UF whey permeate (non-deproteinized, non diluted and non-sterilized) with the average lactose concentration of 50 g L-1 from the Dairy Plant in Nowy Dwor Gdanski, Poland, was used as a fermentation substrate (Table 2).

Continuous fermentation was carried out in the laboratory-scale plant consisted of the two UASB reactors with a working volume of 5 L each (Fig. 8). These two reactors were used to enable parallel test series with and without ultrasound irradiation. The fermentation medium was pumped continuously to the bottom part of the reaction tank by means of the peristaltic pumps. The necessary mixing was achieved through the upward wastewater flow. The reactors were water-jacketed and operated at a constant temperature of 30±1 °С. The pH of mixed liquid in the reactors was controlled automatically at pH 5.1 ± 0.2 with 2 M NaOH.

The reactors were inoculated with 40% (v/ v) solid beads containing the immobilized cells which corresponded to 39.4 g cells dry weight — DW L-1 of working bioreactor volume. After adding the cell beads inoculum to the bioreactors, before starting continuous feeding, a batch fermentation was conducted for 24 h under additional gentle agitation (100 rpm). Next the reactors worked at different HRTs of 12, 24 and 36 h. At each HRT the reactor was operated till it has reached the steady-state (the steady-state conditions were evidenced when the standard deviations of the ethanol and lactose concentrations in the effluent distillate were within 3%), thus 30 days of each fermentation step (step 1 — HRT of 12 h, step 2 — HRT of 24 h, step 3 — HRT of 36 h). The fresh inoculum was added to the reactors before each fermentation step and the aged one was removed.

The ultrasound irradiation of the reactor with yeasts was made by a special ring with a transducer (Intersonic S. C. Poland) that was attached at the bottom of the reactor. The range of the frequency generator was adjustable between 20-25 kHz and the maximum power of 50 W. The experiments were carried out with the stable sonication power of 1 W L-1 and the frequency of 20 kHz.

3.2.1.3 Analytical methods

Lactose and ethanol concentrations in the effluent distillate were determined according to Standard Methods (PN-67/A-86430; PN-A-79528-3:2007). The samples were analyzed in triplicates and results were reproducible within 3% deviation.

All fermentation steps connected with different HRTs were carried out in triplicate. Significant differences between the effects obtained in the two reactors with and without ultrasound exposure were analysed using an ANOVA F-test (Statistica 7.1 software, Statsoft Inc.) A 5% probability level was applied for all the tests. If p<0.05 from an ANOVA F-test, the differences between the effects were considered to be significantly different from one another.