## 3 Results and Discussion
### Biodesulfurization of DBT, 4 MDBT and Their Mixture in a Bubble Column bioreactor  
Previously we developed a two-phase bubble column bioreactor system which was used for desulfurization experiments by resting cells [33]. The application of resting cells in BDS offers many advantages, among them greater desulfurization efficiencies compared with BDS conducted simultaneously with the growth of the microorganism [11]. Biodosulfurization by resting cells requires intermediate steps before the cells can be employed in the biphasic system, such as the harvesting of the cells by centrifugation, and resuspension in a suitable aqueous medium in order to obtain the desired cell concentration. The elimination of those steps could make the BDS process cost effective. Cells derived from the fed-batch culture were used directly for the biodegradation of DBT (1.3 mM), 4-MDBT (0.9 mM) and a mixture of the above (0.7 mM DBT and 0.5 mM 4-MDBT) in the two-phase bubble column bioreactor. The OFP was set at 50% (\(v/v\)) while the biomass concentration in the aqueous phase was 8.35 g DCW/L (as derived from fed-batch culture, Figure 3a). The desulfurization efficiency for DBT and 4-MDBT was expressed as follows [33]:  
\[Y_{BDS}(\%)=\left(\frac{C_{\text{OSC},0}-C_{\text{OSC},t}}{C_{\text{OSC},0}} \right)\cdot 100 \tag{3}\]  
where \(Y_{BDS}\) (%) is the desulfurization efficiency, C\({}_{\text{OSC},0}\) (mM) is the initial OSC concentration and C\({}_{\text{OSC},t}\) (mM) is the concentration at time t  
The maximum percentage of desulfurization of the cells for DBT reaction was calculated according to the following equation [33]:  
\[X_{BDS}(\%)=\frac{C_{2-HBP,\ max}}{C_{\text{DBT},\ 0}}\cdot 100 \tag{4}\]  
where \(X_{BDS}\)(%) is the maximum percentage of desulfurization, C\({}_{\text{DBT},0}\) is the initial concentration of DBT (mM) and C\({}_{\text{2-HBP},\text{max}}\) (mM) is the maximum amount of 2-HBP produced. Figure 5 depicts the time course of the desulfurization of the OSCs studied in the present work. Bacterial cells completely desulfurized DBT and 4-MDBT (\(Y_{\text{BDS}}\) = 100% for both OSCs) within 18 hr (Figure 5a) and 24 h (Figure 5b), respectively, in the single systems. It should be noted though that approximately 70% of the DBT was desulfurized within the first 5 h of reaction, while at the same time point the respected value for 4-MDBT was 42%. Even if DBT was completely desulfurized, the maximum percentage of desulfurization of the cells (\(X_{\text{BDS}}\)) was 88.7% (Table 2). Specific desulfurization rates as well as specific product formation rates (q\({}_{\text{p}}\)) were calculated from the linear parts of the curves (linear part between 0 and 5 h) depicted in Figure 5. DBT exhibited a desulfurization rate approximately 2.5 times higher compared \begin{table}
\begin{tabular}{l c c c c} \hline \hline \multirow{2}{*}{**Substrate Concentration**} & \multirow{2}{*}{\(\mathbf{Y_{BDS}\) (\%)} & \multirow{2}{*}{\(\mathbf{X_{BDS}\) (\%)}} & \multicolumn{2}{c}{**Specific**} & \multirow{2}{*}{\(\mathbf{q_{p}}\) (mmoles} \\  & & & & **Desulfurization Rate** \\  & & & **(mmoles Cx-DBT/kg** \\  & & & **DCW/h)** & **2-HPB/kg** \\ \hline
1.4 mM DBT & 100 & 88.7 \(\pm\) 1.2 & 21.6 \(\pm\) 0.6 & 17.8 \(\pm\) 0.8 \\ \hline
0.7 mM DBT + 0.4 mM 4-MDBT & & & & \\ \hline DBT & 59.6 \(\pm\) 3.2 & 30.6 \(\pm\) 0.9 & 2.8 \(\pm\) 0.3 & 1.3 \(\pm\) 0.1 \\
4-MDBT & 41.2 \(\pm\) 4.4 & - & 3.1 \(\pm\) 0.4 & - \\ Overall & 52.9 \(\pm\) 3.6 & - & 5.8 \(\pm\) 0.3 & - \\ \hline
0.9 mM 4-MDBT & 100 & - & 8.4 \(\pm\) 0.5 & - \\ \hline \hline \end{tabular}
\end{table} 
Table 2: \(\mathbf{Y_{BDS}\), \(\mathbf{X_{BDS}}\), specific desulfurization rate and specific product formation rate (q\({}_{\text{p}}\)) during desulfurization of DBT, 4-MDBT and a mixture of DBT + 4-MDBT in a two-phase bubble column bioreactor. Figure 5: Biodosulfurization of DBT, 4-DMDBT or mixture thereof by _R. qingshengii_ ICTS8 cells derived directly from the fed-batch culture.