Continuous production of n-butanol by Clostridium pasteurianum DSM 525 using suspended and surface-immobilized cells

•Glycerol fermentation by Clostridium pasteurianum DSM 525.•Continuous packed-bed fermentation for n-butanol production.•Comparison of continuous fermentation with suspended and immobilized cells.•Flow cytometric analysis of viability during fermentation.

For n-butanol production by Clostridium pasteurianum DSM 525, a modified reinforced Clostridium medium was used, where glucose was alternated with glycerol and two kinds of continuous fermentation were tested using suspended and surface immobilized cells on corn stover pieces. A steady state, with butanol productivity of 4.2 g/L h, was reached during the packed-bed continuous fermentation at a dilution rate of 0.44 h−1. The average n-butanol concentration, yield and the ratio of n-butanol/liquid by-products were 10.4 g/L, 33 % and 2.5, respectively. Unexpectedly, during continuous fermentation with suspended cells, at a dilution rate of 0.01 h−1, steady-state was not achieved and regular oscillations occurred in all measured variables, i.e. concentrations of glycerol, products and the number of cells stained with the fluorescent dyes carboxy fluorescein diacetate and propidium iodide. A possible explanation for oscillatory/steady-state behavior of suspended/surface-attached cells, respectively, may be specific butanol toxicity (toxicity per cell), which was higher/lower in respective cases, and which might be caused by lower/higher cell numbers respectively in both systems.