Inoculum Preparation

S.cerevisiae (isolated from pressed yeast), maintained on slant tubes containing (per liter) nutrient agar Difco 23.0g and glucose 1.0g, was transferred to test tubes containing 5.0 mL of growth medium (peptone 5.0g, glucose 10.0g, yeast extract 3.0g and distilled water 1.0L; pH adjusted to 4.5) and incubated at 33°C for 48 h. Nine 250 mL-erlenmeyer flasks containing 50 mL molasses medium were inoculated with five tubes of cell suspension, and left incubating at 33°C for 22 h in a rotary shaker (frequency of 120 min−1).

Batch Fermentation

Tests were carried out in a 5-L bench fermenter (NBS-MF 200 coupled with a NBS-dissolved oxygen controller, D0-81) filled with 2.55 L of sterilized blackstrap molasses (Total Reducing Sugars (TRS): 145-150 g/L, clarified (Vitolo et al., 1985) and supplemented with 5.1 g/L (NH4)2SO4, 2.4 g/L Na2HP04.12H2O and 0.075 g/L MgS04.7H2O). The following standard culture conditions were adopted: Impeller speed = 500 min−1; Antifoam: 0.1 mg/L dimethylpolysiloxane (added dropwise as needed); Temperature = 30°C. The mash was inoculated with 0.45 L of S.cerevisiae suspension (10.Og dry matter/L) and left fermenting until TRS was negligible. The pH was controlled during fermentation at 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5. At regular time intervals (1 hour) aliquots (10.0 mL) of the culture medium were drawn for analytical purposes. The cells used for invertase activity determination and protein release from the cell wall were separated by centri fugation (5,000 xg; 15 min) and rinsed twice with distilled water. Then, the cells were resuspended in distilled water in order to obtain a known volume.

Measurement of Invertase Activity

The determination of invertase activity was carried out at 37°C in a mixture of 1.5 mL 0.01M acetate-acetic acid buffer (pH 4.6), 2.5 mL 0.3M sucrose solution and 0.5 mL invertase solution or cell suspension (both adequately diluted to assure that less than 2.0% of the sucrose present in the solution would be hydrolyzed). After 3 min, the hydrolysis was stopped by adding 1.0 mL of the Somogyi alkaline reagent (Somogyi, 1952), quickly followed by immersion in a boiling water bath for 10 min. The TRS concentration was then measured as described previously (Vitolo and Borzani, 1983). One invertase unit (U) was defined as the amount of enzyme catalyzing the formation of 1g of TRS per hour at pH 4.6 and 37°C. Specific invertase activities were expressed as U/mg of protein (soluble invertase, released from yeast cell wall) and U/g dry cell (insoluble invertase).

Measurement of Cell and Ethanol Concentrations

The determination of ethanol and cell concentrations were carried out as described previously (Vitolo et al., 1991).

Protein Release from Cell Wall

Induced release of extracellular protein from the cell wall was achieved as follows: 200mg of cells were suspended in 5mL of a 50mM potassium phosphate buffer (pH 7.0) containing 15 mM 2-mercaptoethanol and 15 mM dithiothreitol. The suspension was incubated for 2 h at 35°C, followed by centrifugation (10,000xg; 15 min) at 4°C. The protein content in the supernatant phase was measured (Bradford, 1976).

Table 1

Variation of invertase activity (v), cell (X) and TRS concentrations in a batch culture carried out at pH 5.5.

Table 2

The specific invertase activity (v’) and protein concentration in the cell-free extract (the cells were harvested from batch culture carried out at pH 5.5).

Table 3

Variation of invertase and ethanol production against the pH of the culture medium. The protein concentration and v’ in the cell-free extract are also presented (the cells were harvested at the end of fermentation).

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