## 3 Results and Discussion  
### Cultivation of R. qingshengii IGTS8 Under Batch and Repeated Batch Modes  
_R. qingshengii_ IGTS8 was cultivated in a 2 L (working volume) stirred tank bioreactor with 20 g/L glycerol as the carbon source and 1.3 mM DMSO as the sulfur source [33]. A wide range of carbon sources have been reported in the literature for the preparation of the biocatalyst for a BDS process. Concerning the bacteria of the genus _Rhodococcus_, carbon sources such as glucose, glycerol, ethanol, acetate, glutamate, succinate, citrate and malate have been employed [31; 47; 49; 50; 51; 52]. The production cost of the biocatalyst is one of the drawbacks for the commercialization of the BDS process. In the present study glycerol was chosen as a carbon source since it is cheap and abundant [53]. DMSO has been used as a sulfur source for the growth of _R. qingshengii_ IGTS8, since it has been reported that DMSO does not repress the expression of deslufurization enzymes in the bacterium [11]. Initially, _R. qingshengii_ IGTS8 was cultivated under batch mode and, following completion of the batch process (at 80 h), the repeated batch fermentation strategy was applied, substituting 80% (\(v/v\)) of the culture broth with fresh medium. Two consecutive substitutions were made: the first (first cycle) at 80 h and the second (second cycle) at 126 h from the onset of the process. Repeated batch culture is a well-known method in fermentation technology [54; 55]. It consists of using a part of the culture as an inoculum into the next batch of fresh medium. Repeated batch culture offers many advantages over microbial batch culture, including the reuse of microbial cells for subsequent fermentation runs which results in a reduction in the labor and time required for inoculum preparation, washing and sterilization of the bioreactor, high cell concentration in the culture, low time required for process operation, and increased process productivity [55]. Figure 2 depicts the evolution of cell mass during the growth of _R. qingshengii_ IGTS8 in the batch and repeated batch modes. The batch cultivation of _R. qingshengii_ IGTS8 resulted in a maximum cell concentration of 2.62 \(\pm\) 0.04 g DCW/L, observed at 80 h of cultivation. The bacterium consumed about 26% of the initial glycerol in the medium with a cell yield (Y\({}_{\mathrm{x/s}}\)) of 0.46 \(\pm\) 0.03 g DCW/g glycerol. The biomass volumetric rate was estimated at the end of the batch process and was found to be equal to 0.03 \(\pm\) 0.001 g DCW/L/h (Table 1). In the first cycle of the repeated batch process, the cultivation period was shortened (45 h) and approximately 15% more cells (3.08 \(\pm\) 0.09 g DCW/L) were obtained and the bacterium consumed 30% of the initial glycerol (Y\({}_{\mathrm{x/s}}\) = 0.43 \(\pm\) 0.02 g DCW/g glycerol), while the productivity of the biomass was 2.3-fold higher than the batch culture (Table 1). The second replacement (second cycle) of the medium did not improve the process. Even though the time required to obtain the maximum biomass concentration was shortened (32.5 h), the biomass concentration was significantly decreased (Table 1). The biomass volumetric productivity in the second cycle decreased compared with the first one. Furthermore, Y\({}_{\mathrm{x/s}}\) declined to 0.24 \(\pm\) 0.02 g DCW/g glycerol despite the fact that the glycerol consumed was in the above range. This phenomenon during repeated batch cultivation could be attributed to the fact that in aerobic cultures, cells are particularly susceptible to senescence and metabolic activity is prone to decline [55]. The experimental data of biomass concentration with time were fitted to the logistic model for growth [10; 31; 52] as follows:  
\[\frac{dX}{dt}=\mu X\bigg{(}1-\frac{X}{X_{max}}\bigg{)} \tag{1}\]