## 3 Results and Discussion
### Cultivation of R. qingshengii IGTS8 Under Fed-Batch Mode  
The fundamental feature of fed-batch cultivation is that the concentrations of nutrients fed into the bioreactor can be controlled by manipulating the feed rate. Fed-batch cultivation can be carried out by the continuous or sequential feeding of the nutrients. Compared with batch cultivation it is advantageous since it can eliminate substrate-associated growth inhibition and/or achieve high cell density cultures. The performance of a fed-batch process is highly dependent on the feeding strategy applied [58]. Several fed-batch strategies, including indirect control methods that couple substrate feeding with the measurement of pH or dissolved oxygen (DO) have been reported in the literature [47; 58; 59]. However, the response of the cells to the changes in pH or DO due to substrate utilization is slow and that can cause over- or under-feeding, affecting the final production. The direct control of the carbon source is one of the most efficient feeding strategies to control commercial-scale bioprocesses. By measuring carbon source concentration regularly, in the bioreactor (offline or online), under- or over-feeding during the cultivation can be prevented [58]. In the present study a fed-batch process was established with _R. qingshengii_ IGTS8 in a stirred tank bioreactor with the following characteristics:  
* Due to the poor assimilation of glycerol (carbon source) in batch and repeated batch cultivation, the initial glycerol concentration in the bioreactor was set at 5 g/L and was maintained relatively constant during the culture process (<6 g/L) by feeding glycerol manually (pulse feeding)
* The growth and desulfurization activity of _R. qingshengii_ IGTS8 is strongly dependent on the availability of oxygen [52; 60], and thus the DO will be maintained above 20% of saturation, adjusting the air flow and agitation rate in the bioreactor. The concentration of glycerol in the feed was 40 g/L. The process performance is shown in Figure 3.  
During the initial stage of batch growth of the bacterium, air flow and agitation speed were maintained constantly (air flow 0.8 L/min and 200 rpm). As shown in Figure 3b, DO rapidly decreased and at the beginning of the fed-batch process was approximately at 35% saturation, indicating active cell metabolism. The cell mass concentration at the end of the batch process was 1.3 \(\pm\) 0.4 g DCW/L (Figure 3a) and Y\({}_{\mathrm{x/s}}\) was estimated at 0.26 \(\pm\) 0.03 g  
Figure 3: Fed-batch culture of _R. qingshengii_ IGTS8. (**a**) Time course of biomass (\(\bullet\)) and residual glycerol (\(\Circle\)) concentrations. (**b**) The curves of parameters monitored online. DCW/g glycerol. CDA increased during the initial batch stage reaching an average of 22.6 \(\pm\) 0.5 U/mg DCW at the onset of the fed-batch process. The fed-batch process started at 21 h of cultivation, when the glycerol was almost exhausted (glycerol concentration in the culture medium \(<\) 1 g/L). The whole process lasted for 93 h (Figure 3a). The biomass concentration during the fed-batch process increased to 8.35 \(\pm\) 0.17 (at 89 h) g DCW/L. After the start of the fed-batch process, the glycerol concentration in the culture broth remained between 0.8 and 6.0 g/L and was randomly distributed (Figure 3a). Contrary to the batch and repeated batch cultivations, where the consumption of glycerol was low, approximately 87% of the glycerol fed to the bioreactors was assimilated by the bacterium. The biomass yield (g DCW/g glycerol) during the initial stages of the fed-batch process was higher compared with the batch stage, while it decreased at the end of the process. More specifically, up to 48 h total cultivation time (20 h from the initiation of the fed-batch process) the biomass yield was 0.55 \(\pm\) 0.05 g DCW/g glycerol, while at the end of the process the respective value declined to 0.41 \(\pm\) 0.04 g DCW/g glycerol.