DOMBEK AND INGRAM
APPL. ENVIRON. MICROBIOL.
FIG. 3. Effects of ethanol exposure on the fermentative activities of 12- and 24-h cells. Fermentative activity is expressed as micromoles of evolved carbon dioxide per hour per milligram of cell protein. Cells were harvested after 12 h (A) or 24 h (B) and suspended in fresh medium containing variotis concentrations of ethanol, and their fermentative activity was measured after 10 min at 300C. A parallel set of samples was exposed to ethanol for 10 min, harvested by centrifugation, washed once, and suspended in fresh medium lacking ethanol. Bars denote a representative standard deviation for an average of three determinations. Symbols: 0, cells in the presence of added ethanol; 0, cells exposed to ethanol and suspended in fresh medium.
mentation, with 90% viability after 48 h as measured by the exclusion of methylene blue dye (12).
In a control experiment, we investigated the inhibition of ethanol production by added ethanol and its reversibility after washing (Fig. 3). Cells were harvested and suspended in fresh medium containing various concentrations of ethanol. Cells from the 12-h period (Fig. 3A) were more active and more sensitive to inhibition by added ethanol than cells from the 24-h period (Fig. 3B). Ethanol caused a progressive, dose-dependent inhibition of fermentation in both, with 12-h cells being more sensitive. Inhibition by ethanol was immediate and appeared complete within the first 10 min. No further decline in activity was observed during a subsequent 2 h of incubation (30°C) with 10% added ethanol (data not shown). The concentrations of ethanol required to inhibit 50% of fermentative activity were 6.5 and 9% (vol/vol), respectively, for 12- and 24-h cells.
The inhibition of fermentation caused by exposure to
aldolase Glycolytic flux (triose)b Triose phosphate isomerase Glyceraldehyde-3-phosphate
2.0 110 (2) 16 (1)
1.0 97 (2) 18 (1)
dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase Pyruvate decarboxylase Alcohol dehydrogenase
11 (1) 6.2 (0.5) 3.0 (0.2) 10 (3) 1.1 (0.2) 4.8 (0.4)
12 (1) 9.0 (0.8) 3.2 (0.4) 8.4 (1.4) 0.92 (0.04) 3.8 (0.4)
Cells were removed from batch fermentations after 12 and 24 h. Standard deviations are based upon determinations from three separate batch fermen- tations. a
b Glycolytic flux for hexose and triose intermediates was estimated from measurements of fermentation rate.
Glycolytic flux (hexose)b
TABLE 1. Specific activities of glycolytic enzymes at the peak of fermentative activity (12 h) and after a 50% decline (24 h)
Phosphoglucose isomerase Phosphofructokinase Fructose diphosphate
Sp act (pLmol/min per mg of protein) (SD)"
concentrations of ethanol above 5% (vol/vol) for 10 min was only partially reversed by resuspension in fresh medium lacking ethanol (Fig. 3), indicating that exposure to ethanol damaged the cells in some way. Again, 12-h cells appeared more sensitive to ethanol damage than 24-h cells. Longer incubation periods with 10% ethanol before washing resulted in further loss of activity, with only 40% of the original activity remaining after 2 h.
Changes in the levels of glycolytic and alcohologenic en- zymes during batch fermentation. The specific activities of the enzymes involved in alcohol production (under substrate saturating conditions) are listed in Table 1 for cells harvested after 12 h (the most active stage of fermentation) and 24 h (50% maximal activity). For comparison, the rates of glycolytic flux for hexose and triose intermediates have been included (based on rates of carbon dioxide evolution). The activities of all but three of these are clearly in excess of that required to support the measured rates of glycolytic flux. The exceptions were hexokinase at 12 h and phosphofructo- kinase and pyruvate decarboxylase at both 12 and 24 h. However, the true in vivo activities must be sufficient to support the measured rates of carbon dioxide evolution except for a small contribution from anabolic processes.
It is of interest to compare the relative activities of each e n z y m e a t t h e s e t w o t i m e s . A f t e r 2 4 h , g l y c o l y t i c f l u x h a d d e c l i n e d b y a p p r o x i m a t e l y h a l f w h i l e t h e s p e c i f i c a c t i v i t i e s o f s i x e n z y m e s r e m a i n e d u n c h a n g e d , f o u r h a d d e c l i n e d b y a p p r o x i m a t e l y 2 0 % , a n d t w o h a d i n c r e a s e d . F i g u r e 4 A illustrates the changes in the specific activities of these enzymes throughout batch fermentation, relative to that of 12-h cells (100%). An analogous plot of fermentation rate is included for comparison. None of the specific activities declined dramatically during batch fermentation. At times beyond 12 h, with the exceptions noted above, all enzymes were in excess of the measured fermentation rates. Phosphofructokinase declined to the greatest extent with a 20% drop in activity after 48 h. The specific activities of three enzymes increased by more than 20%: phosphogluco- mutase (100% increase), hexokinase (50% increase; not shown), and enolase (50% increase; not shown). All other e n z y m e s e x h i b i t e d a s i m i l a r i n c r e a s e o f u p t o 2 0 % . F i g u r e 4 B i l l u s t r a t e s t h e c h a n g e s i n t h e a m o u n t s o f t h e s e e n z y m e s p r e s e n t p e r m i l l i l i t e r o f b r o t h r e l a t i v e t o t h a t a t 1 2 h. Analogous plots of soluble cell protein and fermentative activity per milliliter are included for comparison. The peak
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