Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences

Abstract

Iridium acetohutylicum in continuous culture: strain differences. Journal o/' Applied Bacteriology 69, 71 8-728.Several strains of Clostridium acetobutylicum, including strains ATCC 824 and DSM I73 I, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted. at a low but measurable frequency, to the parental phenotype, indicating that their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent-producing and non-solventogenic colonies of CI. acetobutylicum When Clostridiurn acetobutylicurn NCIB 8052 was grown in chemostat culture under conditions that were unsuitable for solvent production, asporogenous mutant strain@) incapable of producing acetone and butan-1-01 in normal batch culture were quickly selected (Gottschal & Morris 1981). Even when chemostat culture was performed under conditions that were propitious for solvent formation, it was impossible to establish a steady state, and ultimately, irrespective of the nature of the limiting nutrient, solvent production ceased (Stephens et al. 1985). Other strains of CI. acetobutylicurn, e.g. ATCC 824 and DSM 1731, sustained their solvent productivity during prolonged chemostat culture despite the emergence and eventual preponderance of asporogenous mutants (Meinecke et a/. 1984). In contrast, when strain

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

“…One type was small, rounded and opaque, whereas the other was larger, translucent and had an irregular shape. These two distinctive colonial morphologies are characteristic of normal and degenerate bacteria in C. beijerinckii [ 40 ] . The colonies of C. beijerinckii NCIMB 8052, however, retained a uniform morphology.…”

“…[ 40 ] . Culture samples were serially diluted using a holding buffer ( 25 mM Kphosphate, pH 7, 1 mM MgSO 4 ) .…”

“…AA332 and C. beijerinckii NCIMB 8052 were grown on solid CBM medium and harvested after 45 and 110 h. C. beijerinckii NCIMB 8052 was used as its degeneration behaviour has already been studied extensively [ 40]. Clostridium sp.…”

“…The process of degeneration occurs during repeated vegetative subculture [ 21,22 ] , on solid media [ 1,22,40 ] and most especially during continuous cultivation [ 2,3,5,11,30,40 ] . Technological development of the acetone -butanol fermentation is tending toward the establishment of continuous processes for economic reasons [ 12 ] .…”

“…There is good evidence from previous studies that the slow and progressive loss of solvent productivity is due to the formation of a degenerate subpopulation in the culture. Distinct variants can be isolated from continuous cultures, and discriminated on solid medium [ 40 ] . The degenerate population gradually takes over, as these bacteria keep growing, whereas the solvent -producing organisms in the wildtype population slow down or stop growth and initiate sporulation [ 21] .…”