Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum

Folia Microbiol. 55 (4), 340344 (2010) http://www.biomed.cas.cz/mbu/folia/

M. LINHOV, P. PATKOV, J. LIPOVSK, P. FRIBERT, L. PAULOV, M. RYCHTERA, K. MELZOCH

Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology Prague, 166 28 Prague, Czechia fax [phone]e-mail [email protected]

Received 25 March 2010

Revised version 14 April 2010

ABSTRACT. Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, whichis a typical feature of clostridial acetonebutanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G+ bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G bacteria does not allow the uptake and therefore G+ bacteria are stained with higher fluorescence intensity than G bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G+ bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G bacteria is detected after initiation of solvent production.

Abbreviations

FC flow cytometry HI hexidium iodide

FI fluorescence intensity RH123 rhodamine 123 FSC forward scatter SSC side scatter

Clostridium pasteurianum belongs to a solventogenic section of the bacterial genus Clostridium (Drre 2005). Clostridia are rod-shaped, heterotrophic, heterofermentative, mostly spore-forming, Gram-positive/labile bacteria and typically strict anaerobes (Jones and Woods 1986).

A typical feature of the clostridial solvent production is a biphasic course of fermentation. At first, during an exponential growth phase, the bacteria produce as major products acetic and butyric acids, hydrogen, and carbon dioxide. After pH drop, the formation of acids decreases and the solventogenic phase follows, where acetone and 1-butanol become dominant metabolites. Simultaneously, an endospore formation is initiated (Drre 2005; Lee et al. 2008). Jones et al. (2008) hypothesized that the metabolic shift and other morphological changes were coupled with modifications in the cell-wall structure due to different environmental conditions (the presence of solvents and other metabolites). This is supported by the fact that butanol (as the major...