated with spore suspension after heat shock that induced spores to grow and killed present vegetative cells. After the heat shock, the viability at the beginning of the fermentation was very low (Fig. 5B). In the exponential growth phase viability increased to ~78%, as expected. With glucose depletion (Fig. 5B) and reaching the highest concentration of 1-butanol (7.5 g. L-1 see Fig.5A), the viability began to decrease. Relatively rapid viability decline at nutrient depletion conditions has already been observed by Novo et al., (1999) and Jepras et al., (1995) for S. aureus, E. coli and P. aeruginosa. They observed membrane potential decreased within a few minutes after removal of energy resources. Moreover, in our case, elevated 1-butanol concentration contributed to viability decline, too