*2.1. Operating Mode*

A diagram showing the operation of two bioreactors working serially is depicted in Figure 1, where the process involved the following operations:


**Figure 1.** The two serial reactors operating in a semi-continuous mode.

This scheme allowed the following operational variables to be changed: ethanol concentration at the time the first reactor was unloaded (*Eu*1), volume unloaded from the first reactor (*Vu*1), the ethanol concentration while the first and second reactor were loaded (*El*<sup>1</sup> and *El*2, respectively), and the temperature in each reactor (*T*<sup>1</sup> and *T*2, respectively). A total of six operational variables were thus involved. Although the alcoholic strength of the wine and the airflow rate could also have been used as variables, the feed wine usually contains 12% (*v*/*v*) ethanol, and the airflow rate is usually as high as possible (especially when productivity is to be maximized). Also, using gas condensers minimized the sweeping of volatiles.

Except for the temperatures, the working range for the previous variables could not be freely chosen because each variable was subjected to physical limitations, especially because the serial operation of the two reactors imposed constraints arising from the mutual relationships between the variables. This required careful planning of the experimental work in order to define the number of experiments needed to obtain accurate information with a view to predicting the influence of each variable on the resulting system behavior. As described in Section 3.1, a fractional factorial design for this purpose was used [36].
