In all HRTs, significant higher ethanol productions in the ultrasound-assisted fermentation process than in the control fermentation process were recorded (p<0.05). When the HRT was 12 h, the ethanol concentration without ultrasonic treatment was 9.87 g L-1 and it was significant lower by 2.85 g L-1 than the production in the process stimulated with low intensity ultrasounds (p<0.05) (Fig. 9). Lactose consumption was only 62.1%, but application of ultrasound increased it to 69.7% (p<0.05) (Fig. 10). The best results were obtained with the longest HRT of 36 h. Ethanol concentration increased to the value of 26.30 g L-1 when the culture has been sonicated, while in the fermentation process without ultrasound irradiation was only 23.60 g L-1 (p<0.05), (Fig. 9). Lactose consumption was as high as 98.9% in ultrasound-assisted fermentation unit and was significant higher by 6.5% than the consumption in the reactor without ultrasonic irradiation (p<0.05) (Fig. 10). High ethanol production and lactose consumption were observed with shortening HRT to 24 h. S. cerevisiae stimulated with low intensity ultrasound produced 24.85 g ethanol L-1, while the lactose consumption was 95.6% (Fig. 9-10). In the control fermentation unit there was 21.79 g L-1 and 89.5%, respectively. The differences were statistically significant (p<0.05). Under the HRT of 36 h, in the fermentation process with ultrasound irradiation the maximum ethanol yield of 0.532 g g-1 lactose was observed, whereas using biocatalyst S. cerevisiae without ultrasound exposure gave the result as 0.511 g g-1 (Fig. 11) (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated S. cerevisiae, but in the control fermentation process it was as low as 0.487 g g-1 (p<0.05). When the HRT was 12 h the ethanol yield was only 0.365 and 0.318 g g-1, respectively (p<0.05).

There were only few experiments investigating the enhancing ethanol production by ultrasonic stimulation of S. cerevisiae. Schlafer et al. (2000) improved biological activity of S. cerevisiae by low energy ultrasound assisted bioreactors operated at a frequency of 25 kHz and a power input of 0.3 W L-1. The ethanol production without ultrasonic treatment varied between 3-12 g L-1, while ultrasonic stimulation increased it to 30 g L-1. The highest ethanol concentrations were obtained with a cycle regime of ultrasound exposure and a pause, because during continuous ultrasound irradiation no stimulation in the ethanol fermentation process was recorded.

Lanchun et al. (2003) investigated the influence of low intensity ultrasound on physiological characteristic of S. cerevisiae. The results of their study showed, that ultrasounds in the frequency of 24 kHz and the power efficiency of 2 W with 1 s irradiation time every 15 s and 30 min duration cycle, stimulated the material transport and improved the cell’s metabolism by changing the osmotic pressure of membrane. Consequently, transfer of substance was speeded up, enzyme synthesis was driven up and enzyme activity was enhanced.

The positive results of the ultrasound treatment on the ethanol production by co­immobilized S. cerevisiae seemed to be a combination of different processes, including activating the yeast by improving the mass transfer rate of nutrients in the liquid, enhancing the uptake of foreign substances and the release of intracellular products in cells, improving the cell growth and degassing of CO2 (Lanchun et al. 2003; Liu et al., 2007; Liu et al. 2003b). Stimulating enzyme activity is done by increasing in the mass transfer rate of the reagents to the active site (Liu et al., 2007). Ultrasounds irradiation can cause thermal and mechanical stress to biological materials (Liu et al., 2003b). High energy ultrasonic waves break the cells

and denaturate enzymes (Liu et a!., 2007; Pitt & Ross, 2003). Low energy ultrasounds can produce a variety of effects on biological materials, including the inhibition or stimulation cellular metabolisms, enzyme activity, alteration of cell membranes and other cellular structures (Liu et al., 2007; Liu et al. 2003a). According to Xie et al. (2008), cavitation is the primary basis of biological effects of low intensity ultrasound. Cavitation bubbles produced by low intensity ultrasound can cause acoustic microstreaming (Xie et al., 2008). The microstreaming surrounding the cells can cause shear stress and enhance the mass transfer, which may stimulate metabolic activities inside the cells (Liu et al., 2003b; Pitt & Ross, 2003; Xie et al., 2008). When ultrasonic intensity is sufficiently low, a stable cavitation occurs and leads to the enhancement of mass transfer and fluid mixing, which produces positive effects on the rate of biological reactions in the exposure systems (Liu et al., 2007).

The growth activity of yeast cells is hardly changed within the early period of sonication regardless of either damage to cell wall, or complete inactivation of the yeast located in the cavitation zone (Tsukamoto et al., 2004). Short sonication time up to 5 min of irradiation indicated bactericidal effects, but the cells were able to repair the damages. According to Guerrero et al. (2005) yeasts, inclusive with S. cerevisiae, are highly resistant to ultrasound damage. Moreover, at relatively low intensity of ultrasounds, microorganisms can adapt to the irradiation exposure and their biological activity increases (Liu et al., 2007). With relatively short irradiation period, cell damage and membrane permeability induced by ultrasounds appear to be temporary and reversible. Lanchun et al. (2003) also stated that sonication cannot influence on fermentation strength of S. cerevisiae descendants.