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VOL. 53, 1987

ETHANOL PRODUCTION BY S. CEREVISIAE

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FIG. 4. Changes in the levels of glycolytic and alcohologenic enzymes during batch fermentation with 20% glucose. Cells were removeo from various stages of fermentation and disrupted, and the activities of individual enzymes were determined under substrate-saturating conditions. Values are expressed relative to 12-h cells, the time at which the highest activity per milligram of cell protein was observed. Bars denote a representative standard deviation for an average of three determinations. (A) Changes in the specific activities of representative enzymes. (B) Changes in the activities per milliliter of culture of representative enzymes. For comparison, analogous plots of the changes in fermentation rate (A and B) and the changes in the amount of soluble cell protein (B only) have been included. Symbols: A, phosphoglucomutase; r, glyceraldehyde-3-phosphate dehydrogenase; *, triose phosphate isomerase; 0, phosphofructokinase; *, glycolysis; *, soluble cell protein.

of fermentative activity on a volumetric basis occurred after 18 h. Although the rate of fermentation declined beyond 18 h, the activities of all of the glycolytic enzymes continued to increase until 30 h, the peak of soluble proteins. These increases in activities roughly paralleled the increases in soluble proteins. The activities of phosphoglucomutase, enolase (not shown), hexokinase (not shown), and glyceral- dehyde-3-phosphate dehydrogenase increased more rapidly than soluble cell protein, consistent with the observed in- creases in specific activities of these enzymes during fermen- tation. With the exception of phosphoglucomutase, which declined more rapidly, the rates of decline of the glycolytic enzyme activities per milliliter paralleled that of the bulk soluble cell proteins, indicating neither a preferential reten- tion nor degradation of these central catabolic activities.

batch fermentation (Fig. SA). Similarly, At also increased during batch fermentation, resulting in an overall increase in proton motive force (Fig. SB).

These results were somewhat surprising since previous workers have shown that ethanol increases the permeability of yeast suspended in water to hydrogen ions (6). To further examine this point, we determined the effect of added ethanol on the internal pH of cells from various stages of fermentation (Fig. 6). Ethanol concentrations of 15% (vol/vol) or above were required to cause a measurable decline in internal pH. The addition of 20% (vol/vol) ethanol to 12- and 24-h cells caused a complete collapse of ApH. The ApH of cells from 36- and 48-h cultures was considerably more resistant to 20% (vol/vol) added ethanol, consistent with an adaptation of older cells.

Changes in internal pH and membrane energization during batch fermentation. Although ethanol is the principal, re- duced fermentation product from the metabolism of glucose by S. cerevisiae, organic acids are also produced which lower the external pH of the fermentation broth to pH 3.5 (Fig. SA). Since the pH optima for glycolytic enzymes are near neutrality or above (9), the failure of S. cerevisiae to maintain a large ApH during the accumulation of ethanol could explain the rapid decline in the fermentative activities of cells despite the abundance of glycolytic and alcohologenic enzymes. However, this does not appear to be the case. The ApH of yeast cells increases coincident with the decrease in the external pH, maintaining a relatively constant internal pH of between 6.7 and 7.0 throughout

DISCUSSION

The fermentation of glucose to ethanol represents a series of coordinated enzymatic reactions. This process is inter- nally balancing and thermodynamically favorable provided that cellular enzymes consume the net ATP generated from substrate-level phosphorylation. The requirements for this process include glucose, functional enzymes, coenzymes (NAD+, thiamine pyrophosphate, ADP, ATP), cofactors (Mg2+, Zn2+), appropriate internal pH, a functional mem- brane to maintain the concentration of reactants and en- zymes, and a glucose uptake system. Indeed, fermentation can proceed well in concentrated preparations of disrupted cells (14, 27).

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FIG. 5. Changes in internal pH and membrane energization during batch fermentation on 20% glucose. Bars denote a representative standard deviation for an average of three determinations. (A) Internal pH (0), external pH (-), and ApH (A). (B) Membrane energization. Symbols: 0, proton motive force; *, ApH; A, APV.

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