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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1987, p. 1286-1291 0099-2240/87/061286-06$02.00/0 Copyright ©3 1987, American Society for Microbiology

Vol. 53, No. 6

Ethanol Production during Batch Fermentation with Saccharomyces cerevisiae: Changes in Glycolytic Enzymes and Internal pH

K. M. DOMBEK AND L. 0. INGRAM* Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 12 January 1987/Accepted 26 March 1987

During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.

Saccharomyces cerevisiae is used extensively in batch fermentations to convert sugars to ethanol for the production of beverages and biofuels. Despite the obvious importance of this process, the physiological constraints which limit the rate of glycolysis and ethanol production are not fully understood (7, 15, 23). Identification of these constraints represents an important step toward the development of improved organisms and process conditions for more rapid ethanol production. Such improvements could result in an increase in the ethanol production capacity of existing fermentation plants and a reduction in the cost of future facilities.

S. cerevisiae is capable of very rapid rates of glycolysis and ethanol production under optimal conditions, producing over 50 mmol of ethanol per h per g of cell protein (11, 12). However, this high rate is maintained for only a brief period during batch fermentation and declines progressively as ethanol accumulates in the surrounding broth (7, 15, 23). Earlier studies have identified a requirement for lipids (2, 8, 26) or molecular oxygen for lipid biosynthesis (1, 4, 5) in many fermentation broths as being essential for the mainte- nance of high fermentative activity. Magnesium is an essen- tial cofactor for many of the glycolytic enzymes and has also been identified as a limiting nutrient in fermentation broth containing peptone and yeast extract (11, 12). Supplying these nutritional needs reduces but does not eliminate the decline in fermentative activity during batch fermentation.

The basis for the decline in fermentation rate is not fully understood. Since the addition of ethanol to cells in batch cultures and in chemostats causes a dose-dependent inhibi- tion of ethanol production (7, 11, 12), most investigations have focused on ethanol as an inhibitor (7, 15, 22). Ethanol is known to alter membrane permeability and disrupt mem- brane function in a variety of biological systems (7, 15). In yeasts, ethanol causes an increase in hydrogen ion flux across the plasma membrane of cells suspended in water (6). This increased hydrogen ion flux has been proposed as being responsible for the ethanol-induced decline in transport rates observed under similar conditions (2, 17-19).

  • *

    Corresponding author.

Evidence has been accumulating which indicates that the presence of ethanol may not be the only factor responsible for the decline in fermentative activity. The replacement of fermentative broth containing ethanol with fresh medium l a c k i n g e t h a n o l d i d n o t i m m e d i a t e l y r e s t o r e f e r m e n t a t i v e a c t i v i t y ( 1 1 ) . I n a c o m p r e h e n s i v e s t u d y , M i l l a r e t a l . ( 2 2 ) demonstrated that concentrations of ethanol below 12% (vol/vol) do not denature glycolytic enzymes or cause appre- ciable inhibition of activity in vitro under substrate- s a t u r a t i n g c o n d i t i o n s . S i n c e e t h a n o l d o e s n o t a c c u m u l a t e w i t h i n y e a s t c e l l s b u t r a p i d l y d i f f u s e s a c r o s s t h e c e l l m e m - b r a n e ( 1 0 , 1 3 ) , d i r e c t i n h i b i t i o n o f g l y c o l y t i c e n z y m e s b y i n t r a c e l l u l a r e t h a n o l i s u n l i k e l y d u r i n g f e r m e n t a t i o n s w h i c h p r o d u c e 1 2 % ( v o l / v o l ) e t h a n o l o r l e s s . I n t h i s p a p e r , w e h a v e e x a m i n e d t h r e e p h y s i o l o g i c a l factors (glycolytic and alcohologenic enzymes, internal pH, and viability) as possible causes for the decline in fermenta- tive activity during batch fermentation.

MATERIALS AND METHODS

Organism and growth condition. S. cerevisiae KD2 (petite strain) was used in this study and was grown in complex medium supplemented with 0.5 mM magnesium sulfate as previously described (10, 12). Batch fermentations (initial optical density at 550 nm of 0.035; 0.01 mg of cell protein per ml) with 20% glucose were carried out in 300-ml Spinner bottles at 30°C with a 1% inoculum from a 12-h culture.

Analyses of fermentation broth. Cell protein was deter- mined by the method of Lowry et al. (20). Glucose, ethanol, and cell mass were measured as described (12).

Respirometry measurements. The rate of glycolysis and ethanol production was estimated by measuring carbon dioxide evolution with a Gilson differential respirometer (Gilson, Middleton, Wis.). This rate is taken as equivalent to glycolytic flux, assuming the production of 2 mol of ethanol and carbon dioxide per mol of glucose consumed. Results are expressed as micromoles of carbon dioxide evolved per hour per milligram of cell protein (12).

Enzyme analyses. Activities of glycolytic and alcoholo- genic enzymes were determined in 2-ml samples removed at various times during batch fermentation. Cells were har-

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